Genome Instability and Transgenerational Effects [1 ed.] 9781617616631, 9781608768318

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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

GENETICS – RESEARCH AND ISSUES SERIES

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

GENOME INSTABILITY AND TRANSGENERATIONAL EFFECTS

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Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

GENETICS – RESEARCH AND ISSUES SERIES Bacterial DNA, DNA Polymerase and DNA Helicases Walter D. Knudsen and Sam S. Bruns (Editors) 2009. ISBN: 978-1-60741-094-2 Genetic Diversity Conner L. Mahoney and Douglas A. Springer (Editors) 2009. ISBN: 978-1-60741-176-5 Genetic Diversity Conner L. Mahoney and Douglas A. Springer (Editors) 2009. ISBN: 978-1-60876-541-6 (Online)

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Sex Chromosomes: Genetics, Abnormalities, and Disorders Cynthia N. Weingarten and Sally E. Jefferson (Editors) 2009. ISBN: 978-1-60741-304-2 The Human Genome: Features, Variations and Genetic Disorders Akio Matsumoto and Mai Nakano (Editors) 2009. ISBN: 978-1-60741-695-1 Genotoxicity: Evaluation, Testing and Prediction Andor Kocsis and Hajna Molnar (Editors) 2009. ISBN: 978-1-60741-714-9 Cystic Fibrosis: Etiology, Diagnosis and Treatments Paul N. Leatte (Editor) 2009. ISBN: 978-1-60741-833-7 Molecular Polymorphism of Man: Structural and Functional Individual Multiformity of Biomacromolecules Sergei D. Varfolomyev and Gennady E. Zaikov (Editors) 2009. ISBN: 978-1-60741-843-6

Viral Gene Expression Regulation Eli B. Galos (Editor) 2010. ISBN: 978-1-60741-224-3

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Epigenetics: Mechanisms, Functions and Human Effects Balázs Pintér and Zsolt Mészáros (Editors) 2010. ISBN: 978-1-60741-454-4 Genes and Nutrition Viroj Wiwanitkit 2010. ISBN: 978-1-60741-428-5 Metabolomics: Metabolites, Metabonomics, and Analytical Technologies Justin S. Knapp and William L. Cabrera (Editors) 2010. ISBN: 978-1-61668-006-0 Phylogeography: Concepts, Intraspecific Patterns and Speciation Processes Damien S. Rutgers (Editor) 2010. ISBN: 978-1-60692-954-4

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Phylogeography: Concepts, Intraspecific Patterns and Speciation Processes Damien S. Rutgers (Editor) 2010. ISBN: 978-1-61668-869-1 (Online) Discrete Optimization for TSP-like Genome Mapping Problems D. Mester, D. Ronin, M. Frenkel, A. Korol, Z. Bräysy, O. Dullaert, and W. Raa 2010. ISBN: 978-1-61668-170-8 Discrete Optimization for TSP-like Genome Mapping Problems D. Mester, D. Ronin, M. Frenkel, A. Korol, Z. Bräysy, O. Dullaert, and W. Raa 2010. ISBN: 978-1-61668-374-0 (Online) The Power of the Gene: The Origin and Impact of Genetic Disorders Chris Murgatroyd 2010. ISBN: 978-1-60876-949-0 Genetic Screening of Newborns: An Ethical Inquiry Carlos Valverde (Editor) 2010. ISBN: 978-1-60876-068-8 Genome Instability and Transgenerational Effects Igor Kovalchuk and Olga Kovalchuk (Editors) 2010. ISBN: 978-1-60876-831-8

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

GENETICS – RESEARCH AND ISSUES SERIES

GENOME INSTABILITY AND TRANSGENERATIONAL EFFECTS

IGOR KOVALCHUK AND Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

OLGA KOVALCHUK EDITORS

Nova Science Publishers, Inc. New York Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Transgenerational genome instability / editors, Igor Kovalchuk and Olga Kovalchuk. p. ; cm. Includes bibliographical references. ISBN:  (eBook)

1. Mutation (Biology) 2. Variation (Biology) 3. Epigenesis. I. Kovalchuk, Igor. II. Kovalchuk, Olga, MD. [DNLM: 1. Genomic Instability--genetics. 2. Heredity--genetics. QS 677 T772 2009] QH460.T73 2009 572.8'77--dc22 2009040795

† New York

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CONTENTS Preface Chapter 1

Chapter 2

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Chapter 3

ix Genetic and Epigenetic Regulation of Transgenerational Changes in Genome Stability: An Overview Olga Kovalchuk and Igor Kovalchuk

1

Genetic and Epigenetic Mechanisms Ensuring Stability of Mammalian Genomes Jody Filkowski and Olga Kovalchuk

7

Genetic and Epigenetic Regulation of Genome Stability in Plants Alex Boyko and Igor Kovalchuk

33

Chapter 4

Evolution of the Fpg/Nei Family of DNA Glycosylases Dorothy E. Pumo, Ramiro Barrantes-Reynolds, Scott Kathe, Susan S. Wallace and Jeffrey P. Bond

71

Chapter 5

Stress-Induced Mutagenesis in Bacteria Andrey Golubov

89

Chapter 6

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Loci as an Example of Bacterial Adaptive Immunity Against Phages Igor Kovalchuk and Andrey Golubov

Chapter 7

Chapter 8

Chapter 9

White Tail Tip Inheritance in Mice: From the ―Kit Locus‖ to a New Mode of Heredity Minoo Rassoulzadegan Role of Epigenetic Changes in the Non-Targeted Radiation-Induced Effects: Understanding the Epigenetics of Bystander and Transgenerational Effects Jody Filkowski, Yaroslav Ilnytsky and Olga Kovalchuk A Review of Tandem Repeat Sequences: Markers of Genetic Instability Carole Yauk and John D. H. Stead

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105

113

131

151

viii Chapter 10

Transgenerational Genomic Instability in Fish Carmel Mothersill and Colin Seymour

Chapter 11

Germ Cell-Mediated Mutagenesis: Insights from the  Transgenic Medaka Richard N. Winn

Chapter 12

Transgenerational Genomic Instability in Mammals Carmel Mothersill and Colin Seymour

Chapter 13

The Epigenetic (Re)Programming of Phenotypic Differences in Behavior Patrick O. McGowan, Michael J. Meaney and Moshe Szyf

Chapter 14

Lifestyle Factors, Epigenetic Abnormalities, and Genomic Instability: Implications for Carcinogenesis Igor P. Pogribny

Chapter 15

Paramutations in Plants Palak Kathiria and Igor Kovalchuk

Chapter 16

Transgenerational Response to Stress in Plants Involves Genome Destabilization, Changes in Methylation and Increase in Stress Tolerance Igor Kovalchuk, Palak Kathiria and Alex Boyko

Chapter 17

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Contents

Chapter 18

Chapter 19

169

199 225

263

291 309

321

Plant Transposable Elements and Plasticity of Host Genomes: The Stress Connection Marie-Angèle Grandbastien

343

Intra- and Interplant Stress Signalling: Bystander-Like Effects Found in Plants Franz J. Zemp

377

Systemic Heuristic Approaches Guide the Interaction of Enhanced Genetic Diversity and Complex Stresses to Generate Better Wheat Germplasm Faster and at Lower Cost A. Comeau, V. R. Caetano, S. Haber, F. Langevin, M. Lévesque and J. Gilbert

Index

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447

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PREFACE Chapter 1 - Genome stability of every species depends on complex interaction of predefined and environmentally induced genetic and epigenetic states. Predefined states consist of chromatin structure and cell metabolic processes such as DNA repair, radical scavenging and cell signalling, whereas induced states depend on interactions with the environment. Organisms are able to respond to a changing environment by various alterations in their somatic cells as well as in their germline and progeny. This book, will describe various phenomena associated with the maintenance of genome stability. These include genetic and epigenetic responses to various stresses in exposed cells and organisms, bystander and, bystander-like effects, transgenerational changes in genome stability and stress tolerance in bacteria, plants and animals. Chapter 2 - Maintenance of genome stability is a key to a disease-free survival of mammalian organisms. Mammalian cells are affected by a wide variety of endogenous and exogenous DNA damaging agents. To effectively deal with DNA damage, mammalian cells harbor a versatile network of repair, cell cycle control and genome maintenance mechanisms, since failure to eradicate DNA damage may lead to genome instability. Yet, the correct function of these and other cellular mechanisms heavily depends upon gene expression and organization as well as on the accessibility of DNA for DNA-protein interactions. These domains are governed by epigenetic processes—meiotically heritable and mitotically stable alterations in gene expression that include DNA methylation, histone modification, and RNA-associated silencing. This chapter will introduce some of the key strategies mammalian cells have evolved to maintain the integrity of their heritable information and will describe some interconnected genetic and epigenetic tactics that frequently work in concert to achieve a common goal of maximum stability of the genome. Chapter 3 - Maintaining genome stability is one of the most critical tasks for each and every living organism. During their life span, organisms are constantly exposed to a variety of internal and external stimuli, namely stresses, which can alter genome stability and lead to heritable changes. Over the past several years, a number of naturally occurring stresses were revealed to affect genome stability. They include: light spectrum and the day length, various types of ultraviolet and ionizing radiation, chemical mutagens and herbicides, temperature, salt, water stress and pathogen attacks. In fact, many of those stresses can change genome stability at both genetic and epigenetic levels, inducing rearrangements and altering existing DNA methylation patterns. Control and/or prevention of these changes is an issue of vital importance as they exert their influence on organisms experiencing stress, and they can be

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Igor Kovalchuk and Olga Kovalchuk

transmitted to progeny altering genome stability for several generations. The epigenetic regulation, including DNA methylation, histone acetylation/methylation and chromatin remodeling, play a key role not only in controlling gene expression but also in preventing undesirable rearrangements and transposon activation. The presence of high amounts of heterochromatic DNA containing various repetitive elements, pseudo genes, and virusderived sequences that often carry strong promoters and/or enhancers emphasizes the importance of epigenetic mechanisms in controlling genome stability. The system of epigenetic DNA modifications relies on a complex network of various protein factors, including numerous DNA and histone methyltransferases, histone deacetylases, methyl-CpGbinding domain proteins etc., many of which are guided via multifaceted systems of small RNA molecules. Overall, these systems permit sequence-specific changes in DNA epigenetic modifications, which allow adjusting transcriptional profiles of cells to new conditions and also modifying genome stability, if needed. This chapter is focused on various aspects and mechanisms involved in the maintenance of genome stability. It also discusses how changing the stability of a given locus may contribute to genome evolution and the ability to adapt to stress. Chapter 4 - Oxidative damage to DNA results in lesions such as thymine glycol, 5hydroxycytosine, formamidopyrimidine and 8-oxoguanine that have very different structures. Base excision repair is the main biological process for repairing DNA damage caused by oxidative stress. During the first step of base excision repair, an oxidative DNA lesion is recognized and excised by a DNA glycosylase. The Fpg/Nei family of DNA glycosylases is one of two families, each containing members that have distinct substrate specificities which collectively excise the oxidative lesions listed above. Events in the Fpg/Nei phylogeny include acquisition of novel substrate specificity, horizontal transfer, changes in a major structural motif (a zinc finger), and expansion within a bacterial clade. Importantly, the function of one clade, Fpg2, remains unknown. Chapter 5 - Stress-induced mutagenesis in bacteria has been puzzling researchers for decades. Bacteria regularly encounter a variety of chemical, physical and biological stressors. Some bacteria manage to survive and proliferate, very often giving rise to cell populations with new phenotypes. Numerous attempts have been made to study mutagenic factors and their influence on microbial physiology, genome stability and evolution. These attempts have raised more questions than answers. A classical example is the fact that until now there is no unambiguous theory regarding mechanisms of adaptive mutation in a Lac- strain of Escherichia coli. This chapter will attempt to outline the influence of main mutagenic factors on bacterial physiology and the bacteria‘s ability to adapt to these factors. Chapter 6 - Bacteria and archaea lacking sexual reproduction need to use a variety of mechanisms for gene exchange/gene acquisition. These mechanisms of horizontal gene transfer (HGT) in bacteria and archaea include phage transduction, transformation, and conjugation. At the same time, bacteria require protection against their ancient enemies, phages. One of the most interesting mechanisms of protection is based on the function of regulatory RNAs encoded by clustered regularly interspaced short palindromic repeat (CRISPR) loci to confer sequence-directed immunity against phages. According to recent publications, this is an active protection mechanism by which prokaryotes integrate short fragments of phage/viral nucleic acids into clusters of CRISPRs. As such, this process represents an ancient adaptive mechanism of protection against pathogens.

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Chapter 7 - In contrast with a wide definition of ‗epigenetic variation‘, including all changes in gene expression that do not result from the alteration of gene structure, a more restricted class has been defined, initially in plants, under the name ‗paramutation‘. It corresponds to epigenetic modifications distinct from regulatory interactions of cell differentiation pathways, which are meiotically stable and sexually transmitted by nonMendelian ratios. This class of epigenetic changes appeared for some time restricted to the plant world, but examples of epigenetic inheritance progressively accumulate in organisms ranging from mice to humans. Occurrence and possible mechanisms of paramutation in the mouse were first established with a tail color alteration in the paradigmatic case of a mutant phenotype maintained and hereditarily transmitted by genotypically wild-type homozygotes. Studies in the mouse point to a new role of RNA as an inducer and hereditary determinant of epigenetic variation. Given the known presence of a wide range of RNAs in human spermatozoa as well as a number of unexplained cases of familial disease predisposition and transgenerational maintenance, one may consider a role of RNA-mediated inheritance in mammals. A possible involvement in evolutionary processes makes for interesting speculations. Chapter 8 - Ionizing radiation, in addition to being an important treatment modality, is a potent tumor causing agent. The resultant risk of secondary radiation treatment-related cancers is a growing clinical problem. Some studies link secondary radiation-induced cancers to an enigmatic phenomenon of bystander effects whereby exposed cells signal damage and distress to their naïve neighbors resulting in genome instability. There is also welldocumented evidence that radiation exposure leads to transgenerational genome instability in the offspring of exposed parents. The exact molecular mechanisms of these indirect/nontargeted radiation effects have yet to be defined; however, recent evidence suggests that they may be epigenetic in nature. Epigenetic phenomena seem to be mediators of indirect radiation effects, including radiation-induced genome instability, bystander and transgenerational effects. DNA methylation and histone modification changes directly impact chromatin packaging and therefore influence gene expression and susceptibility of DNA to rearrangements. Short RNAs (such as microRNAs and piRNAs) may potentially be acting as key mediators of these non-targeted effects due to their small size, relative stability, their roles in maintenance of gene expression, stability of transposable elements, and the genome as a whole. This chapter will summarize the current knowledge of the existence of non-targeted radiation-induced bystander and transgenerational effects and the roles of epigenetic changes in their initiation and maintenance. Chapter 9 - The study of germline mutations is extremely difficult because rates of mutation are extremely low for gene sequence DNA. An alternative approach uses highly variable non-coding regions of the genome that possess rates of mutations that are orders of magnitude greater than protein-coding sequences. Mutations result from gains and losses of repeat units that arise during replication, recombination and repair. Germline mutation frequencies range from 1 – 20% and facilitate the measurement of induced mutations in small sample sizes. Mutations can be detected in pedigrees using Southern blotting, or by PCR analysis of sperm DNA. Expanded simple tandem repeat loci (ESTR) and minisatellites have been shown to undergo elevated rates of mutation following exposure to radiation and chemicals. As such, the study of repeat mutations in populations exposed to toxicants in the environment showed that induced germline mutations may result following exposure to

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ambient levels of contaminants. This chapter reviews the current state of knowledge in this field of study and provide an overview of repeat types and structure, the mechanisms of repeat mutations, the methods used to study mutations and discuss some experiments applying repeat analysis to study germline mutations. Chapter 10 - This chapter will review the data for somatic and germ line transgenerational effects in fish. The emphasis will be on radiation studies with reference, where appropriate, to chemical or mixed exposure studies. The terms will be defined as used in most of the literature with discussion of confusing elements. There are two main sources of data for fish transgenerational studies. These are the use of fish in the laboratory as models for human studies and the study of impacts of radiological accidents or planned exposures on fish as critical elements in the aquatic ecosystem. Both data sets will be reviewed. Comparative studies of mammalian and fish radiation responses are rare but important for both mechanistic understanding and environmental risk assessment. Common assumptions are that fish are ―radioresistent‖. The basis for this will be reviewed and discussed in detail. Work from the authors‘ laboratory concerning bystander signaling and associated induction of genomic instability will also be reviewed. Finally, reasons for these studies will be discussed in terms of relevance for evolutionary biology and environmental protection of seeking a mechanistic understanding of vertebrate responses to low doses of environmental stressors. Chapter 11 - Transgenic animal models that carry mutation target genes have proven invaluable in addressing the need for improved approaches to study mutations induced in somatic cells. Recently, a small laboratory fish model, the  transgenic medaka, was introduced as a new model for germ cell-mediated mutagenesis. Attributes of medaka as an animal model and the cII transgene as a mutation target gene combine to provide numerous practical and scientific benefits for such investigations. In this chapter, features of using the  transgenic medaka and the cII mutation assay, and results from this recent investigation using the germ cell mutagen ethylnitrosourea (ENU) are reviewed with a focus on comparisons with investigations using mouse models. The ability to characterize both the frequencies and specific types of cII mutations carried by individual mutant offspring proved invaluable in distinguishing non-mutant, whole body and mosaic mutant offspring. The frequencies of mutant offspring derived from ENU-treated spermatogonial stem cells of fish were remarkably similar to those reported for transgenes and endogenous genes of mice, thereby supporting the use of medaka as a comparative animal model for germline mutagenesis. The prevalence of mosaic mutant offspring and the distinctive spectra of mutations they carried revealed that, in addition to the role germ cells play as direct transmitters of mutations, germ cells carrying persistent DNA damage act as mediators of indirect mutagenesis in cells of early stage embryos. Emerging evidence suggests that error-prone DNA repair processes and likely, epigenetic processes acting independently or together contribute to a phase of hypermutagenesis manifested as genomic instability in offspring of mutagen-exposed germ cells. Whereas the processes of mutagenesis mediated by germ cells remain incompletely characterized, a model for the mechanism(s) will incorporate the interplay between persistent DNA damage, delayed and untargeted mutations, and constraints of rapidly dividing cells of early stage embryos in responding to damage contributed by germ cells. Chapter 12 - The field of transgenerational genomic instability in mammals, including humans, is a confusing area of contradictory results, generalizations based on limited data sets

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xiii

and confusion about terminology. This chapter will focus on radiation-induced effects on genome stability, with some discussion of the literature on chemical-induced effects where relevant. The old and recent radiation literature will be summarized, and controversial issues will be highlighted. The relevance of non-targeted effects, such as bystander signaling and stress-induced instability, will also be discussed. An emphasis will be placed on the recent data from the authors‘ laboratory concerning medium- and blood-borne signals, which appear to regulate behaviors of lower hierarchical levels to achieve coordination of tissue, organism and ecosystem response at higher levels of organization. The hypothesis is put forward that such signal-mediated coordination represents a natural response to stress such as radiation exposure, which enables an appropriate reaction to ensure an optimal outcome for irradiated organisms. Data sources will be reviewed, and limitations of these studies will be discussed. Most of the discussion will relate to experimental studies in (mainly) rodents, but available data on wild mammals (e.g., post Chernobyl) will also be discussed. Chapter 13 - Phenotypic diversity is shaped by both genetic and epigenetic mechanisms that program tissue specific patterns of gene expression. Cells, including neurons, undergo massive epigenetic reprogramming during development through modifications to chromatin structure and by covalent modifications of DNA through methylation. There is evidence that these changes are sensitive to environmental influences, leading to sustained differences in phenotype. As in humans, variations in maternal behavior of rat mothers have long-term consequences for the behavior of their offspring. For example, in rats, species-typical differences in maternal behavior have long-term effects on the Hypothalamic-PituitaryAdrenal (HPA) axis, cognitive and emotional function of offspring. Thus, the offspring of mothers that engage in high amounts of licking and grooming and arched-back nursing exhibit less stress reactivity than do the offspring of mothers that exhibit low licking and grooming and arched-back nursing. These differences induce long-term changes in gene expression, including those in the glucocorticoid receptor, which is associated with altered histone acetylation, DNA methylation, and NGFI-A transcription factor binding. Thus, maternal care plays a critical role as a mediator of the relationship between early-life events and health in adulthood. Interestingly, these effects can be reversed by early post-natal cross fostering, and by pharmacological manipulations in adulthood, including Trichostatin A and l-methionine, that influence the epigenetic status of critical loci in the brain. Recent evidence suggests analogous mechanisms may be at work in humans. These findings provide evidence for a stable yet dynamic epigenome capable of regulating phenotypic plasticity through behavioral programming. Chapter 14- Classically, the development of cancer in humans has been viewed as a progressive multistep process of transformation of normal cells into malignant cells driven by genetic alterations. However, a wealth of data in the past decade indicating the importance of epigenetic mechanisms has largely changed the view on cancer as being a solely genetic disease. Currently, cancer is recognized as a disease provoked by both genetic and epigenetic alterations, and both of these components cooperate and complement each other at every stage of cancer development. It is widely believed that the major causes of human cancer are due to environmental exposure to natural and man-made chemical and physical agents. However, during recent years, an interest to the causative role of personal lifestyle factors and nutrition in the origin of cancer has increased significantly. This is driven by growing evidence that lifestyle may substantially compromise the stability of the genome by inducing

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genetic and epigenetic alterations that predispose cells to tumorigenesis. Therefore, the elucidation of the effects of personal lifestyle factors on epigenetic processes during carcinogenesis is of great importance, as these epigenetic alterations may not only have been potential early-detection and prognostic markers but also targets for cancer prevention. Chapter 15 - Epigenetic regulation of genes serves as a flexible way for variation in expression without alterations in existing genetic sequences of genes. Paramutation is one such manifestation of epigenetic regulation. Paramutation leads to heritable alterations in the expression status of one allele in accordance with the second paramutagenic allele. Such examples exist in a wide range of organisms including plants, mammals and fungi. Causes and mechanisms responsible for paramutation have long been sought for. A siRNA-based model is emerging as the most promising candidate for paramutations. Yet, exact details of paramutations are inconclusive. Here, two well-studied examples of paramutations in plants are discussed. Also, the most evident mechanisms of paramutation are discussed in relation to the two examples. Chapter 16 - Plants are sedentary organisms that respond to stress with reprogramming of patterns of gene expression, thus allowing fast acclimation and adaptation in response to specific environmental conditions. Mechanisms underlying the ability of plants to respond to stress so rapidly and efficiently could be broadly classified into the mechanisms associated with physiological changes in exposed plants and accumulation of newly synthesized metabolites in formed seeds and into those that can be associated with epigenetic mechanisms of reprogramming of gene expression. The latter ones are the mechanisms of inheritance that do not involve permanent changes in DNA sequence. However, epigenetic modifications may also trigger permanent changes in the genome. This chapter describes what changes occur in progeny of plants exposed to stress and will discuss epigenetic mechanisms regulating a transgenerational response to stress, describe what is known from current literature reports, and present several sets of data accumulated in our lab that show various examples of heritable changes in response to stress. Cgapter 17 - Transposable elements (TEs) are ubiquitous mobile DNA sequences that have the ability to insert at different positions in the genome and sometimes to amplify at high levels. The activity of TEs can deeply modify host genome structure, it also creates genetic variation and affects gene expression and function. In plants, TEs represent a major genome component, and their activity is often associated with stress conditions. The expression of LTR-retrotransposons in particular is tightly linked to plant stress response pathways. Several evidences of heritable genome restructuring associated with the plant TE activity have been reported in response to environmental challenges and genome shocks such as interspecific crosses. More importantly, TEs may affect gene functions in multiple ways, from contributing to proteic sequences or even metamorphosing into new genes to relocating genes and genic sequence to new positions. They also provide new or alternative expression patterns and act as mediators of epigenetic regulations. Retrotransposon LTRs, in particular, may act as promoter capsules modulating neighbouring gene expression in response to their own transcriptional and epigenetic responses to specific stress stimuli. An emerging paradigm thus considers TEs as sensors of specific challenges that are able to both restructure genomes and modulate gene expression and function in response to these challenges, thus playing a significant role in the generation of phenotypic plasticity. Chapter 18 - Bystander effects can be defined as unexposed ‗units‘ that exhibit molecular symptoms of stress exposure when adjacent or nearby ‗units‘ are subject to stress. Here a unit

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may be referred to as a cell, organ, or even another organism. Plants have a variety of bystander-like effects, most of which involve local pathogen infection and pest infestation that result in a systemic resistance response to current and future attacks. These bystander effects are critical for plants to survive in their environment. The following chapter will outline some of the known inter- and intraplant signalling events that occur in response to pathogen or pest detection. As such, it will involve an overview of the plant immune system as it is understoon today, and how a plant signals resistance to the rest of the plant, or in some cases to other plants. By no means does the chapter mean to cover all of what is known in each of these areas, only the most accepted paradigms and the ideas behind how they function. This chapter may be utilized by undergraduate and graduate students who want a taste of the plant signalling field or for those with little experience in plant signalling biology. More experienced molecular botanists can use this chapter as a ‗jumping off‘ point, as the chapter directs readers to numerous current reviews in each of the discussed areas. Chapter 19 - Since the time man has domesticated plants for food, this chapter seeks to expand the range of benefits they can provide. Starting in the late 19th century, we began to apply the principles of modern science in order to achieve these aims faster, more consistently and on a wider scale. In modern times, the goals included higher, more sustainable yields in the face of changing environments. Recently increasing emphasis is on improving aesthetic and sensory experience and addressing health concerns. The complexity of the goals is clear. In this review, attempts are made to make the case that geneticists are ready to exploit new possibilities arising from a better appreciation of the complexity of inheritance and its interactions with the environment. In doing so, one should seek to preserve and consolidate the undeniable extensive gains made by what might be now regarded as classical or conventional plant breeding approaches. Standing on such a solid foundation,will show that by recognizing and more fully respecting the complex, adaptive and newly-discovered, selfmodifying nature of biological information, will have developed novel, complementary approaches to current plant breeding practice. With the benefit of this new perspective, one may better see the way to reach our goals faster, with less costly screening, and for a wider range of environments.

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In: Genome Instability and Transgenerational Effects ISBN: 978-1-60876-831-8 Editors: I. Kovalchuk, O. Kovalchuk, pp. 1-5 © 2010 Nova Science Publishers, Inc.

Chapter 1

GENETIC AND EPIGENETIC REGULATION OF TRANSGENERATIONAL CHANGES IN GENOME STABILITY: AN OVERVIEW Olga Kovalchuk and Igor Kovalchuk

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Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, CANADA

Genome stability of every species depends on complex interaction of predefined and environmentally induced genetic and epigenetic states. Predefined states consist of chromatin structure and cell metabolic processes such as DNA repair, radical scavenging and cell signalling, whereas induced states depend on interactions with the environment. Organisms are able to respond to a changing environment by various alterations in their somatic cells as well as in their germline and progeny. In this book, we will describe various phenomena associated with the maintenance of genome stability. These include genetic and epigenetic responses to various stresses in exposed cells and organisms, bystander and bystander-like effects, transgenerational changes in genome stability and stress tolerance in bacteria, plants and animals. The history of studies of the environment‘s influence on the phenotypic appearance of organisms and inheritance goes all the way back to Jean-Baptiste Lamarck and Charles Darwin. Their ideas laid an important foundation for the development of the field of research that we are going to introduce you to. With this in mind, we would like to briefly recall how our understanding of interactions between the genetic make-up of organisms and their environment have been formed and developed over time. The theory of pangenesis suggests that every cell in every organism not only experiences environmental changes and responds to them but also generates molecules capable of contributing to the development of new traits and new organisms. Darwin called these molecules ―gemmules‖ [1]. Darwin‘s ideas and views were strongly influenced by the work of another great scientist of 19th century, the French biologist Jean-Baptiste Lamarck. Lamarckism (often referred to as Lamarckian evolution) is a widely accepted idea that an organism can pass on characteristics that it acquired during its lifetime to its offspring. This

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theory which is often referred to as a ―soft inheritance‖ is named for its founder Jean-Baptiste Lamarck, who was the first to suggest that acquired characteristics can be passed to progeny. Specifically, he proposed that individual adaptational efforts during the lifetime of the organisms were the main mechanism driving species to adaptation to a changing environment. Neo-Darwinism is another theory of evolution that represents the ―modern synthesis‖ that combines Darwin's theory of natural selection as a major factor of evolution and population genetics. The term Neo-Darwinism was first used by George Romanes in 1895 and referred to the idea that evolution occurs solely through natural selection, as proposed by Alfred Russel Wallace and August Weismann. Neo-Darwinism suggests that evolution occurs without mechanisms involving the inheritance of acquired characteristics resulting from their use or disuse. Thus, this ‗modernized‘ Darwinism accepted some ideas that were developed by Darwin's original theory of evolution via natural selection, but at the same time it separated them from Darwin‘s hypothesis of Pangenesis as a Lamarckian source of variation involving blending inheritance. Complete rejection of Lamarckian theory of inheritance was based on the germ plasm theory proposed by Weissmann in 1893. He discovered that the cells that produce the germ plasm (gametes) separate from the somatic cells at an early stage of organismal development. Weissman could not understand how somatic and gametic cells would communicate with each other and declared that the inheritance of acquired characteristics was therefore impossible. Overall, many scientists further tried to prove or disprove Darwin‘s hypothetical theory of Pangenesis. Francis Galton conducted many experiments that led him to refute this theory. Initially he believed in the pangenesis doctrine and theory, and in consultation with Darwin, he attempted to detect how gemmules were transported in the blood. His hypothesis was very simple. He hypothesized that if gemmules were transferred to gametic cells though the blood, then blood transfusion between various breeds of animals would allow obtaining new traits in offspring. In a long series of experiments initiated around 1870, he transfused the blood between dissimilar breeds of rabbits, and found no evidence of characteristics transmitted in the transfused blood. Darwin challenged the validity of Galton's experiment. In his article published in 1871, he wrote: "Now, in the chapter on Pangenesis in my "Variation of Animals and Plants under Domestication," I have not said one word about the blood, or about any fluid proper to any circulating system. It is, indeed, obvious that the presence of gemmules in the blood can form no necessary part of my hypothesis; for I refer in illustration of it to the lowest animals, such as the Protozoa, which do not possess blood or any vessels; and I refer to plants in which the fluid, when present in the vessels, cannot be considered as true blood." Until the end of the 19th century, the scientific world still believed in Darwin‘s theory of pangenesis. The work of Gregor Johann Mendel on plant hybridization completely changed the scientific approach to understanding the mechanism of inheritance. Although Mendel published his work in 1866, it was not until 1900 that the importance of his work has been reanalyzed. Once Mendel‘s work was re-discovered, a new era of ‘Mendelian‘ genetics began which rejected the possibility of transmission of information from somatic cells to gametes and thus to the progeny.

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It should be noted that many scientists still considered the possibility of environmentinduced heritable changes. The Russian scientist, Ivan Michurin was one of the founders of scientific agricultural selection. He worked on hybridization of plants of similar and different origins and on development of new cultivating methods in connection with the natural course of ontogenesis. He was also interested in directing the process of predominance, evaluation and selection and in working out methods of acceleration of selection process with the help of physical and chemical factors. In early 20th century, he proved that dominant traits in generation of hybrids depend on heredity, ontogenesis, and phylogenesis of the initial cell structure as well as on individual features of hybrids. Like Lamark and Darwin, Michurin in his works assumed a possibility of changing genotype under external influence. The fact that phenotypical characteristics can be inherited during hybridization/grafting experiments using different plant cultivars has been reported by many famous plant breeders. Over the past decades independent scientists have repeatedly shown that graft-induced phenotype variations are stable and heritable [2]. In 1928, Trofim Lysenko, one of the proponents of Michurin‘s ideas of hybridization and grafting, claimed to have developed an agricultural technique of vernalization which used the combination of humidity and low temperatures to make wheat grow in spring. He suggested that using this technique would increase crop yields by several times, which turned out to be impossible. Lysenko attempted to further develop the Lamarckian idea of inheritance of acquired characteristics in an organism. At the same time, he had some fairly strange ideas, suggesting for example, that the state of being leafless as a result of having been plucked could be inherited by the organism's descendants. Some time ago, the ideas of Lamark and Michurin seemed to be pseudo-scientific and impossible to believe in, did not they? Recently, a breakthrough publication describing changes in genetic make-up of grafted plants was an eye-opener that brought many new possibilities for transmission of genetic material. Sandra Stegemann and Ralph Bock reported transfer of genetic material from stock to scion upon grafting of tobacco plants [3]. The study showed that recipient plants acquired tolerance to an antibiotic in the same manner as donor plants, and it also confirmed the existence of transfer of genetic material from a donor to a recipient. Although it is still unclear whether it occurs via plastid transfer through plasmodesmata or via transfer of a large portion of the plastid genome from a donor cell to a recipient cell, it can definitely be considered an example of changes not only in phenotypic appearance but also in a genetic make-up of a grafted plant. Ideas of epigenetic regulation of organism development and cell fate have been developing very actively throughout the entire 20th century. The actual name ‗epigenetics‘ did not, however, emerge until 1942 when Conrad Hal Waddington used it to describe how genes might interact with their surroundings to produce a phenotype. During the past 50 years, interest in epigenetics has fallen and risen many times. In 1990, Robin Holliday defined epigenetics as "the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms" [4]. The current definition of epigenetics is not so broad; it refers to heritable traits that do not involve changes in DNA sequences. These changes occur during somatic cell divisions and sometimes can be transmitted transgenerationally through the germline. The field of epigenetics has again exploded onto the scientific scene in the recent years. Numerous articles have been published in top-ranking journals. Nowadays, epigenetics is perhaps one of the most popular fields of studies. Genome sequences of model organisms

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such as C. elegans, Drosophilla, Arabidopsis, human, mice, rice, etc. having become available, more and more studies appeared that attempted to understand genome and chromatin organization and explain mechanisms of inheritance, maintenance of genome stability, and regulation of gene expression. What has become clear is that these mechanisms are of genetic and epigenetic nature. In this book, we will discuss various genetic and epigenetic mechanisms of genome stability regulation in animals and plants. We will also describe the influence of environment on genome stability and epigenetic changes; evidence of inheritance of such changes will also be presented. We will also introduce concepts of non-targeted events, bystander and bystander-like effects, paramutations, and other epigenetics-related phenomena. Several introductory chapters will describe normal DNA repair and genome maintenance processes in bacteria, plants and animals. Specifically, the chapters by Jody Filkowski and Olga Kovalchuk will introduce the reader to cellular mechanisms of genome stability regulation, including DNA repair and epigenetic regulation of chromatin structure and gene expression in animals. Alex Boyko and Igor Kovalchuk will cover these processes in plants. The chapter by Susan Wallace and colleagues will give a specific example of evolution of DNA repair enzymes - glycosylases in various organisms and underscore their role in maintaining genome stability and species evolution. The influence of stress on genome stability and genome plasticity will be demonstrated by Grandbastien on the examples of transposon activation in plants. Several chapters will discuss targeted and non-targeted mutagenesis, stress-induced communication between cells and organisms, and even evidences of transgenerational changes induced by stress. The chapter by Andrey Golubov will introduce the reader to stress-induced mutagenesis in bacteria and will attempt to analyze whether such events are random or directed. The chapter by Igor Kovalchuk and Andrey Golubov will describe a specific case of sequencespecific DNA degradation used by bacteria for the protection against bacteriophages and will discuss some recent examples of how bacteria can rapidly acquire tolerance to phages. One of the most interesting examples of an epigenetically controlled process - the nonlinear response to DNA damaging agents – will be described by Carol Yauk and John Stead. It has already been known that a higher dose of mutagen does not necessarily result in a higher level of damage to DNA. In fact, low doses of ionizing radiation often result in disproportionally high levels of DNA damage. Doses of ionizing radiation that are believed to have a negligible effect on a cell often exert dramatic influence on DNA damage and cell viability. The chapter focuses on the stability of tandemly repeated sequences and analyzes the phenomenon of non-targeted instability of these repeats. Cell-to-cell communication between neighbouring cells and communication between cells of different tissues and organs of multicellular organisms is well-known and occurs regularly. These communications involve hormonal signalling, neurotransmission, etc. It is believed that damaged tissues are able to communicate with non-damaged tissues - a phenomenon known as bystander effect. The chapter by Jody Filkowski, Yaroslav Ilnytsky and Olga Kovalchuk will present several examples of such bystander effects triggered by ionizing radiation in vivo in an animal model. The chapter by Carmel Mothersill and Colin Seymour will introduce the reader to the phenomenon of transgenerational genome instability in somatic and germ cells in mice.

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Organisms are also able to communicate danger to each other through water and air. These communications are often chemical in nature, although in many cases an exact nature of signals is not known. Another chapter by Carmel Mothersill and Colin Seymour will introduce the reader to bystander effect between irradiated and non-irradiated fish. In plants, mechanisms of intra- and inter-plant communication are often used for pathogen response. Molecules involved in this process include small peptides, volatile organic compounds (methyl salicylate, methyl jasmonate) and even small non-coding RNAs. The chapter by Franz Zemp will introduce the reader to various bystander-like phenomena used by plants to communicate danger between treated and non-treated tissues. Can organisms communicate memory of stress across generations? According to Darwin, organisms evolve through the process of natural selection from a pool of individuals with spontaneous changes/mutations. The process of mutagenesis is believed to be random and the majority of mutations are deleterious. The rare mutations that become beneficial under certain environmental conditions have a chance to be fixed in a population. Since mutagenesis is a rare process, the fixation of a desired trait would be extremely rare. In contrast, processes of acclimation and adaptation are rapid ones that allow organisms to acquire protection against stress in a single generation after exposure. These processes cannot be explained by the laws of Mendelian genetics. Several chapters that include works by Igor Kovalchuk, Richard Winn, McGowan, Meaney and Szcyf, Olga Kovalchuk, Carmel Mothersill, Andere Comeau present the data demonstrating the inheritance of stress memory across generations in various organisms. The chapter by Igor Pogribny shows how the epigenetic machinery of the cell is influenced by a diet and life style. Chapters by Palak Kathiria and Minoo Rassoulzadegan will describe paramutation in plants and animals, the phenonon that is based on allelic interactions and changes in the phenotype influenced by the environment. In sum, the current book covers a variety of topics associated with direct and indirect damage to DNA, genetic and epigenetic control of genome stability, local and systemic responses to mutagens, phenomena of non-targeted effects including bystander effect and transgenerational genome instability.

REFERENCES [1] [2] [3] [4]

Darwin, C. R. 1871. Pangenesis. Nature. A Weekly Illustrated Journal of Science 3 (27 April): 502-503. Liu Y. Like father like son. A fresh review of the inheritance of acquired characteristics. EMBO Rep. 2007 Sep;8(9):798-803. Stegemann S, Bock R: Exchange of genetic material between cells in plant tissue grafts. Science 2009, 324:649-651. Holliday R: Mechanisms for the control of gene activity during development. Biol Rev Camb Philos Soc 1990, 65:431-471.

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Chapter 2

GENETIC AND EPIGENETIC MECHANISMS ENSURING STABILITY OF MAMMALIAN GENOMES Jody Filkowski and Olga Kovalchuk Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, CANADA

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ABSTRACT Maintenance of genome stability is a key to a disease-free survival of mammalian organisms. Mammalian cells are affected by a wide variety of endogenous and exogenous DNA damaging agents. To effectively deal with DNA damage, mammalian cells harbor a versatile network of repair, cell cycle control and genome maintenance mechanisms, since failure to eradicate DNA damage may lead to genome instability. Yet, the correct function of these and other cellular mechanisms heavily depends upon gene expression and organization as well as on the accessibility of DNA for DNAprotein interactions. These domains are governed by epigenetic processes—meiotically heritable and mitotically stable alterations in gene expression that include DNA methylation, histone modification, and RNA-associated silencing. This chapter will introduce some of the key strategies mammalian cells have evolved to maintain the integrity of their heritable information. We will describe some interconnected genetic and epigenetic tactics that frequently work in concert to achieve a common goal of maximum stability of the genome.

Keywords: genome stability, mutations, DNA repair, epigenetics, chromatin, DNA methylation, histones modifications, telomeres, short RNA

INTRODUCTION Every cell possesses a quantum of heritable material that provides instructions for how that cell is to carry out its life processes. This material is known as DNA, and collectively the

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entire DNA within a given cell is termed a genome. Although there are many common features of DNA found within all genomes of living organisms, a wide range of variations observed between organisms belonging to different kingdoms, species, and even between specialized tissues is due to intense micro-management of the expression of information from their genomes. The cellular epigenome partakes in control of gene expression in cells. Historically, the concept of the epigenome and the term ‗epigenetics‘ (‗outside of genetics‘) was introduced by developmental biologist Conrad Hal Waddington well before the discovery of genes and the molecular structure of DNA itself. The Waddington‘s model proposed that epigenetics describes how genes within a multicellular organism interact with other genes and their environment to yield a certain phenotype [1, 2]. In later works by Robin Holliday, epigenetics was characterized as cellular ―mechanisms of temporal and spatial control of gene activity during development‖[3]. After the discovery of DNA as the genetic material, the epigenome field was overshadowed by studies of the role of changes in DNA sequences under various normal and pathological conditions. It was not until the near completion of the human and several other genome projects that interest in epigenetics and the epigenome research was ―reborn.‖ At that time, many researchers understood that genome sequences failed to fully explain the complexity of cellular processes, regulation and, most importantly, the fine-tuning of cell-organism-environment interactions [4]. Modern science defines the epigenome and epigenetic changes as meiotically and mitotically stable alterations in gene expression that are not based on DNA sequence changes and involve processes that impact chromatin structure such as DNA methylation, histone modifications and genomic imprinting [5-7]. More recently, RNA-mediated silencing was proposed as another epigenetic phenomenon [8]. Nowadays it is well-accepted that expression of genetic information is dictated not only by the genetic sequence of a cell but also by the accessibility of this sequence for the transcription machinery and the translational potential of given transcripts. Therefore, the genome and epigenome are equally responsible for maintaining proper expression levels. The genetic sequence and epigenetic status of a cell provide the necessary information for proper maintenance of cellular differentiation and homeostatic balance. Therefore, it is absolutely essential that a cell is able to maintain its genomic integrity. The ability to maintain genomic integrity is referred to as genome stability. Consequences of failing to maintain this integrity manifest as mutations which may ultimately culminate in diseased phenotypes [9]. Although initially, lapses in this maintenance affect a single cell, the nature of DNA replication and cellular reproduction provide a strong potential to create a lineage of altered cells. This consequence is further compounded when mutations are acquired in germ cells as the mutation and/or diseased phenotype is propagated to future generations. Since cells are subject to a wide range of genetic insults that cause various types of DNA damage, the necessity for a surveillance and repair system is high [10]. The mammalian genome exists as linear DNA molecules that code for all genes required throughout embryonic development and adult life. These molecules called chromosomes are wrapped around structural proteins called histones in a predictable manner. DNA is constantly challenged by several types of insults from endogenous sources (hydrolysis, oxidation alkylation and erroneous DNA replication) and exogenous sources (ionizing radiation, ultraviolet radiation and chemical agents). These insults may result in several types of lesions that modify bases, affect nucleic acids or break the DNA backbone— DNA strand breaks [9, 10]. However, as evidenced by evolution and a wide range of life that has existed

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throughout time, a certain level of genomic instability obviously persists. In these instances, we refer to genomic instability as an evolutionary process and typically use the term ―adaptation‖: any change in the functioning of an organism that makes it better suited to its environment. Therefore, evolution indeed is a cumulative result of mutations acquired over time. The goal of this chapter is to introduce a number of mechanisms that exist to stabilize the mammalian genome and epigenome. As mentioned above, DNA damage is inevitable, and therefore repair and reversal of damage are key factors in maintaining genomic stability. These mechanisms fall into two broad categories of regulation: genetic and epigenetic. Genetic mechanisms may be attributed to the functions of specific products of DNA repair genes that act directly and fix the altered DNA sequence. Epigenetic mechanisms are associated with control of gene expression at transcriptional, translational and posttranslational levels and at the level of genome organization. These include: DNA methylation, histone modifications, and RNA associated silencing (RNAi). The epigenetic status is heritable and frequently responsible for defining the expressed genetic profile of a cell [5].

GENETIC MECHANISMS OF GENOME STABILITY

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Genetic mechanisms involved in genome stability include several key DNA repair pathways. These pathways are characterized by the type of damage repaired and may be broadly classified into systems for: 1.direct reversal of damage, 2. mismatch repair, 3. nucleotide excision repair, 4. base excision repair, 5. homologous recombination, and 6. nonhomologous end joining. Here we will briefly outline each of these pathways and identify the major genes involved.

Direct Reversal of Damage Direct reversal of damage is usually employed to fix rare nicks and most importantly to repair modified bases. It is the simplest pathway as it involves a single step with a single protein. Modified bases are caused by exposure to chemicals such as alkylating agents. The result is the transfer of extra methyl or ethyl groups to nucleotide bases forming changes bases such as O6-alkylguanine, 1-methyladenine, and 3-methylcytosine. These types of lesions are highly mutagenic and can be cytotoxic as the extra moiety alters base pairing characteristics of nucleotides, thus creating a large potential for introduction of sequence errors during replication [11]. Interestingly, this property is exploited in chemotherapeutic drugs that promote the formation of O6-alkylguanine in an attempt to reduce tumor survival [12]. During direct reversal, the erroneous alkyl group is transferred to a recipient protein restoring a proper structure of the nucleotide base. Examples of these include O6methylguanine-DNA methyltransferase (MGMT) and dioxygenase protein alkylation repair homologs 2 and 3 (ABH2 and ABH3) [12, 13]. The importance of these proteins in maintaining genome integrity is demonstrated in the mouse over-expression and knockout models. Transgenic mice over-expressing human MGMT demonstrated a reduced susceptibility to developing tumors after exposure to alkylating agents, while Mgmt-/-, Abh2-

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/- and Abh3-/- knockout mutants experienced an increased sensitivity to alkylating agents and in the case of Mgmt-/-, induced lung and liver cancer were more frequent [14-16].

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Base Excision Repair In another DNA sequence maintenance strategy, the modified base(s) are removed and the strand is partially degraded and replaced. This pathway known as base excision repair (BER) has two approaches: short-patch BER which repairs only a single nucleotide and longpatch BER where 2-23 nucleotides are removed and replaced [17]. The initial removal of the modified base is carried out by DNA glycosylases that cleave the glycosylic bond leaving an apurinic/apyrimidinic (AP) site [18]. The DNA backbone adjacent to the AP site (if it has not already been cut by certain glycosylases) is nicked by AP endonuclease (APE1). This nick allows for DNA polymerases β, δ or ε to fill the resulting gap [19, 20]. This is where the two BER pathways diverge. During short-patch BER, the gap is filled using polymerase β followed by XRCC1/DNA ligase III-mediated ligation. During long-patch BER, POLδ and ε— polymerases employed during this strategy require Replication factor C (RFC), proliferating cell nuclear antigen (PCNA) as well as flap endonuclease1 (FEN1) [21]. Unlike other repair pathways which, when defective, display a strong disposition to cancer, a link to cancer is not (yet) obvious in BER compromised individuals. An explanation for the above is likely two-fold: first, an abundant number of glycosylases available (in mammals there are several of them: Myh, Nth1, Ogg1, Ung and Aag) may provide a redundant function in initiating the process; second, loss-of-function mutants of genes responsible for post-recognition steps (as demonstrated in mice) are either embryonic or postnatally lethal [22]. An enormous variety of chemical and physical DNA-damaging agents in our environment can generate a massive array of DNA damage types. While BER is a very important repair mechanism, it is clearly insufficient to deal with a wide variety of different DNA damage types since DNA glycosylases are substrate-specific and capable of only recognizing specific types of damages.

Nucleotide Excision Repair Nucleotide excision repair (NER) is a much more flexible damage repair mechanism. It recognizes damaged regions based on their abnormal structure and chemistry, excises and replaces them. Although the process is similar to base excision repair, it uses a different mechanism (i.e., a diferent set of enzymes) and typically repairs more bulky adducts. The NER mechanism removes small stretches of single-stranded DNA (ssDNA) that contain various DNA lesions in a multi-step fashion. These lesions may result from alkylation by chemical agents, pyrimidine dimers caused by UV exposure, oxidative damage initiated by radical oxygen species (ROS) (either exo- or endogenous), or bulky adducts formed as a result of cellular detoxification of highly reactive chemicals such as aflotoxins. NERrecognized lesions usually cause significant distortions in a spacing, size and shape of the DNA helix [9]. In short, the process involves recognizing damage, marking the affected area, and creating ssDNA nicks up- and downstream of the lesion. A small stretch of the affected

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oligonucleotide is excised, and the gap is filled in. Upwards of 30 proteins are involved in the process. NER is grouped into two main categories: global genomic repair (GGR or GG-NER) that occurs throughout the genome in both transcriptionally active and inactive regions and transcription coupled repair (TCR) that occurs at transcriptionally active sites. The primary difference between these mechanisms is at the recognition step. In GG-NER, XPC constantly scans the genome searching for helix-distorting lesions. When identified, it binds along with RAD23B (HR23B) and centrin2 (CETN2) [23]. This is facilitated by the DDB complex (DDB1 and XPE/DDB2), specifically in the case of damage caused by UV-irradiation [24]. Damage to bases which block transcription triggers transcription-coupled NER (TCR or TC-NER). CSA, CSB and XAB2 proteins facilitate TC-NER by stabilizing RNA polymerase II near the site of damage [25]. CSA and CSB serve to displace the polymerase and facilitate further recruitment of the repair machinery [26]. Once the lesion is identified, GG-NER and TC-NER continue by the same excision mechanism [24]. The DNA helix is unraveled, and ssDNA is stabilized by Replication protein A (RPA), XPA and by a transcription factorIIH (TFIIH) multiprotein complex [27]. TFIIH contains 10 subunits including XPB,XPD and TTD-A [28]. The XPB and XPD subunits of TFIIH are helicases that unwind DNA around a lesion. XPA and RPA stabilize the open structure and recruit the ERCC1–XPF nuclease. This nuclease incises the damaged DNA strand 20-22 nucleotides upstream of the lesion. The XPG nuclease makes the 3′ incision 5-6 nucleotides downstream of the damaged site [24, 29]. The gap is then filled with polymerase ε or δ [30, 31]. There are three major human syndromes that display compromised NER: Xeroderma pigmentosum (XP), Cockayne Syndrome (CS) and trichothiodystrophy (TTD). XP arises from loss of function of any one of seven XP genes (A through G); CS patients have deficient CSA or CSB, while TTD patients have faulty TTD-A, XPA and XPD [32]. Numerous knockout XP, CS and TTD mice have been generated. These mice show varying degrees of cancerous disposition, sunlight sensitivity, developmental defects, growth retardation as well as shortened life spans, thus demonstrating severe consequences of a faulty NER pathway [22]. The absence of GG-NER leads to the accumulation of DNA damage and, as a consequence, mutations and cancer predisposition [33]. Contrarily, TC-NER defects result in disrupted and stalled transcription which is highly cytotoxic [24].

Mismatch Repair An endogenous process of DNA replication is not an error-free process. Replication mistakes, if not repaired, are the main contributors to the accumulation of mutation. During replication, base substitutions and small insertions and deletions (IDL) create mismatches within the base sequence. Frequently, these mismatches lead to microsatellite instability—a key factor in numerous cancers. The mismatch repair (MMR) mechanism serves to identify and direct repair of mismatched bases and small loops[34]. A wealth of initial knowledge about MMR stemmed from analysis of the E.coli MMR system in which monomeric proteins MutS and MutL are the key players. In mammalian cells, MMR is carried out by means of multiple protein heterodimeric complexes. One of two complexes, MutSα or MutSβ, makes the initial recognition of a lesion. MutSα is comprised of

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MutS homologs 2 and -6 (MSH2, MSH6) and MutSβ is composed of MutS homologs 2 and 3 (MSH2, MSH3). MutSα is involved in repair of single-base mismatches and shorter insertion/deletion loops (IDLs), while MutSβ is responsible for longer IDLs [35]. However, a certain degree of overlap and redundancy exists [36]. Upon recognition of a lesion by either of the MutS complexes, a second MutLα complex is recruited (MLH1 and PMS2). MutLα cleaves DNA either 5‘ and 3‘ to the lesion. The resulting nicks are used as excision initiation sites allowing for the exonucleolytic excision via EXOI, at which point DNA polymerase δ (POLδ) loads, synthesizing a new strand across the lesion site. Besides POLδ, another nonMMR specific protein, PCNA, is involved. PCNA is tightly coupled with the polymerase during replication and recruits the MMR machinery to lesion sites [34]. The MMR machinery has a multi-faceted purpose. Besides recognizing and repairing mismatches, it is capable of sensing other types of DNA lesions, activating cell cycle checkpoints and initiating apotosis [37, 38]. In this instance, the mismatch is identified by MutS and MutL homologs that stall the 5‘-3‘ progressing polymerase and signal DNA damage signaling pathways through activation of ATM or ATRIP/ATR proteins. From this point, the signaled pathway determines the fate of the lesion [39]. Furthermore, other MSH proteins, i.e., MSH4 -5 and MLH3, are involved in processing meiotic recombination intermediates: homozygous mutants of these genes are infertile [40-42]. The importance and necessity of this system is illustrated by one of the most common types of human cancers—hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome. 80-90% of people with Lynch syndrome possess mutations in one of three genes employed during mismatch repair, including MLH1, MSH2 or MSH6; mutations in other MMR genes such as PMS1, PMS2, MLH3 and EXO1 are also observed, albeit at a lower frequency [43, 44]. Besides colon cancer, Lynch syndrome sufferers demonstrate a predisposition to many other types of malignancies. Yet, the high incidence of colon cancer in MMR deficient individuals demonstrates the importance of MMR during replication. Colon cells are the fastest dividing cells in animals and humans. There is almost a continuous demand for new cells to be differentiated from stem cells and, thus there are many more cells undergoing DNA replication. With higher frequency of replication comes a larger number of mismatch errors. Therefore, any compromise in MMR creates a large potential for errors to be maintained and propagated.

Repair of DNA Strand Breaks by Homologous Recombination DNA strand breaks are the most hazardous forms of DNA damage, as they can lead to chromosomal aberration, loss of heterozygosity, cancer and apoptosis [10] . Double-strand breaks usually result from ionizing radiation, contacts with hydroxyl radicals or stalls, and breaks during replication [45]. Even one unrepaired DNA double-strand break is lethal for cells. This type of damage is rectified through one of two pathways: homologous recombination (HR) or non-homologous end joining (NHEJ). Homologous recombination accounts for about 10% of repair in the mammalian genome and occurs during the S and G2 phases of the cell cycle. The importance of a proper function of HR is well illustrated by lethality of mutants of numerous members of the pathway [45]. HR is the predominant repair mechanism during replication when chromatin is in its most open and accessible form. The open chromatin structure during S phase facilitates homology

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searching factors. Furthermore, other usual HR substrates (single strand gaps, nicks, and fork collapses) are produced during the DNA replication process. Although chromatin has recondensed in the G2 phase making strands less accessible, the presence of sister chromatids provides an easy access to a template with matching homology [46]. DNA strand breaks are recognized and marked by a protein complex known as the MRN (MRE11, RAD50 and NBS1) complex [45]. It is important to note that this step is not unique to HR as the same protein complex also recognizes breaks that will be fixed by NHEJ. Furthermore, this step is accompanied by extensive chromatin remodeling at histone H2AX within 5-10kbp of the lesion—a process that will be discussed in further detail later in this chapter. The decision of HR over NHEJ is likely due to the interfering action of RAD18 and poly(ADP-ribose) polymerase (PARP) with KU70/80 binding (to be discussed later) [47]. Proteins in the MRN complex are indispensable for life, and null mutations for MRE11, RAD50 or NBS1 are embryonic lethal in mice [48-50]. Lesions having been identified, several proteins begin to associate with ssDNA. These proteins including RAD52, RAD54 and BRCA2 act as mediators and ultimately lead to the presence of RAD51—a protein responsible for conducting a homology search [51, 52]. Individuals with mutated BRCA2 show a predisposition to cancer, while in mice both the RAD51 and BRCA2 null mutations lead to embryonic lethality [53, 54]. In contrast, RAD52 or RAD54 mutants are viable and demonstrate no disposition to cancer compared to wild type individuals [55, 56], suggesting that RAD52 and -54 are less required for completing the HR process.

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Non-Homologous End Joining In mammalian cells, NHEJ, being the most common way of repairing strand breaks, is employed more often than HR because it can occur throughout the cell cycle and is a quicker and more efficient process [46]. However, this repair process has a potential to reduce genome integrity as it is somewhat random with respect to the two ends it joins, and it can involve removal or addition of several end nucleotides while remodeling strands into a ―ligatable‖ profile. As mentioned above, strand breaks are initially demarcated by the MRN complex followed by association of the KU70/80 complex at the break ends. KU70/80 acts to stabilize downstream processes that include the action by a nuclease, polymerase and ligase [57, 58]. DNA-PKcs, the nuclease involved in the process, co-loads with Artemis, an endo/exonuclease that trims the 5‘ or 3‘ end of the strand to create overhangs. These overhangs are then brought into an overlapping position with the opposite strand, at which point the polymerase X family proteins - polymerase μ or λ or terminal deoxynucleotidyltransferase- fill in the resulting gaps [58]. The nature of the POL X family polymerase further adds to the acquisition of sequence errors during NHEJ, since they can add random nucleotides and act in a template-independent manner that frequently results in the addition of direct or inverted repeats [59]. In a rare instance of a cellular process favoring genome instability, this large capacity for NHEJ this NHEJ capacity to create novel sequences is exploited during the production of T-cell receptors and V(D)J recombination [60]. The nicks and/or gaps left upon polymerase extension are ligated by DNA ligaseIV in conjuction with XRCC4. LigaseIV is required for ligation of blunt ends, overhangs, ssDNA, dsDNA and across gaps of several nucleotides[58, 61].

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Although NHEJ is the predominant pathway for repair of strand breaks in mammalian cells, it is interesting to note that mutant mice of most of the NHEJ individual proteins are viable. DNA-PKCS, KU70, KU80, and Artemis mutants all show compromised immune- cell development and some growth retardation (in KU70, KU80). The only mutants that are embryonic lethal are the LigaseIV and Xrcc4 mutants [22].

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Cell Cycle Checkpoints The DNA damage response is an intricate matrix of pathways which are integrated with cell cycle stages to keep genomic integrity in living cells under tight control. Checkpoints that exist between cell cycle transitions to survey, stall and signal DNA repair processes are governed by checkpoint proteins which respond to DNA lesions. When appropriate, one or several DNA repair pathways are employed to take care of DNA lesions prior to further progression of the cell cycle. In the instances where repair is not feasible, this response culminates in either a dormant state known as senescence or in cellular death via apoptosis [62]. The major checkpoints exist at the G1/ S and G2/ M transitions. The first confirmes that engaging in the replication process is favorable, while the second ensures that it is ―safe‖ for cell division to occur. The apex of several checkpoint cascades starts with protein members of the phosphoinositide 3-kinase-related family: DNA-dependent protein kinase (DNA-PK), Ataxia telangiectasia mutated (ATM) and ATM-related (ATR). These kinases recognize the presense of DNA lesions and signal to initiate their repair. DNA-PK and ATM respond to strand breaks that have been flagged by the KU70/80 or the MRN complex, respectively; while ATR responds to ssDNA and stalled forks flagged by ATRIP (ATR interacting protein). For some lesions, such as those repaired by NER, it is not well understood how communication between lesion identifiers and cell cycle control proteins occurs. It is possible that surveyors in other pathways, e.g., MMR, can alert checkpoint proteins [62]. Overall, cross-talk between the DNA repair machinery and the cell cycle and replication control machinery is crucially important for maintaining genome stability of any given cell and organism.

EPIGENETIC REGULATION OF GENOMIC STABILITY As it has been mentioned, genome stability is achieved through resisting and reversing changes that occur to the genomic sequence of the cell. Up until now, this chapter has illustrated numerous genetic mechanisms that center on identifying and repairing damage. Yet, the correct function of these and other cellular mechanisms heavily depends upon gene expression and organization, as well as on the accessibility of DNA for DNA-protein interactions. These domains are governed by epigenetic processes—meiotically heritable and mitotically stable alterations in gene expression that include DNA methylation, histone modification, and RNA-associated silencing [63]. Now, we will cover some interconnected epigenetic strategies that affect genome stability: DNA methylation, histone modifications and the role of short interfering RNAs.

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DNA Methylation Mammalian DNA methylation has only been described as a covalent addition of a methyl group at the 5-carbon position of the cytosine base, and it has a well investigated role in controlling gene expression, genetic imprinting, and tissue- or temporal- specific gene expression [64]. DNA methylation is a stable and heritable yet reversible epigenetic trait of mammalian genomes [6, 7, 63-68]. There are three main contributing proteins involved in establishing and maintaining DNA methylation patterns within mammalian cells: DNA methyltransferase (DNMT) 1, DNMT3a, and DNMT3b [64],[69, 70]. DNMT3a and DNMT3b are responsible for de novo methylation of sequences and appear to hold an indispensable function, as mutant mice lacking either of these genes die within weeks of birth or are not viable through embryonic stages, respectively [64]. Not surprisingly, DNMT3a and DNMT3b are extremely active during development where cellular differentiation is achieved through high levels of de novo DNA methylation in promoter regions of pluripotency genes [71, 72]. In contrast, DNMT1 is responsible for restoring methylation patterns of hemi-methylated DNA following replication. It localizes to the replication fork where it could directly modify nascent DNA immediately after replication [6, 67, 70]. Its function is also mandatory as DMNT1-/- mice are embryonic lethal [73]. A correlation between methylation status and expression of endogenous genes is apparent [74]. Cytosine methylation is observed at CpG dinucleotides that tend to cluster into islands containing the GC content of >55% in a 500-bp region [6]. These CpG islands are observed within promoter regions of about ~72% of human genes, and methylation of CpG-rich promoters frequently coincides with reduced gene activity [75]. Reduced expression is achieved directly through disruption of the transcription factor and RNA polymerase binding and indirectly through the recruitment of methyl-CpG binding domain proteins as subsequent chromatin remodeling [76]. Interestingly, CpG rich promoters are most often associated with housekeeping genes that require some constant levels of expression and therefore are not heavily methylated [75]. Furthermore, there is a considerable number of unmethylated CpGrich promoters associated with unexpressed DNA. The correlation between the methylation status of CpG-poor promoters (representing ~28% of all promoters [75]) and gene expression is not so strong. The relative amount of methylation experienced by CpG-poor promoters is much larger than that of the CpG-rich class (owing to a fewer number of potentially methylated CpGs), yet this does not correlate with lower gene activity. Indeed, there is no difference between DNA methylation values of expressed and non-expressed CpG-poor genes, which demonstrates that low concentrations of methylated cytosines do not prevent gene activity [77]. It has been suggested that CpG-poor promoter regions are prone to instability due to deamination of 5-methylcytosine to thymine. In fact, the mutation rate of methylated cytosines is 2.2-fold higher than that of their unmethylated alternative which is deaminated to uracil [78]. The resulting erroneous thymine, being an expected base in the genetic code, may not be recognized and reversed by the DNA repair machinery and thus results in a mutation to the original DNA sequence while the uracil is efficiently rectified through the BER pathway. Overtime, a few CpGs that exist are lost along with any impact that DNA methylation may have on the genome. Sequence analysis of species demonstrated that in higher primates relative to lower ones methylated CpGs were lost more frequently in promoter regions, while CpG-poor promoters were more prone to CpG loss than CpG-rich ones [77]. Collectively, this

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suggests a propensity for decreased genome stability in species of a lesser order with respect to their CpG sequences, with a potential evolutionary impact on epigenetic profiles and gene expression in future generations and species. Besides controlling gene expression, DNA methylation is complicit in suppressing parasitic DNA sequences such as transposons and endogenous retroviruses [79]. It is postulated that up to 35% of the human genome is composed of parasitic sequences and that most of them are methylated in an attempt to quarantine these sequences [80]. Active transposable elements are highly mutagenic as they tend to insert within expressed genes disrupting their normal function and can cause illegitimate recombination events and genomic rearrangements [81]. Interestingly, global hypomethylation is a hallmark of all stages of tumor cells with a 20%-60% decrease in methylated cytosines. This decrease in methylated DNA coincides with the reactivation of transposable elements, mitotic recombination (leading to loss of heterozygosity) and aneuploidy [6, 7, 82, 83]. Furthermore, cells lacking the activity of DNMT3b display high levels of chromosome aberrations [84]. Therefore, in a hypomethylated environment chromosomal instability increases and genome integrity is challenged.

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Histone Modifications In the nucleus, eukaryotic DNA closely interacts with histones [7, 63]. Histones are structural proteins that provide scaffolding for DNA molecules to wrap around in a predictable manner forming many nucleosomes in a beads-on-a-string manner. The nucleosome contains 4 core histones (H2A, H2B, H3 and H4) which exist in duplicate forming an octamer around which approximately 150 base pairs of DNA is wrapped [85]. The most obvious role of epigenetic characteristics providing genomic stability is the stabilizing effect of histones on DNA. This association provides a mechanical support that protects the linear DNA molecule from becoming tangled with other molecules and from breaking during the regular DNA movement experienced throughout the cell cycle. However, over the last few decades it has become apparent that histones are subject to numerous posttranslational modifications that can affect their interactions with DNA as wells as interaction of DNA with surrounding non-histone proteins. These modifications have a profound impact on transcriptional expression, DNA repair and genome stability [86, 87]. The combination of DNA wrapped around histones is referred to as chromatin, and it generally falls into two categories: heterochromatin (a tightly wound inaccessible state) and euchromatin (an open accessible form). Post-translational modifications to the N-terminal ends of histones include phosphorylation, acetylation, methylation, sumoylation and ubiquitination events that define and/or change the chromatin state. Collectively, histone modifications are known as the histone code and constitute part of the epigenome of a given cell [86, 88-90]. This review will focus primarily on histone acetylation, methylation and phosphorylation. As mentioned earlier, chromatin remodeling frequently occurs to modify the transcriptional activity of a gene. Therefore, chromatin remodeling is a change between chromatin states. It is achieved through acetylation and methylation of key amino acid residues within specific core histones. Unlike DNA methylation where a single chemical modification occurs at a single position on a single base, histone modifications are much more complex. The general school of thought is that these chemical modifications on histones alter the shape of chromatin, thus affecting the

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ease with which non-histone proteins may access and bind altering the transcriptional outcome. Histone modifications are achieved through the work of several different enzymes including histone methyltransferases, histone acetylatases, and histone deacetylases [86]. Amino acid residues that are involved in acetylation are: lysine(K) 9, K14, K18, K23 of H3, K5, K8, K12, K16 of H4, and lysines of H2A and H2B; while methylation may occur at residues K4, K9, K27, arginine(R) 2, R17, R26 of H3 and K20, R3 of H4. The acetylated histone tails lose their positive charge reducing the affinity to the negatively charged DNA and leading to the packaging of relaxed chromatin. Generally, histone acetylation is linked to transcriptional activation, and histone deacetylation is considered to be a repression event associated with silencing [88]. This was effectively demonstrated in the human IFN-β gene where the recruitment of a chromatin remodeling complex and subsequent upregulation of transcription relied upon acetylation of three lysine residues [91]. Furthermore, constitutive heterochromatin that is devoid of transcriptional activity (for instance, centromeres, and the silenced X chromosome in females) exist in a hypoacetylated state [92, 93]. Certain histone modifications, acetylation in particular, create an ―open‖ state that makes DNA more accessible to the DNA repair machinery, thus promoting increased stability. An example of this is NER that coincides with a global relaxation of chromatin through a more equal distribution of acetylation at the 9th lysine residue of H3. The same shift in acetylated H3K9 is observed following UV exposure. Interestingly, several types of UV-induced DNA damage employ the NER repair pathway [94]. These chromatin alterations are also likely to serve as communication between TC-NER and GG-NER. The damage detected during transcription is fixed via the TCR mechanism. This causes a local relaxation of chromatin (triggered by the stalled RNA polymerase) which in turn causes a global relaxation, thus permitting GGR to proceed [87]. Histone methylation occurs as a mono-, di-, or tri- event on lysine residues and a monoor di- event on arginines [86]. It can result in different transcriptional consequences depending upon the residue affected [95, 96]. Methylation of K9 of H3 is associated with chromatin compaction and gene silencing, while methylation of K4 and K27 of H3 results in transcription activation and chromatin relaxation. Since these residues may experience three methylation states (mono-, dior tri-methylated), an enormous complexity exists to the yet unexplored histone code [7, 95, 96]. Effects of histone modifications may be compounded as a hypoacetylated state allows for the action of histone methyl transferases (HMTs) that methylate K9 on H3, thus recruiting the non-histone protein heterochromatin protein1 (HP1)—a hallmark of constitutive heterochromatic regions. Moreover, constitutive heterochromatin that is devoid of transcriptional activity and rich in repetitive sequences as well as the silenced X chromosome in females exists in a hypoacetylated and hypermethylated state [92, 93]. Histone modifications that are indicative of silencing commonly occur along with DNA methylation. Furthermore, it was recently shown that tumors undergo a massive loss of tri-methylation at K20 of H4 [97-99]. This loss occurs along with DNA hypomethylation and is linked to chromatin relaxation and aberrant expression. It was suggested to be a universal marker for malignant transformation and genome instability [97]. It has been stipulated that one event may beget the other two. Furthermore, it is not obligatory that DNA methylation changes coincide with silencing histone modifications or vice versa [77]. With the constant flux of genomic stability comes a concomitant change in chromatin profiles. Although the benefits gained by altering the transcriptional activity of a gene may outweigh the risks of congruent changes of stability, there are instances (for example, in

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highly repetitive regions such as telomeric and centromeric DNA or transposons) where shifts away from a highly stable heterochromatic state can present significant challenges to genome integrity. Histone modifications are crucially important for maintenaning stability of telomeric regions. Telomeres are comprised of short tandem repeats of guanine-rich DNA sequences that may be repeated spanning for thousands of base pairs. Maintaining genomic integrity of chromosome ends is mandatory as shortening of telomeric regions results in cell aging and eventual senescence. Furthermore, the structural nature of telomeric regions makes them good substrates for DNA repair processes of NHEJ and HR—events that would lead to gross chromosomal aberrations, aneuploidy and/or gene duplication [100]. To prevent this from happening, telomeres are wrapped into a tight, highly stable, silent, constitutive heterochromatic state characterized by tri-methylated K9 on H3, K20 on H4 and heavy DNA methylation. Interestingly, mice devoid of telomerase (the enzyme responsible for adding telomeric repeats to the 3‘ end of the chromosome following replication) demonstrate shortened telomeres, with decreased tri-methylation and increased acetylation of H3 and H4 and decreased DNA methylation leading to a more open chromatic state. Collectively, these results suggest that there is a communication link between the length of the repeated region and the maintenance of the heterochromtic state—perhaps an example of a DNA sequence involved in dictating the epigenetic profile [101]. Regardless of a tightly condensed chromatin state during interphase, this protection is compromised during mitosis when the genetic structure must be relaxed and accessed by the replication machinery. Therefore, it is reasonable to assume that additional protective mechanisms exist during the relaxed state where relaxed telomeres are recognized as dsDNA breaks, and the signaling and repair machinery localize to them. Nevertheless, the cell cycle proceeds without stalling, and the perceived ―breaks‖ are not processed. This additional protection is likely achieved through the presence of a telomere protection complex called Shelterin. Shelterin is comprised of six telomere-binding proteins that associate with telomeres at all times during the cell cycle. The presence of at least some of these proteins is likely to communicate to the DNA repair machinery and prevent its action [100]. Like telomeres, centromeric DNA sequences are highly repetitive heterochromatic regions. Although devoid of transcriptional activity, their sequence stability is important in achieving a normal un-diseased state for an organism. During replication, sister chromatids are fused at the centromere region of the chromosome The centromere is a dense region of heterchromatin that provides support to keep the sister chromatids attached until metaphase is reached and until the mechanical force applied by the spindles is able to separate the sister chromatids. Thus, the relative strength of centromere stability is in flux when metaphase is reached and a more instable state is displayed [102]. Although centromere functions and the protein machinery that associate with them are highly conserved, the actual sequence and the number of repeats flanking the centromere are not well conserved between mammalian orders [103]. Nevertheless, all species face the challenge of maintaining stability in a region whose sequence renders it an excellent candidate for polymerase slippage (leading to amplifications and deletions) and homolgous recombination [104]. High stability of the centromere proper is a result of its organization into a specialized type of chromatin called centromeric (CEN) chromatin. It is comprised of usual histone proteins plus a special histone and three additional centromeric specific proteins. CEN chromatin is immediately flanked by usual heterchromatin displaying methylation of lysine9 of H3. It appears that the presence and

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length of heterochromatin in the centromeric region may dictate the activity at the centromere proper most likely through the recruitment of non-histone-binding proteins such as HP1. Hence, heterochromatin in the centromeric region regulates the strength and stability of CEN chromatin; the lack of heterochromatin increases the strength of CEN chromatin, while too much chromatin weakens it. Mice embryos lacking the histone methyltransferase responsible for methylating lysine9 demonstrate frequent polyploidy and G1 chromosomes consisting of four sister chromatids. Boundaries between CEN chromatin and heterochromatin are likely controlled by the presence of heterochromatin. CEN chromatin harbors specific proteins and di-methylation of lysine 4 of H3. The spread of these histone markers is prevented by constitutive heterochromatin [103]. Transposon management is another responsibility of histone modifications. As mentioned before, histone modifications work in conjunction with DNA methylation to maintain transposons in a heterochromatic state. Silenced transposons exist in the regions of chromatin that display high levels of tri-methylation at lysine9 of H3 [81]. Typically, they are observed in constitutive heterochromatin at both telomeric and pericentromeric regions. Both these regions of the chromosome display high levels of repeated sequences, and the maintenance of these sequences is crucial for multiple rounds of proper DNA replication and cell division [101, 103]. The ability of transposons to recruit the necessary chromatin remodeling proteins that institute and maintain these regions as facultative heterochromatin may aid in achieving genome stability. Under this premise, the activation of transposons would lead to a more relaxed chromatin state that would further facilitate the loss of repetitiveness in telomeric and percentromeric regions. This may further relax chromatin permitting erroneous repair events and causing senescence or recombination between repeat regions of other chromosomes. Support for this theory lies in the mouse models where transposon re-activation was associated with chromosomal segregation defects [81]. Although influencing transcription and expression is considered to be a paramount purpose of the epigenome, it has also been implicated in identifying DNA lesions, recruiting repair complexes and facilitating repair processes. The most intensely investigated histone modification in response to DNA damage is phosphorylation of histone H2AX at serine residue 139 [105]. H2AX is a member of the H2A histone family. Following damage, it becomes phosphorylated at serine 139 (γH2AX) possibly as one of the earliest cellular responses to double-strand breaks (DSBs) [106-108]. This phosphorylation is carried out by the PI3K-like kinases, a family including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK); these proteins get activated in the presense of DNA damage and phosphorylate H2AX as well as many other DNA repair and checkpoint proteins [105]. γH2AX accumulates in the nucleus at DSBs forming γH2AX foci [109]. This phosphorylation is an early event that promotes the concentration of the repair machinery at the site of a strand break and recruits histone acetyl transferase (HATs) to relax neighboring chromatin. As in the context of strand breaks, phosphorylation of H2AX may be carried out by either ATM or DNA-PK (initiators of HR and NHEJ, respectively), it is likely that γH2AX plays a role in both repair pathways [110]. Although the formation of γH2AX does not appear to favor only one repair mechanism, subsequent histone modifications may act in a mutually exclusive manner by promoting one repair pathway over another [87]. In addition, γH2AX has also been observed in response to replication damage (e.g., collapsed forks) that are primarily detected by the ATR checkpoint protein [111]. Therefore, γH2AX is not only a central component of numerous signalling

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pathways in response to DNA double-strand breaks and other damages but also an important modification that may act as an initiator of further modifications of other histones. Interestingly, the human H2AX gene maps to chromosome 11q23, and this region is frequently deleted or mutated in a large number of human cancers [105]. These cancer cells exhibit a significant destabilization of the genome and are deficient in repair of DNA strand breaks. Analysis of H2AX knockout animals further supported its role in genome stability and DNA repair maintenance. H2AX–/– mice survive well under unstressed conditions, but they are much less efficient at DNA DSB repair and therefore accumulate significant levels of chromosomal abnormalities [105]. Since histone modifications are established by different histone-modifying enzymes, altered levels and functions of these enzymes may directly result in changes in histone modification levels and therefore may affect the chromatin structure and genome stability. Recent reports implicate the histone deacetylase SIRT 1,the yeast Sir2 homologue, in chromatin silencing and genome stability. Mouse knockout studies revealed that a vast majority of SIRT1 null embryos die between E9.5 and E14.5 due to altered histone modification, impaired DNA damage response, and a reduced ability to repair DNA damage [112].

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Short RNA-Mediated Silencing It has been recently discovered that small RNA molecules can act in a potent gene silencing mechanism. This mechanism, termed RNA inhibition (RNAi), involves two classes of short RNA molecules: siRNA (silencing RNA) and miRNA (microRNA) that are 21 to 25 nucleotides in length. These small RNA molecules are generated through a two-step cleavage of long primary RNA transcripts that form a secondary hairpin structure. The first cleavage is performed in the nucleus by the RNase III protein Drosha and results in a shortened hairpin that is transported to the cytoplasm where a second RNase III protein, Dicer, performs a second cleavage. Dicer-dependent cleavage results in short dsRNA molecules that are 19 to 23 base pairs in length. These short dsRNA molecules are loaded onto a RNA-induced silencing complex (RISC) where they are further processed and matched with their complimentary sequences within the 3‘ untranslated region (UTR) of mRNA transcripts. If it happens, two outcomes are possible. In the instance of siRNA, a perfect complimentary match between siRNA and 3‘UTR leads to cleavage of the message by a member of the RISC machinery, the protein Argonaute2. If a small RNA molecule is miRNA, an imperfect match between miRNA and 3‘UTR exists resulting in inhibition of the translational machinery and eventual sequestering and destruction of the messenger molecule. Interference with the translation of transcripts is termed post-transcriptional gene silencing (PTGS) which is a widely conserved mechanism for controlling gene expression across several kingdoms. However, it is also employed in plants, fungi and some invertebrates as an effective mechanism for achieving transcriptional gene silencing (TGS). During TGS, transcribed siRNAs are transported back to the nucleus to guide the acquisition of DNA methylation patterns and/or silencing histone modifications at their complimentary sequences. The result is a localized change in the epigenetic profile of that cell [113].

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In mammals, the potential of siRNA to partake in a TGS mechanism and induce heterochromatin has been described several times in different contexts. When siRNAs harboring a sequence complimentary to the promoter of a stable GFP transgene were transiently introduced in human cells, Morris et al. noted a 93% drop in transcription of the transgene and notably elevated levels of methylation of its promoter region. Furthermore, when siRNA transfected cells were treated with drugs that acted as inhibitors of HDACs (trichostatin) or DNMTs (5-azacytidine), transgene expression was rescued [114]. Suzuki et al. found similar results in human cells infected with HIV-1 and transiently transfected with siRNA complimentary to the promoter region of the viral gene [115]. Once again, a 90% decrease in viral gene transcription coincided with DNA methylation at the promoter region. Similarly, methylation was prevented when cells were treated with 5-azacytidine [115]. Interestingly, Suzuki et al. also reported that the silencing effect lasted in excess of 14 days, considerably longer than the 8 days reported by Morris et al. [114, 115]. Finally, Castanotto et al. were able to diminish transcription of the endogenous gene in human cells by 30 – 60% using stably integrated small hairpin RNAs [116]. Initially, these reports suggest that siRNA-mediated TGS indeed exists in mammalian cells, which means that the genome has the potential to affect changes in its own stability through expression of specific anti-sense short RNA sequences. However, during the five years that have passed since these reports have been communicated, a self-sustaining mechanism geared towards achieving selective transcriptional gene silencing has not been realized. Such a mechanism would require genomes to produce a double-stranded RNAs that are perfectly complimentary to the promoter regions to be silenced. It will also need to develop conditions for efficient processing of these transcripts so that a functionally active type of 21-25- nucleotide-long RNA exists in the nucleus. Furthermore, it will heavily rely on the protein machinery capable of unwinding and loading ssRNA onto its targeted promoter and some sort of amplifying or sustaining mechanisms to keep a gene silenced even after the initial transcription that led to the establishment of silencing in the first place stops. There are several difficulties for the existence of such TGS mechanism in mammalian cells. Firstly, long double-stranded RNA does survive well in mammalian cells. The innate immune system of mammals has evolved to recognize double-stranded RNA molecules over 30 base pairs as unwanted foreign viral invaders. Mammalian cells possess a cytoplasmic dsRNA binding protein, termed protein kinase R (PKR), that, when bound to RNA, initiates a global repression of translation and transcription of numerous genes (such as interferon genes), eventually leading to apoptosis. Although this deleterious effect may be accompanied by some level of dicer-dependent sequence-specific silencing effects, it is a moot point due to imminent death of cells [117, 118]. Since PKR is a cytoplasmic protein, it is possible that long double-stranded RNA confined to the nucleus may avoid its effects. However, nuclei contain an RNA-editing protein ADAR (adenosine deaminase, RNA specific) that acts to destabilize dsRNA by converting adenosine residues to inosine (read as a guanine by the translational machinery), thus causing bulges and mutations in the sequence. These base changes would likely quash any silencing potential the siRNA may have had as complete complementarity was absolutely necessary to achieve any level of silencing and methylation in the above describe reports [114-116, 119]. Furthermore, any surviving long dsRNA would have to be processed into functional 21-25 nt long ones. Since Dicer is cytoplasmic, it is unlikely it could be shuttled to the nucleus without activating PKR and, as of now, there has not been a report of Dicer or a Dicer-like protein isolated from mammalian nuclei. Finally,

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unlike plants, fungi and some invertebrates that possess an RNA-dependent RNA polymerase for amplification of siRNA during TGS, a mammalian homolog has not be isolated [113]. However, it is not necessarily mandatory if, in mammals, pairing of siRNA with its cognate mRNA could initiate chromatin remodeling and the formation of stably silent chromatin. Nevertheless, additional evidence for a role of RNAi in establishing and maintaining genome stability does exist. Dicer-knockout mouse embryonic stem (ES) cells expressed high levels of centromeric repeat sequences that are transcribed from both strands of the centromeric region forming dsRNA, the substrate that Dicer requires. The dsRNA content of cells was shifted from predominatly long to predominantly short dsRNAs in knockout cells compared to Dicer-containing cells, indicating that long dsRNAs were indeed processed by Dicer. DNA methylation was significantly less in knockout cells as was methylation at lysine9 of H3 (reduced by~70%). In addition, when treated with TSA, an inhibitor of HDACs capable of diminishing the heterochromatin content of a cell, the volume of repeated transcripts increased. Finally, reintroducing Dicer into knockout cells rescued all the phenotypes described. Taken together, these data suggest that a Dicer-dependent mechanism for maintaining the heterchromatic state at the centromeric regions may exist [120]. In contrast, it was recently shown that Dicer deficinecy in mouse ES cells was indirectly responsible for telomeric instability as characterized by aberrant elongation of and recombination between telomeres. Benetti et al. have alleged that this is the result of significant DNA demethylation of telomeres and is not due to loss of heterochromatin as usual methylation at lysine9 of H3 was present, and the authors were able to rescue DNA methylation patterns when DNMT expression was restored [121]. The authors went on to demonstrate that telomere instability actually resulted from the depleted presence of miR-290 which in turn allowed for an increased expression of Rb proteins. These Rb proteins bind to promoters of DNMTs, thus suppressing DNMT expression. Transfection of miR-290 alleviated Rb-mediated repression and DNMT expression. Furthermore, DNA methylation and telomere stability were restored. This work suggests that Dicer and Dicer-dependent small RNAs are not directly required for the establishment of heterochromatin at telomeric regions [121]. Another class of recently identified short RNAs plays a role in silencing of transposable elements in genomes. These are small RNA molecules are known as Piwi-interacting RNAs (piRNA). piRNAs were originally discovered in Drosophila. There they are expressed only in germ cells and associated with one of three proteins belonging to the Argonaute subclass of proteins known as Piwi-like proteins. piRNA are transcribed from long transcripts that are typically rich in transposable elements inserted in both orientations, and their production is Dicer-independent. However, all members of the Piwi-like protein class demonstrate the cleavage ability and thus are capable of slicing long transcripts into their functionally active length of 24-30 nt. Smaller piRNAs can then seek out and cleave mRNA transcripts of cognate transposable elements, silencing them effectively. Alternatively, they can interact with genomic sequences promoting the formation of heterochromatin [122, 123]. In mice, there exist three Drosholia Piwi-homologues - Mili, Miwi and Miwi2. Mili- and Miwi2knockout male mice experience a significant increase in the expression of transposable elements, impaired spermatogenesis and depleted DNA methylation at transposable elements in the germline. Interestingly, no phenotype was noted in female lines where only Mili is expressed [124, 125].

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Figure 1. Common types of DNA lesions and mechanisms employed to fix the damage. Several key genes employed in each pathway are listed.

Figure 2. A mechanism of chromatin alteration: histone modification and DNA methylation. Several proteins are involved in maintaining chromatin and converting it from an open accessible euchromatic state to closed heterochromatin. In the presence of histone acetylases (HAT), key residues on particular histones (e.g., K9H3, green cylinders) are acetylated giving rise to a more relaxed chromatin state that is readily transcribed. This euchromatin is typically associated with unmethylated cytosine residues (white circles). Deacetylation of key residues by histone deacetylases (HDAC) promote tighter binding of the DNA duplex resulting in a more condensed chromatin state with diminished transcriptional activity. The hypermethylated CpG dinucleotides (black circles) within the heterochromatic region attract methyl-binding proteins (blue arcs) that are involved in the heterochromatic protein (HP1) complex associated with silenced regions of the genome. The chromatin profile of a cell is fluid as changes in histone acetylation and DNA methylation are commonly observed at particular chromosomal loci (e.g., telomeres and centromers). These changes are considered to be hallmarks of certain pathologies (e.g., cancer). However, it is not clear which event - histone modifications or DNA methylation - occurs first.

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Figure 3. In the post-transcriptional gene-silencing (PTGS) mechanism, double stranded RNA (dsRNA) transcribed as long imperfectly paired hairpins are initially processed within the nucleus by the Drosha/Pasha protein complex exported to the cytoplasm and further processed to a functional length (miR) by an RNAase enzyme—Dicer. With the aid of the RNA-induced silencing complex (RISC), functional dsRNA imperfectly pairs with the 3‘-untranslated region (3‘UTR) of a cognate transcript resulting in repression of translation. It is not yet clear whether these small RNA molecules have epigenetic effects. However, one postulated mechanism of transcriptional gene silencing (TGS) suggests that long RNA is involved in chromatin remodeling. In this mechanism, small RNA molecules (siRNA) are generated from longer and perfectly paired precursors and directly interact with cognate sequences at the genome level. This interaction elicits histone modifications and/or DNA methylation changes that lead to remodeling of chromatin. The processing of the long dsRNA precursor molecules has several possibilities. It may be processed within the nucleus by Dicer or a Dicer-like enzyme or exported and processed in the cytoplasm before being imported back into the nucleus. In addition, editing by the adenosine deamination of RNA (ADAR) protein is also possible, and it may even be necessary to allow the long dsRNA molecule to elude the activation of protein kinase R (PKR). PKR activation is a facet of a cellular innate immune response and ultimately results in apoptosis.

CONCLUSION The genomes of living organisms are constantly under attack from DNA damaging/altering agents—both exogenous and endogenous ones. Deleterious effects of these genetic modifications manifest as diseases that occur as a result of genetic instability. This chapter has briefly introduced a myriad of strategies that mammalian cells have evolved to maintain their genetic integrity. The nuclear organization of DNA is such that it can to some extent protect the genome from damage and resist changes. Mammalian cells also have numerous mechanisms to repair the damage. They include both genetic and epigenetic strategies that frequently work in concert with a goal to achieve maximum genome stability, thus demonstrating that the maintenance of genome integrity is of utmost importance to the cell.

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REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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[13]

[14]

[15]

[16]

[17] [18] [19]

Waddington C: Canalization of development and the inheritance of acquired characteristics. Nature 1942, 3811:563-565. Van Speybroeck L: From epigenesis to epigenetics: the case of C. H. Waddington. Ann. N. Y. Acad. Sci. 2002, 981:61-81. Holliday R: Mechanisms for the control of gene activity during development. Biol. Rev. Camb. Philos. Soc. 1990, 65:431-471. Kovalchuk O: Epigenetic research sheds new light on the nature of interactions between organisms and their environment. Environ. Mol. Mutagen 2008, 49:1-3. Bird A: Perceptions of epigenetics. Nature 2007, 447:396-398. Weber M, Schubeler D: Genomic patterns of DNA methylation: targets and function of an epigenetic mark. Curr. Opin. Cell Biol. 2007, 19:273-280. Weidman JR, Dolinoy DC, Murphy SK, Jirtle RL: Cancer susceptibility: epigenetic manifestation of environmental exposures. Cancer J. 2007, 13:9-16. Bernstein E, Allis CD: RNA meets chromatin. Genes Dev. 2005, 19:1635-1655. Lindahl T: Instability and decay of the primary structure of DNA. Nature 1993, 362:709-715. Hoeijmakers JH: Genome maintenance mechanisms for preventing cancer. Nature 2001, 411:366-374. Sedgwick B, Bates PA, Paik J, Jacobs SC, Lindahl T: Repair of alkylated DNA: recent advances. DNA Repair (Amst) 2007, 6:429-442. Margison GP, Santibanez-Koref MF: O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy. Bioessays 2002, 24:255-266. Aas PA, Otterlei M, Falnes PO, Vagbo CB, Skorpen F, Akbari M, Sundheim O, Bjoras M, Slupphaug G, Seeberg E, Krokan HE: Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 2003, 421:859-863. Glassner BJ, Weeda G, Allan JM, Broekhof JL, Carls NH, Donker I, Engelward BP, Hampson RJ, Hersmus R, Hickman MJ, et al: DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents. Mutagenesis 1999, 14:339-347. Becker K, Gregel CM, Kaina B: The DNA repair protein O6-methylguanine-DNA methyltransferase protects against skin tumor formation induced by antineoplastic chloroethylnitrosourea. Cancer Res. 1997, 57:3335-3338. Ringvoll J, Nordstrand LM, Vagbo CB, Talstad V, Reite K, Aas PA, Lauritzen KH, Liabakk NB, Bjork A, Doughty RW, et al: Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA. Embo J. 2006, 25:2189-2198. Wilson DM, 3rd, Bohr VA: The mechanics of base excision repair, and its relationship to aging and disease. DNA Repair (Amst) 2007, 6:544-559. Dizdaroglu M: Base-excision repair of oxidative DNA damage by DNA glycosylases. Mutat Res. 2005, 591:45-59. Fortini P, Pascucci B, Parlanti E, Sobol RW, Wilson SH, Dogliotti E: Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells. Biochemistry 1998, 37:3575-3580.

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[20] Stucki M, Pascucci B, Parlanti E, Fortini P, Wilson SH, Hubscher U, Dogliotti E: Mammalian base excision repair by DNA polymerases delta and epsilon. Oncogene 1998, 17:835-843. [21] Matsumoto Y, Kim K, Hurwitz J, Gary R, Levin DS, Tomkinson AE, Park MS: Reconstitution of proliferating cell nuclear antigen-dependent repair of apurinic/apyrimidinic sites with purified human proteins. J. Biol. Chem. 1999, 274: 33703-33708. [22] Hakem R: DNA-damage repair; the good, the bad, and the ugly. Embo J. 2008, 27:589605. [23] Sugasawa K, Ng JM, Masutani C, Iwai S, van der Spek PJ, Eker AP, Hanaoka F, Bootsma D, Hoeijmakers JH: Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol. Cell 1998, 2:223-232. [24] Niedernhofer LJ: Nucleotide excision repair deficient mouse models and neurological disease. DNA Repair (Amst) 2008, 7:1180-1189. [25] Laine JP, Egly JM: When transcription and repair meet: a complex system. Trends Genet. 2006, 22:430-436. [26] Laine JP, Egly JM: Initiation of DNA repair mediated by a stalled RNA polymerase IIO. Embo J. 2006, 25:387-397. [27] Araujo SJ, Tirode F, Coin F, Pospiech H, Syvaoja JE, Stucki M, Hubscher U, Egly JM, Wood RD: Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes. Dev. 2000, 14:349-359. [28] Coin F, Proietti De Santis L, Nardo T, Zlobinskaya O, Stefanini M, Egly JM: p8/TTDA as a repair-specific TFIIH subunit. Mol. Cell 2006, 21:215-226. [29] Aboussekhra A, Biggerstaff M, Shivji MK, Vilpo JA, Moncollin V, Podust VN, Protic M, Hubscher U, Egly JM, Wood RD: Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 1995, 80:859-868. [30] Shivji MK, Podust VN, Hubscher U, Wood RD: Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA. Biochemistry 1995, 34:5011-5017. [31] Moser J, Kool H, Giakzidis I, Caldecott K, Mullenders LH, Fousteri MI: Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner. Mol. Cell 2007, 27:311-323. [32] Thoms KM, Kuschal C, Emmert S: Lessons learned from DNA repair defective syndromes. Exp Dermatol 2007, 16:532-544. [33] Mitchell JR, Hoeijmakers JH, Niedernhofer LJ: Divide and conquer: nucleotide excision repair battles cancer and ageing. Curr. Opin. Cell Biol. 2003, 15:232-240. [34] Jiricny J: The multifaceted mismatch-repair system. Nat. Rev. Mol. Cell Biol. 2006, 7:335-346. [35] McCulloch SD, Gu L, Li GM: Bi-directional processing of DNA loops by mismatch repair-dependent and -independent pathways in human cells. J. Biol. Chem. 2003, 278: 3891-3896. [36] Edelmann W, Umar A, Yang K, Heyer J, Kucherlapati M, Lia M, Kneitz B, Avdievich E, Fan K, Wong E, et al: The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression. Cancer Res. 2000, 60:803-807.

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[37] Brown KD, Rathi A, Kamath R, Beardsley DI, Zhan Q, Mannino JL, Baskaran R: The mismatch repair system is required for S-phase checkpoint activation. Nat. Genet. 2003, 33:80-84. [38] Lin DP, Wang Y, Scherer SJ, Clark AB, Yang K, Avdievich E, Jin B, Werling U, Parris T, Kurihara N, et al: An Msh2 point mutation uncouples DNA mismatch repair and apoptosis. Cancer Res. 2004, 64:517-522. [39] O'Brien V, Brown R: Signalling cell cycle arrest and cell death through the MMR System. Carcinogenesis 2006, 27:682-692. [40] Kneitz B, Cohen PE, Avdievich E, Zhu L, Kane MF, Hou H, Jr., Kolodner RD, Kucherlapati R, Pollard JW, Edelmann W: MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice. Genes. Dev. 2000, 14:1085-1097. [41] de Vries SS, Baart EB, Dekker M, Siezen A, de Rooij DG, de Boer P, te Riele H: Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male and female meiosis. Genes. Dev. 1999, 13:523-531. [42] Lipkin SM, Moens PB, Wang V, Lenzi M, Shanmugarajah D, Gilgeous A, Thomas J, Cheng J, Touchman JW, Green ED, et al: Meiotic arrest and aneuploidy in MLH3deficient mice. Nat. Genet. 2002, 31:385-390. [43] Peltomaki P, Vasen HF: Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology 1997, 113:1146-1158. [44] Vasen HF, Moslein G, Alonso A, Bernstein I, Bertario L, Blanco I, Burn J, Capella G, Engel C, Frayling I, et al: Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer). J. Med. Genet. 2007, 44:353-362. [45] San Filippo J, Sung P, Klein H: Mechanism of Eukaryotic Homologous Recombination. Annu. Rev. Biochem. 2008, 77:229-257. [46] Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S: Differential usage of nonhomologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst) 2006, 5:1021-1029. [47] Saberi A, Hochegger H, Szuts D, Lan L, Yasui A, Sale JE, Taniguchi Y, Murakawa Y, Zeng W, Yokomori K, et al: RAD18 and poly(ADP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair. Mol. Cell Biol. 2007, 27:25622571. [48] Xiao Y, Weaver DT: Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. Nucleic Acids Res. 1997, 25:2985-2991. [49] Zhu J, Petersen S, Tessarollo L, Nussenzweig A: Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. Curr. Biol. 2001, 11:105-109. [50] Luo G, Santoro IM, McDaniel LD, Nishijima I, Mills M, Youssoufian H, Vogel H, Schultz RA, Bradley A: Cancer predisposition caused by elevated mitotic recombination in Bloom mice. Nat. Genet. 2000, 26:424-429.

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[51] Shinohara A, Ogawa H, Matsuda Y, Ushio N, Ikeo K, Ogawa T: Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA. Nat. Genet. 1993, 4:239-243. [52] Esashi F, Christ N, Gannon J, Liu Y, Hunt T, Jasin M, West SC: CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature 2005, 434:598-604. [53] Lim DS, Hasty P: A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol. Cell Biol. 1996, 16:7133-7143. [54] Ludwig T, Chapman DL, Papaioannou VE, Efstratiadis A: Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos. Genes. Dev. 1997, 11:1226-1241. [55] Wesoly J, Agarwal S, Sigurdsson S, Bussen W, Van Komen S, Qin J, van Steeg H, van Benthem J, Wassenaar E, Baarends WM, et al: Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis. Mol. Cell Biol. 2006, 26:976-989. [56] Rijkers T, Van Den Ouweland J, Morolli B, Rolink AG, Baarends WM, Van Sloun PP, Lohman PH, Pastink A: Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation. Mol. Cell Biol. 1998, 18:64236429. [57] Lieber MR: The mechanism of human nonhomologous DNA end joining. J. Biol. Chem. 2008, 283:1-5. [58] Ma Y, Lu H, Tippin B, Goodman MF, Shimazaki N, Koiwai O, Hsieh CL, Schwarz K, Lieber MR: A biochemically defined system for mammalian nonhomologous DNA end joining. Mol. Cell 2004, 16:701-713. [59] Jager U, Bocskor S, Le T, Mitterbauer G, Bolz I, Chott A, Kneba M, Mannhalter C, Nadel B: Follicular lymphomas' BCL-2/IgH junctions contain templated nucleotide insertions: novel insights into the mechanism of t(14;18) translocation. Blood 2000, 95:3520-3529. [60] Lieber MR, Ma Y, Pannicke U, Schwarz K: The mechanism of vertebrate nonhomologous DNA end joining and its role in V(D)J recombination. DNA Repair (Amst) 2004, 3:817-826. [61] Gu J, Lu H, Tippin B, Shimazaki N, Goodman MF, Lieber MR: XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps. Embo J. 2007, 26:1010-1023. [62] Branzei D, Foiani M: Regulation of DNA repair throughout the cell cycle. Nat. Rev. Mol. Cell Biol. 2008, 9:297-308. [63] Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 2003, 33 Suppl:245-254. [64] Gopalakrishnan S, Van Emburgh BO, Robertson KD: DNA methylation in development and human disease. Mutat Res. 2008, 647:30-38. [65] Baylin SB: DNA methylation and gene silencing in cancer. Nat. Clin. Pract. Oncol. 2005, 2 Suppl 1:S4-11.

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[66] Baylin SB, Ohm JE: Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat. Rev. Cancer 2006, 6:107-116. [67] Jirtle RL, Skinner MK: Environmental epigenomics and disease susceptibility. Nat. Rev. Genet. 2007, 8:253-262. [68] Ooi SK, Bestor TH: Cytosine methylation: remaining faithful. Curr Biol 2008, 18:R174-176. [69] Brenner C, Fuks F: DNA methyltransferases: facts, clues, mysteries. Curr. Top. Microbiol. Immunol. 2006, 301:45-66. [70] Goll MG, Bestor TH: Eukaryotic cytosine methyltransferases. Annu. Rev. Biochem. 2005, 74:481-514. [71] Okano M, Bell DW, Haber DA, Li E: DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999, 99:247257. [72] Watanabe D, Suetake I, Tada T, Tajima S: Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech. Dev. 2002, 118:187-190. [73] Li E, Bestor TH, Jaenisch R: Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992, 69:915-926. [74] Mohn F, Schubeler D: Genetics and epigenetics: stability and plasticity during cellular differentiation. Trends Genet. 2009, 25:129-136. [75] Saxonov S, Berg P, Brutlag DL: A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc. Natl. Acad. Sci. USA 2006, 103:1412-1417. [76] Klose RJ, Bird AP: Genomic DNA methylation: the mark and its mediators. Trends Biochem. Sci. 2006, 31:89-97. [77] Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D: Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 2007, 39:457-466. [78] Shen JC, Rideout WM, 3rd, Jones PA: The rate of hydrolytic deamination of 5methylcytosine in double-stranded DNA. Nucleic Acids Res. 1994, 22:972-976. [79] Esteller M: Aberrant DNA methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicol. 2005, 45:629-656. [80] Yoder JA, Walsh CP, Bestor TH: Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 1997, 13:335-340. [81] Slotkin RK, Martienssen R: Transposable elements and the epigenetic regulation of the genome. Nat. Rev. Genet. 2007, 8:272-285. [82] Robertson KD: DNA methylation and chromatin - unraveling the tangled web. Oncogene 2002, 21:5361-5379. [83] Robertson KD, Wolffe AP: DNA methylation in health and disease. Nat. Rev. Genet. 2000, 1:11-19. [84] Xu GL, Bestor TH, Bourc'his D, Hsieh CL, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ, Viegas-Pequignot E: Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 1999, 402:187-191.

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[85] Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997, 389:251-260. [86] Munshi A, Shafi G, Aliya N, Jyothy A: Histone modifications dictate specific biological readouts. J. Genet. Genomics 2009, 36:75-88. [87] Escargueil AE, Soares DG, Salvador M, Larsen AK, Henriques JA: What histone code for DNA repair? Mutat Res. 2008, 658:259-270. [88] Jenuwein T, Allis CD: Translating the histone code. Science 2001, 293:1074-1080. [89] Cosgrove MS, Wolberger C: How does the histone code work? Biochem. Cell Biol. 2005, 83:468-476. [90] Kouzarides T: Chromatin modifications and their function. Cell 2007, 128:693-705. [91] Agalioti T, Chen G, Thanos D: Deciphering the transcriptional histone acetylation code for a human gene. Cell 2002, 111:381-392. [92] Ekwall K, Olsson T, Turner BM, Cranston G, Allshire RC: Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell 1997, 91:1021-1032. [93] Jeppesen P, Turner BM: The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell 1993, 74:281-289. [94] Rubbi CP, Milner J: p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage. Embo J. 2003, 22:975-986. [95] Cheung P, Lau P: Epigenetic regulation by histone methylation and histone variants. Mol. Endocrinol. 2005, 19:563-573. [96] Saha A, Wittmeyer J, Cairns BR: Chromatin remodelling: the industrial revolution of DNA around histones. Nat. Rev. Mol. Cell Biol. 2006, 7:437-447. [97] Fraga MF, Esteller M: Towards the human cancer epigenome: a first draft of histone modifications. Cell Cycle 2005, 4:1377-1381. [98] Tryndyak V, Kovalchuk O, Pogribny IP: Identification of differentially methylated sites within unmethylated DNA domains in normal and cancer cells. Anal. Biochem. 2006, 356:202-207. [99] Tryndyak VP, Muskhelishvili L, Kovalchuk O, Rodriguez-Juarez R, Montgomery B, Churchwell MI, Ross SA, Beland FA, Pogribny IP: Effect of long-term tamoxifen exposure on genotoxic and epigenetic changes in rat liver: implications for tamoxifeninduced hepatocarcinogenesis. Carcinogenesis 2006, 27:1713-1720. [100] Matulic M, Sopta M, Rubelj I: Telomere dynamics: the means to an end. Cell Prolif. 2007, 40:462-474. [101] Benetti R, Garcia-Cao M, Blasco MA: Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres. Nat. Genet. 2007, 39:243-250. [102] Bernard P, Allshire R: Centromeres become unstuck without heterochromatin. Trends Cell Biol. 2002, 12:419-424. [103] Schueler MG, Sullivan BA: Structural and functional dynamics of human centromeric chromatin. Annu. Rev. Genomics Hum. Genet. 2006, 7:301-313. [104] Warburton PE, Willard HF: PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA. Nucleic Acids Res. 1992, 20:6033-6042.

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[105] Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, Pommier Y: GammaH2AX and cancer. Nat. Rev. Cancer 2008, 8:957-967. [106] Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM: Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem. Cell Biol. 2003, 81:123-129. [107] Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM: DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 1998, 273:5858-5868. [108] Sedelnikova OA, Pilch DR, Redon C, Bonner WM: Histone H2AX in DNA damage and repair. Cancer Biol. Ther. 2003, 2:233-235. [109] Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, et al: Genomic instability in mice lacking histone H2AX. Science 2002, 296:922-927. [110] Stiff T, O'Driscoll M, Rief N, Iwabuchi K, Lobrich M, Jeggo PA: ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res. 2004, 64:2390-2396. [111] Ward IM, Chen J: Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 2001, 276:47759-47762. [112] Wang RH, Sengupta K, Li C, Kim HS, Cao L, Xiao C, Kim S, Xu X, Zheng Y, Chilton B, et al: Impaired DNA damage response, genome instability, and tumorigenesis in SIRT1 mutant mice. Cancer Cell 2008, 14:312-323. [113] Siomi HaS, MC.: On the road to reading the RNA-interference code. Nature 2009, 457:396-404. [114] Morris KV, Chan SW, Jacobsen SE, Looney DJ: Small interfering RNA-induced transcriptional gene silencing in human cells. Science 2004, 305:1289-1292. [115] Suzuki Kea: Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1promoter region. Journal of RNAi and Gene Silencing 2005, 1:66-78. [116] Castanotto D, Tommasi S, Li M, Li H, Yanow S, Pfeifer GP, Rossi JJ: Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol Ther 2005, 12:179-183. [117] Cole JL: Activation of PKR: an open and shut case? Trends Biochem. Sci. 2007, 32:5762. [118] Lemaire PA, Anderson E, Lary J, Cole JL: Mechanism of PKR Activation by dsRNA. J. Mol. Biol. 2008, 381:351-360. [119] Ohman M: A-to-I editing challenger or ally to the microRNA process. Biochimie 2007, 89:1171-1176. [120] Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T, Livingston DM, Rajewsky K: Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes. Dev. 2005, 19:489-501. [121] Benetti R, Gonzalo S, Jaco I, Munoz P, Gonzalez S, Schoeftner S, Murchison E, Andl T, Chen T, Klatt P, et al: A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases. Nat. Struct. Mol. Biol. 2008, 15:998.

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[122] Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Hannon GJ: Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 2007, 128:1089-1103. [123] Girard A, Sachidanandam R, Hannon GJ, Carmell MA: A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 2006, 442:199-202. [124] Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, Asada N, Kojima K, Yamaguchi Y, Ijiri TW, et al: DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes. Dev. 2008, 22:908-917. [125] Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T, Nakano T, et al: Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 2008, 453:539-543.

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In: Genome Instability and Transgenerational Effects ISBN: 978-1-60876-831-8 Editors: I. Kovalchuk, O. Kovalchuk, pp. 33-69 © 2010 Nova Science Publishers, Inc.

Chapter 3

GENETIC AND EPIGENETIC REGULATION OF GENOME STABILITY IN PLANTS Alex Boyko and Igor Kovalchuk Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, CANADA

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ABSTRACT Maintaining genome stability is one of the most critical tasks for each and every living organism. During their life span, organisms are constantly exposed to a variety of internal and external stimuli, namely stresses, which can alter genome stability and lead to heritable changes. Over the past several years, a number of naturally occurring stresses were revealed to affect genome stability. They include: light spectrum and the day length, various types of ultraviolet and ionizing radiation, chemical mutagens and herbicides, temperature, salt, water stress and pathogen attacks. In fact, many of those stresses can change genome stability at both genetic and epigenetic levels, inducing rearrangements and altering existing DNA methylation patterns. Control and/or prevention of these changes is an issue of vital importance as they exert their influence on organisms experiencing stress, and they can be transmitted to progeny altering genome stability for several generations. The epigenetic regulation, including DNA methylation, histone acetylation/methylation and chromatin remodeling, play a key role not only in controlling gene expression but also in preventing undesirable rearrangements and transposon activation. The presence of high amounts of heterochromatic DNA containing various repetitive elements, pseudo genes, and virus-derived sequences that often carry strong promoters and/or enhancers emphasizes the importance of epigenetic mechanisms in controlling genome stability. The system of epigenetic DNA modifications relies on a complex network of various protein factors, including numerous DNA and histone methyltransferases, histone deacetylases, methyl-CpG-binding domain proteins etc., many of which are guided via multifaceted systems of small RNA molecules. Overall, these systems permit sequence-specific changes in DNA epigenetic modifications, which allow adjusting transcriptional profiles of cells to new conditions and also modifying genome stability, if needed. This chapter is focused on various aspects and mechanisms involved in the maintenance of genome stability. It also discusses how changing the stability of a given locus may contribute to genome evolution and the ability to adapt to stress.

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INTRODUCTION The maintenance of genome stability and integrity is a vital task for every species. Organisms are continuously interacting with the environment which imposes numerous stimuli upon them. In order to survive the influence of the environment, organisms must effectively respond and, if possible, develop adaptive changes that can be advantageous under new conditions. The majority of these stimuli are commonly described as stresses, and they can be subdivided in several categories. Internal stresses usually result from various metabolic activities undergoing in a cell, such as photosynthesis, cellular respiration and other physiological activities associated with functions of lysosomes and peroxisomes. These cellular processes lead to the internal formation of radicals that cells have to deal with. During DNA replication, polymerase errors can also be placed into the category of internal stresses. In contrast, external stresses are imposed by the environment surrounding organisms, and they can be further subdivided into two subclasses based on their abiotic or biotic nature (reviewed by Madlung and Comai, 2004) [1]. A sedentary life style that is typical for plants confines their stress-surviving strategies to tolerance and resistance mechanisms and demands introducing effective means directed at the maintenance of genome stability in an ever-changing growth environment. Genome stability is maintained through a variety of different mechanisms, including direct repair of DNA damage, genetic and epigenetic regulation of gene activity under physiological conditions and stress, the development of stress tolerance, and the epigenetically-mediated establishment and stable inheritance of gene transcription profiles conferring memory of stress. If needed, genome stability can be relaxed by choosing different DNA repair pathways and/or introducing epigenetic modifications such as DNA methylation into the genome. This relaxation of genome stability can occur on a whole genome scale, or it can be directed to specifically defined DNA regions, thus curving the plant genome and mediating its evolution. In this chapter, we discuss various factors and regulation mechanisms that can affect and modify genome stability in response to environmental stimuli. Based on our findings and recent reports, we hypothesize that these modifications can play a role in genome evolution and in the development of stress tolerance in plants.

1. ENDOGENOUS AND EXOGENOUS FACTORS THAT AFFECT GENOME STABILITY 1.1. Reactive Oxygen Species Challenge DNA the Repair Machinery and Decrease Genome Stability Despite the existence of many differences among stresses, a vast majority of them share some major similarities in the way they affect plants. Frequently, primary stress itself may be associated with increased reactive oxygen species (ROS) production (for example, ionizing radiation), and it may also lead to a secondary oxidative stress (for example, high light, salinity). Both scenarios are equally dangerous to plants. All ROS are moderate or highly reactive molecules which may react with cell proteins, unsaturated fatty acids in the plasma

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membrane, carbohydrates, and nucleic acids, thus leading to oxidative damage [2]. Ultimately, ROS over-accumulation may trigger cell apoptosis [3]. Oxidative damage to DNA caused by the interaction with ROS is of special concern, as its powerful mutagenic properties threaten genome stability. Superoxide anion and hydrogen peroxide are not normally reactive towards DNA. However, in the presence of a ferrous or cuprous ion, they can be converted to the highly reactive hydroxyl radical via the HaberWeiss and Fenton reaction. DNA exposure to the hydroxyl radical results in the release of free bases from DNA. It also generates DNA single and double strand breaks (SSBs and DSBs), DNA-protein crosslinks, and simple apurinic/apyrimidinic sites (AP) [4]. DNA replication through AP sites results mainly in dATP insertion (reviewed by Loeb and Preston 1986) [5]. In contrast, repair of abasic sites by base excision repair leads to the introduction of transient DNA strand breaks [6]. If left unrepaired, SSBs and DSBs caused by ROS can block DNA replication, and therefore they can be lethal to a cell [7]. Moreover, repair of strand breaks frequently results in deletions, insertions, point mutations, and may lead to gene conversion and gene translocation events, as well as to duplication events [5, 811]. Importantly, no hydroxyl radical scavengers are produced in plant cells [12]. Therefore, it indicates that the avoidance of oxidative DNA damage and associated genotoxic effects is possible only by precise control over the reactions that lead to the generation of hydroxyl radicals. To avoid a negative influence of ROS and achieve a high degree of flexible control over ROS accumulation, plants have developed complex and efficient enzymatic and nonenzymatic antioxidant defense systems that allow scavenging ROS and protecting cells from oxidative damage [2, 13, 14]. Overall, an equilibrium exists between ROS production and ROS scavenging under physiological conditions. This delicate balance can be shifted toward a rapid production of ROS by a number of stress factors. A rapid increase in ROS accumulation is known as an oxidative burst, and it was shown to be a general response to various biotic and abiotic stresses [14]. Indeed, a number of studies demonstrated that ROS production was triggered by various stress conditions, including drought and desiccation, high temperature, chilling, salt, heavy metals, UV, intensive light, air pollutants (ozone and SO2), mechanical and nutrient deprivation, and pathogen attacks [3, 15-17]. In fact, ROS play a critical role as secondary messengers in stress-response signal transduction for a vast majority of stresses [17, 18]. Different stresses such as cold, drought and salinity result in the activation of similar signaling pathways [19, 20]. Overall, a tightly regulated balance between different ROSproducing and ROS-scavenging mechanisms existing in a cell allows ROS to play an important role as cellular stress indicators and secondary messengers associated with stressresponse signal transduction pathways [13, 17, 18].

1.2. The Choice of DNA Repair Pathway May Regulate Plant Genome Stability Maintaining genome stability requires the presence of efficient repair mechanisms of oxidative DNA damage. In fact, these mechanisms are the keystones for successful plant defense against stress. They help preserve genome integrity by reducing the risk of heritable mutations. DNA DSBs are among the most critical types of DNA damage which result from

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DNA interactions with ROS. If left unrepaired, DNA DSBs may lead to loss of genetic information and chromosomal translocations. They may cause replication arrest and even trigger apoptosis, if cells suffer extensive DNA damage [7, 21, 22]. In contrast, the presence of DSBs is absolutely required for the initiation of DNA strand exchange via homologous recombination (HR) and for generation of genetic diversity in gametes during meiosis [23]. There are two major evolutionary conserved DNA repair pathways available in an eukaryotic cell that have an impact on DSBs and insure the maintenance of genome stability: non-homologous end-joining (NHEJ) and HR. HR requires extensive sequence homology for repair events to occur, whereas in NHEJ repair, significant homology between interacting DNA molecules is not required. NHEJ involves the direct rejoining of break ends. If breaks are not rejoined by a simple ligation reaction, proteins involved in NHEJ search for microhomology, align one or a few complementary bases and remove the DNA in between [22, 24]. Consequently, NHEJ is a relatively inaccurate DNA repair pathway which is frequently associated with small and large-scale deletions (from 1bp up to >1kb), insertions (up to 1.2kp long) or point mutations [9]. In contrast, HR is considered to be a more precise DNA repair pathway with a 100% accuracy, if perfectly homologous templates such as sister chromatids, homologous chromosomes, or repeated regions are used to prime repair synthesis. Alternatively, if a template with imperfect homology is used for repair, HR may result in gene conversion events leading to loss of heterozygosity. HR may result in gene translocation and duplication events and in generation of large deletions, if intrachromosomal recombination occurs within the DNA region containing multiple repeats [10, 11, 25]. Finally, compared to replicative polymerases, lower fidelity of repair polymerases causes an increase in the point mutation frequency at the sites of DSBs, even if a perfectly homologous template is used for repair [26]. To maintain the required level of genome stability, cells can discriminate in favor of using one of these two DSB repair pathways. In general, the balance between HR and NHEJ is tightly controlled, and it depends on the availability of repair templates, cell cycle phases, proliferation rate, and functions of specific cell types (reviewed in Shrivastav et al., 2008) [22]. Whereas NHEJ is dominant during the G1 phase of the cell cycle, upregulation of HR can be observed during the S and G2 phases, if sister chromatids are available [22]. This shift can be achieved via increased expression of RAD52 and RAD51 during the S phase [27]. Moreover, the balance between these two DSB repair pathways changes during plant development and differs in various plant tissues [28, 29]. These changes correlate with different types of cell genome content and ploidy levels in different plant tissues during different developmental stages [30, 31]. High activity of HR in plant cells containing large amounts of repetitive DNA may pose a threat to genome stability and cause large-scale deletions and chromosomal translocations [9, 11, 32]. Genome stability is regulated not only by a non-random selection of either one of the two, HR and NHEJ, pathways. Different mechanisms representing the same pathway and resulting in different repair outcomes can be preferentially used by a cell at different stages of its life. In fact, repair of DSBs via a HR pathway in somatic tissues mainly occurs via the SDSA (synthesis-dependent strand annealing) and SSA (single-strand annealing) mechanisms. In contrast, cells undergoing meiosis repair DSBs preferentially via the DSBR (double strand break repair) mechanism [9, 11]. This difference can be explained by the fact that the DSBR mechanism involving the formation of a double Holiday junction and its resolution leads to gene conversion and crossover events [9]. Hence, to reduce the risk of

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crossovers and translocations, the SDSA mechanism is predominantly used in somatic cells. Moreover, the prevalence of the SDSA mechanism over the DSBR one in somatic plant cells may be critical, since plants contain high amounts of repetitive DNA in their nucleus, which can increase the risk of unpredictable deleterious genome rearrangements by crossovers. Therefore, the SDSA mechanism represents a safer option, since it may result only in gene conversion events [9, 11]. A very interesting difference can be noticed, if we compare the involvement of HR and NHEJ pathways in DSB repair in yeast and higher eukaryotes. While HR is the dominant mode of DSB repair in yeast [33, 34], a vast majority of breaks in higher eukaryotes are processed by NHEJ [35, 36]. This selective processing of DSBs via one of the two aforementioned pathways has important consequences for genome stability in each species and can be explained by their genome content. In contrast to the small yeast genome comprised mainly of coding sequences, higher eukaryotes have large genomes with a very high content of non-coding sequences [37]. HR as the dominant mode of repair allows yeast to prevent generation of random changes in coding DNA which can occur if the same damage is repaired using NHEJ. In contrast, small-scale deletions and insertions caused by NHEJ [9, 38, 39] can be easily accommodated by higher eukaryotes due to their large size genomes with a low content of coding sequences. Furthermore, NHEJ as the dominant mode of repair helps resolve the challenge of locating a homologous template in a large size genome [39, 40].

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1.3. The Importance of Plant Genome Plasticity for Stress Tolerance and Genome Evolution Plant adaptation to changing environmental conditions represents a complex array of biochemical, molecular, and metabolic responses, and all of them are orchestrated by the genetic material of a cell. High plasticity of the plant genome is one of the key determinants that are required for quick acclimation to ever-changing growth conditions [41, 42]. Plants rely on a complex network of stress perception and stress signaling pathways, many of which may cross-talk at various steps [19, 20, 41, 43]. This causes the activation of hundreds of genes, changes in cell transcription and metabolome profiles followed by adaptive changes to new growth conditions [44-47]. Continuous exposure to stress may not always have a negative impact on plants and their metabolism. In many cases, exposure to mild stress is followed by acquired acclimation to more severe conditions. To date, much experimental evidence has been accumulated suggesting the existence of a phenomenon of fast stress-mediated acclimation to drought, cold and freezing, high temperature, high light and UV-B radiation stresses [42, 48, 49]. Successful adaptation to the new environment does not completely rely on physiological changes, it also requires effective mechanisms protecting the integrity of the plant genome under a negative influence of stress [1, 50]. In fact, a number of natural stresses and various chemicals were shown to affect genome stability. Among them are light spectrum [51], day length [52], various types of ultraviolet (UV) [53-56] and ionizing radiation [57], chemical mutagens [58, 59], toxic substances such as herbicides [60], temperature [52, 61, 62], salt [56, 63], water stress [151], and pathogen attacks [64-66].

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Recent studies suggested that the activity of HR pathways may not only affect genome stability but also contribute to genome evolution and development of stress tolerance. Mistakes generated during DSB repair provide the raw material for evolution and also increase intragenome diversity and plasticity [9, 11, 36, 64]. In plant genomes that contain high amounts of repetitive DNA, the elevated activity of HR may mediate rearrangements between repetitive sequences, and these rearrangements may generate new alleles of existing genes. In fact, many disease-resistance genes usually show high polymorphism and can be located in clusters, thereby being a good target for HR-mediated rearrangements [67-69]. This hypothesis is consistent with a recently reported increase in the frequency of somatic HR in the progeny of virus-infected plants [64]. Another important component of a system that controls genome stability can be recognized if the negative correlation between the frequency of HR occurrence and methylation levels of repair templates is considered [70, 71]. This indicates that DNA methylation can be used to protect important genes from undesirable rearrangements, or in contrast, DNA demethylation can encourage recombination in plants exposed to certain stress [64, 72]. Importantly, such correlation between HR frequency and DNA methylation at a specific locus was demonstrated for the progeny of virus-infected plants [64]. The complex interplay between DNA methylation, genome stability, and gene expression patterns induced by stress raises an important question about the role of epigenetic regulations in the maintenance of genome stability and response to stress.

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2. MECHANISMS AND FUNCTIONS OF EPIGENETIC REGULATIONS IN THE PLANT GENOME A number of abiotic stress conditions can persist at a given site for a prolonged period of time, thereby continuously challenging the defense system of plants during many generations. As it has already been mentioned, plants have a sedentary lifestyle and cannot use an escape as a strategy to minimize the influence of stress. Hence, plants require the presence of efficient short-term defense strategies for a fast development of stress tolerance. These strategies can be based on the manipulation of existing genetic information, and they may include alterations in plant homeostasis during somatic growth [73, 74] and heritable (also called transgenerational) modifications of gene expression [75]. These modifications can occur without changing the original DNA sequence, and they are known as epigenetic modifications. They can be achieved at several interdependent levels, including heritable but reversible methylation of DNA sequences, numerous histone modifications and chromatin remodeling [76, 77]. All these modifications can be regulated by a number of physiological and developmental stimuli including stress [78]. The spectrum of external and internal influences experienced by an organism during its lifespan may lead to the generation of specific changes in gene expression that could be epigenetically (without changing the DNA sequence) fixed and passed to the progeny forming an epigenetic memory. Epigenetic memory is initially fixed in the form of DNA methylation and can be further reinforced via numerous histone modification and chromatin remodelling processes that lead to the formation of heritable epialleles and paramutations [78].

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The maintenance of changes in gene expression in prokaryotic and eukaryotic organisms over several cell generations is well documented [79]. In fact, the notion that transgenerational changes in DNA methylation are more frequently observed in plants than in animals [80] is consistent with a sedentary lifestyle of plants. In contrast to animals, plants establish the germ line late during the development, thus allowing the transmission of epigenetic memory accumulated during their life period to the following generations.

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2.1. DNA Methylation Is a Critical Component in the System of Epigenetic Modifications in Plants DNA methylation plays a major role in various functions of the cell: regulation of gene expression, transposable element activity, in the defense against foreign DNA, and inheritance of specific gene expression patterns [81]. The major differences in methylation patterns between plants and animals include a substantially higher percentage of modified cytosines (30% in plants versus 2-8% in animals), significantly higher levels of CpNpG, and the presence of asymmetrical cytosine methylation [79, 82-84]. Symmetrical CpG and CpNpG methylation is inherited during DNA replication in the form of hemimethylated sequences. Hence, it provides a methylation imprint memory on the parental DNA and also guides the activity of methyltransferases [79]. On the contrary, asymmetrical cytosine methylation must be reestablished de novo after each replication cycle, since there is no complementary methylated sequence available to guide remethylation [85, 86]. Finally, symmetric and asymmetric DNA methylation can exist independently of each other playing a significant role in gene silencing in plants [87]. Experimental evidence suggests the existence of three distinct classes of enzymes responsible for cytosine methylation (Table 1). The first class is represented by a plant homologue of mammalian Dnmt1 methyltransferases, METHYLTRANSFERASE1 (MET1) (Table 1). Plants defective in MET1 activity show lack of widespread CpG methylation [88]. While dnmt1 mutations are lethal for animals, plants deficient in MET1 activity are viable [89]. However, met1 plants inbred for several generations exhibit progression of abnormalities [90, 91]. Overall, experimental evidence exists that MET1 may not be required for establishing new methylation imprints [92, 93]. In contrast, the establishment and maintenance of de novo CpG methylation of targeted sequences during RNA-directed DNA methylation (RdDM) requires MET1 activity [94, 95]. The second class of methyltransferases, CHROMOMETHYLASE3, is unique to plants (Table 1). A cmt3 loss-of-function mutant is characterized by a genome-wide loss of CpNpG methylation, especially at centromeric repeats and transposons [88, 96]. A decrease in asymmetric methylation at several genomic loci was also observed [88, 97]. Recent studies on the activation of a normally silenced CACTA transposon in the met1 and cmt3 single and double mutants indicated redundancy in the function of CMT3 with MET1 in CpG and CpNpG methylation [98]. This could explain why dnmt1 mutations are lethal for animals and homozygous met1 plant mutants are viable.

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Table 1. Plant factors involved in the maintenance of DNA methylation Effects on chromatin

Effects of mutation and the involvement in stress response

Modification/ Transcription

Refs

METHYLTRANSFERAS E1 (MET1)/ Methyltransferase

Methylation of symmetrical CpG sites; post replicative de novo CpG methylation; not required to establish new methylation imprints; mediates the establishment and maintenance of de novo CpG methylation in the RdDM pathway

Lack of CpG methylation; passive loss of DNA methylation throughout generations. MET1 is repressed in response to stress, leading to activation of repressed genes

Global/ Repression

[89, 91, 94, 95, 142, 145]

CHROMOMETHYLASE 3 (CMT3)/ Methyltransferase

CpNpG methylation; functionally redundant with MET1 and DRM in methylation of CpG and asymmetrical sites, respectively; targets centromeric repeats and transposons

Loss of CpNpG methylation

Global/ Repression

[88, 96, 97]

DOMAIN REARRANGED METHYLTRANSFERAS ES (DRM1, DRM2)/ Methyltransferase

de novo methylation of asymmetric sites; functionally redundant with CMT3 in CpNpG methylation; possibly reinforces preexisting methylation; DRM2 mediates the establishment of de novo CpG methylation in the RdDM pathway

Loss of de novo asymmetric methylation at non-CpG sites

Global/ Repression

[88, 9497, 99101, 165]

Local, promoters/ Activation

[108-110]

Local, promoters/ Activation

[106, 109, 112, 113]

Local, 5′ and 3′ ends of genes/ Mostly unaffected

[109, 114]

Name and function

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Cytosine methylation

Cytosine demethylation DEMETER (DME)/ DNA glycosylase

Demethylation of previously silenced sequences, possibly in a tissue-specific manner

REPRESSOR OF SILENCING1 (ROS1)/ DNA glycosylase/lyase

Demethylation activity on methylated and not on demethylated DNA substrates

DEMETER-LIKE (DML) proteins: DML2 and DML3/ DNA glycosylase/lyase

Demethylation activity is primarily localized at the 5′ and 3′ ends of genes, thus preventing the accumulation of methylation at or near genes. DML proteins remove this aberrant methylation and prevent the formation of highly methylated stable epialleles.

Inability to activate imprinted genes; inheritance of a mutant maternal allele results in seeds abortion Hypermethylation and transcriptional silencing of specific genes; enhanced sensitivity to genotoxic agents Hypermethylation of gene sequences at either the 5′ or 3′ end; dml mutant hypermethylation has a negligible effect on gene expression

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The last known class, DOMAIN REARRANGED METHYLTRANSFERASES, is composed of DRM1 and DRM2 (Table 1); it shows homology to the mammalian Dnmt3 methyltransferase [99]. DRM1 and DRM2 are mainly directed to de novo methylation of asymmetric sites [100], and they are capable of methylating CpNpG sites along with CMT3 [101]. Consistently, DRM2 is required for de novo non-CpG methylation at RdDM-targeted sequences [94, 95]. While the presence of DNA methylating enzymes is well proven, the existence of direct DNA demethylation mechanisms remains controversial. In fact, enzyme-mediated active DNA demethylation previously reported and observed in cell extracts [102, 103] has not been confirmed. It was also suggested that nucleotide excision repair could actively demethylate DNA [104, 105]. However, no demethylase involved in this pathway has been cloned and described yet. In contrast, a passive loss of DNA methylation may occur due to the inhibition of de novo DNA methylation or inability to maintain the parental imprint after DNA replication as it is observed in met1 mutants [89]. Alternatively, active demethylation may occur via the glycosylase activity by removing 5-methylcytosins from DNA [106-108]. It may play a critical role in preventing the formation of stable hypermethylated epialleles in the plant genome [109]. Indeed, the demethylation activity of Arabidopsis DNA glycosylase DEMETER (DME) regulates the gametophytespecific activation of flowering time (FWA) gene expression [110] (Table 1). It also reverses imprinting of maternal copies of a MEDEA allele in the endosperm [111]. Gong et al. (2002) isolated a REPRESSOR OF SILENCING1 (ROS1) gene that encodes a DNA glycosylase/lyase functioning on methylated and not demethylated DNA substrates [112] (Table 1). Transcriptional gene silencing in ros1 mutants can be released by the ddm1 mutation and application of DNA methylation inhibitors [112]. Also, ROS1 exhibits high similarity in protein domains with DME [113]. To date, four members of the DEMETER DNA glycosylase family involved in DNA demethylation have been found in Arabidopsis. They are DME and three DME-LIKE proteins, DEMETER-LIKE2 (DML2), DML3, and ROS1 [109, 114].

2.2. Histone Modifications and DNA Methylation Are Interdependent Transcription of DNA templates occurs within nucleosomes containing DNA wrapped around an octamer histone core. This offers yet another opportunity to control genome activity via modifications of histone proteins. These modifications include (de)methylation and (de)acetylation of histone proteins and may regulate gene expression. It was demonstrated that the euchromatin state is dependent on hyperacetylation of histones H3 and H4 along with methylation of H3 at the lysine K4 position [79]. In contrast, the formation of heterochromatin structure requires underacetylation of H3 and H4, methylation of K9, and demethylation of K4 residues of H3 [79]. It was suggested, however, that different methylation states of histone H3 might also result from deposition of two independent H3 variants that differ in sequences and posttranslational modifications, particularly in an enrichment of methylated K9 and K27 [115]. There are several experimental evidences suggesting the interdependence of DNA and histone methylation. It was shown that CpG methylation loss in the met1 mutant results in H3K9 methylation loss [116, 117]. In contrast, H3K9 methylation loss in the KRYPTONITE

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(KYP) histone methyltransferase kyp mutant does not affect CpG methylation [118] (Table 2). This suggests that H3K9 methylation acts downstream of CpG methylation and reinforces heterochromatin. On the contrary, DNA methylation at CpNpG sites appears to be partially dependent on the activity of KYP [119]. Indeed, inactivation of KYP in kyp mutants resulted in a loss of methylation at Ta2 and Ta3 retrotransposons followed by their transcriptional activation [120]. Consistently, the maintenance of CpNpG methylation at sequences targeted by RdDM-induced transcriptional silencing requires the activity of KYP [119]. Interestingly, the Arabidopsis mutant of another histone modifying enzyme, a histone deacetylase HDA6, was isolated based on reactivation of the previously methylated transgene reporter [121] (Table 2). HDA6 was required for the maintenance of RdDM-induced CpG methylation [121]. Histone methylation can recruit other proteins such as HETEROCHROMATIN PROTEIN1 (HP1) that binds to methylated H3K9 [122] and helps propagate heterochromatin to adjacent regions on chromosome [123]. An Arabidopsis homologue of HP1, HETEROCHROMATIN PROTEIN1 (LHP1), is also involved in regulating flowering time in response to environmental stimuli [124, 125] (Table 2). Methylated DNA can serve as a substrate for binding nuclear proteins named methylCpG-binding domain proteins or MBDs (Table 2). These proteins bind to 5-methylcytosins, recruit enzymes which modify core histone proteins, and change local chromatin structure [126]. However, it must be noted that not all MBDs are able to bind methylated CpG in vitro. These MDBs can possibly be involved in control of chromatin structure through other mechanisms [127].

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2.3. Chromatin Remodeling Shapes Chromatin Structure and Complements DNA and Histone Modifications Control of gene expression through DNA methylation and histone modifications is complemented by the activity of chromatin remodeling proteins. Among them, there are members of the SWI2/SNF2 DNA helicase family that are of crucial importance in DNA repair, recombination, gene expression and replication [128]. The SWI2/SNF2 family proteins alter chromatin structure through the disruption of DNA-histone interactions [129]. The DECREASED DNA METHYLATION1 (DDM1) protein is the first described member of this family that controls methylation directly and indirectly by changing histone methylation [120] (Table 2). Recently, Zemach et al. (2005) demonstrated that AtMBDs bind DDM1 [130]. They also reported a disrupted localization of AtMBDs at chromocenters in the ddm1 mutant. This suggests that DDM1 may facilitate the localization of MBDs at specific nuclear domains. The ddm1 mutant shows a 70% reduction in global genome methylation [131], activation of transposable elements [132], and phenotypical instability [133]. The ddm1-induced hypomethylation also results in transcriptional activation of a previously silent disease-resistance gene array [134], and activates a number of retrotransposons [135]. Consistently, DDM1 is involved in the maintenance of CpG methylation at RdDM-targeted sequences after an RNA signal is removed [110, 136]. Finally, the ddm1-induced hypomethylation gene states can be stably inherited through mitotic and meiotic cell divisions [137].

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Table 2. Plant factors involved in histone modifications and chromatin remodeling Effects on chromatin

Effects of mutation and the involvement in stress response

Modification/ Transcription

Refs

SUVH1/ Histone methyltransferase

Methylation of histone H3K9; has a minor impact on heterochromatin reinforcement.

Loss of H3K9 methylation

Global/ Repression

[226]

SUVH2/ Histone methyltransferase

Methylation of histones H3K9, H3K27, H4K20; heterochromatin reinforcement; SUVH2 mediated gene silencing depends on MET1 and DDM1

Loss of H3K9; H3K27 and H4K20 methylation; reduction of DNA methylation in heterochromatin

Global/ Repression

[226]

SUVH4 (KRYPTONITE) (SUVH4/KYP)/ Histone methyltransferase

Methylation of histone H3K9; the activity is dependent on CpG DNA methylation at the given loci; has a minor impact on heterochromatin reinforcement; KYP reinforces RdDM-induced CpNpG methylation

Loss of H3K9 methylation; a negative effect on CpNpG methylation

Global/ Repression

[118120, 226, 227]

HISTONE DEACETYLASE6 (HDA6)/ Histone deacetylase

Reinforcing CpNpG methylation induced by RNAdirected transcriptional silencing

Reactivation of previously silenced transgenes

Local/ Repression

[121]

METHYL-CpG-BINDING DOMAIN PROTEINS (AtMBD1 – AtMBD13)/ 5-methylcytosin binding proteins

Bind methylated CpG and change local chromatin structure through recruiting enzymes involved in modification of core histone proteins; promote heterochromatin formation and repeat silencing

Late flowering and reduced fertility (mbd11); shoot branching and early flowering due to transcriptional repression of FLC (mbd9)

Local/ Repression, activation

[126, 127]

LIKE HETEROCHROMATIN PROTEIN1 (LHP1) Chromodomain protein

Binds to histone H3K9; chromatin condensation and coating

Global/ Repression

[124, 125]

DECREASED DNA METHYLATION1 (DDM1)/ SWI2/SNF2 DNA helicase

Controls DNA methylation, possibly binds methylCpG binding domain proteins and affects their subnuclear localization; aids in maintaining CpG methylation induced by RdRM

Global/ Repression

[116, 120, 130, 134, 156]

MAINTENANCE OF METHYLATION1 (MOM1)/ Similar to SWI2/SNF2

Regulation of silent heterochromatic regions transcription; transgene silencing; preventing transcription of 180-bp satellite repeats and not of transposons

Global/ Repression

[138, 139]

Name and function Histone modifications

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Chromatin remodeling

Inability to repress expression of euchromatic genes associated with a specific developmental stage Decondensation of centromeric heterochromatin, redistribution of remaining DNA methylation, changes in a pattern of histone methylation Silencing of R genes and retrotransposons; involved in DNA damage response Release of transcriptional gene silencing and 5S repeat silencing; no effect on heterochromatin organization and DNA methylation.

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Another control mechanism of gene expression is represented by the nuclear MAINTENANCE OF METHYLATION (MOM1) protein with limited homology to DDM1 [138], and it is involved in DNA-methylation-independent silencing of repetitive sequences in Arabidopsis [139] (Table 2). MOM1 prevents transcription of 180bp satellite repeats of transposons [139]. In mom1 mutants, a release of transgene silencing [138] and 5S repeat repression [139] occurs without reducing/alternating their DNA and histone methylation patterns (Table 2). This suggests the existence of two distinct epigenetic silencing pathways, DNA-methylation-dependent and DNA-methylation-independent.

3. EPIGENETIC MODIFICATIONS – A STRESS-RESPONSIVE MECHANISM CONTROLLING GENE REGULATION AND GENOME STABILITY

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3.1. Effects of Stress on Epigenetic Regulations Until recently, the link between stress exposure and sequence-specific changes in DNA methylation was hypothetical. It was demonstrated that prolong exposure to cold triggers stable transcriptional silencing of FLC that leads to flowering inhibition [140]. Moreover, flowering time directly correlates with the level of DNA methylation in MET1 antisense knockouts [141]. Therefore, met1 mutants do not require cold treatment to initiate flowering. This proves that a developmental switch was epigenetically controlled. The activity of MET1 was also shown to be affected by other stresses. Exposure of maize seedlings to wounding, desiccation and salinity reduced DNA replication activity in roots and decreased transcription of the MET1 gene [142]. Cold exposure of root tissues of maize seedlings resulted in DNA demethylation at nucleosome core regions [142]. In fact, DNA replication was strongly reduced in chilled tissues, allowing speculations that genome hypomethylation was caused by active rather than passive demethylation. Cold-induced demethylation of a nucleosome core and relaxation of chromatin structure could serve as a stress-induced transcriptional switch for many stress-regulated genes [142]. Several other papers suggest that changes in DNA methylation are required for stress protection. Dyachenko et al. (2006) demonstrated a two-fold increase in the level of CpNpG methylation in the nuclear genome of M. crystallinum plants exposed to high salinity [143]. An increase in methylation was associated with switching from C3- to C4-type photosynthesis. Similarly, Sha et al. (2005) reported that an age-dependent increase in methylation confers resistance to the blight pathogen X. oryzae in rice [144]. Methylation contributes greatly to the plant‘s ability to respond to stress. Hypomethylation found in met1 results in specific expression of 31 genes, most of which being related to stress response [145]. Demethylation the NtAlix1gene occurs also as a result of viral infection, thus confirming that the induction of this gene under natural stress conditions requires sequence demethylation. Steward et al. (2000) also showed that transcriptional activation of the ZmMI1 gene in maize seedlings was dependent on coldinduced sequence demethylation [142]. The ZmMI1 gene contains a retrotransposon-like sequence, and its activation mirrored cold-induced root-specific demethylation in the Ac/Ds transposon regions followed by their activation [142].

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Transposon activation in response to stress is a common phenomenon that affects genome stability. Low temperature treatment decreases methylation and increases the rate of transposon Tam3 excision by binding its transposase to GCHCG (H=not G) sites immediately after DNA replication, and thus preventing de novo sequence methylation [146, 147]. Moreover, three different subfamilies of Tnt1 retrotransposons were shown to be activated by stresses associated with plant defense reactions. Interestingly, these retrotransposons displayed tissue-specific activation and demonstrated different inducibility by stress elicitors [148]. Stress induced the activity of Tos17 (rice) [149], Tto1 (tobacco) [150], Tnt1 (tobacco) [148] and BARE-1 (barley) [151] retrotransposons. This stress-mediated Tnt1 and Tto1 activation can be explained by the interaction of their promoters with host transcription factors, due to sequence similarities found in retrotransposons and plant defense gene promoters [148]. An intriguing hypothesis that stress-activated transposons could positively contribute to genome adaptation to colder climates was supported by the detection of mPing transposition into a rice homologue of the flowering time gene CONSTANS in stressed cultivars [62]. Indeed, Song et al. (1997) suggested that a number of transposable elements and their derivatives that are present in resistance gene (R-gene) loci played a significant role in a rapid diversification of this gene family [152]. These publications support the longstanding hypothesis proposed by Barbara McClintock. She suggested that all kinds of stresses could potentially reshape the plant genome via transposon activation [153]. Histone modifications represent another stress response mechanism in the system of epigenetic control over gene expression. Chua et al. (2003) established a link between the light-dependent transcriptional induction of the pea plastocyanin gene and histone acetylation [154]. It was suggested that binding an enhancer to a nuclear matrix activates transcription through alteration of local chromatin structure, thus increasing acetylation of the promoter and 5‘ coding region [154]. Tsuji et al. (2006) demonstrated that transcriptional activation of the ADH1 and PDC1 submergence-inducible genes in rice was reversibly mediated through histone H3K4 methylation and H3 acetylation [155]. In consistence with the previously mentioned types of epigenetic modifications, the involvement of chromatin remodeling factors in stress response was supported by extensive studies on plants impaired in their functions. DDM1 deficient plants were shown to be more sensitive to UV-C and γ-radiation than wild type and met1 mutant plants [156]. This indicates that increased radiation sensitivity can be mediated by disrupting chromatin remodeling functions rather than cytosine methylation. It should be noted, however, that Shaked et al. (2006) did not analyze the double met1cmt3 mutant [156]. If they considered any possible functional redundancy of MET1 and CMT3 [98], they could have made different conclusions. Other reports also indicated the link between chromatin maintenance and stress response. Mutants of the nuclear protein BRU1 involved in the maintenance of chromatin structure were highly sensitive to genotoxic stress and were characterized by increased intrachromosomal HR [157]. Similarly, the expression of the MIM1 gene involved in the maintenance of chromosome structure and required for efficient HR was significantly increased by DNA-damaging agents [158]. Another SWI/SNF-like protein, DRD1, represents a novel plant-specific chromatin remodelling protein that is required for RNA-directed de novo methylation of target promoters [159] (Table 3).

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Table 3. Plant factors involved in siRNA-directed DNA methylation Name and function

Effects on chromatin

Effects of mutation and the involvement in stress response

Modification/ Transcription

Refs

nrpd1a and nrpd2a mutants show loss of cytosine methylation and the formation of disrupted facultative heterochromatin, and they display loss of siRNAs corresponding to targeted loci; both NRPD1a and NRPD2a are required for endogenous retroelements and transgenes silencing

Local, heterochromati n maintenance/ Repression

[95, 177, 207]

Loss of NRPD1b leads to loss of cytosine methylation mainly in the non-CpG sequence context and does not affect heterochromatic siRNAs production; methylation loss is primarily observed in previously silenced euchromatic promoters and transposons

Local, euchomatic promoters/ Repression, possibly activation

[94, 95, 161, 162, 206, 207]

Mutants do not show significant defects in CpG methylation but exhibit loss of non-CpG methylation in previously silenced euchromatic promoters and transposons; drd1 mutation leads to down regulation of ROS1 and DME

Local, euchomatic promoters/ Repression, activation

[95, 159, 160, 162]

RdDM pathway RNA POLYMERASE IVa (pol IVb) (subunits NRPD1a and NRPD2a)/ Nuclear RNA polymerase

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RNA POLYMERASE IVb (pol IVb) (subunits NRPD1b and NRPD2a)/ Nuclear RNA polymerase

DEFECTIVE FOR RNADIRECTED DNA METHYLATION1 (DRD1)/ SWI/SNF-like protein

pol IVa is localized in the nucleus and required for siRNA production; pol IVa acts upstream from RDR2 that uses its products as templates for dsRNA synthesis in the nucleolus; these dsRNAs are processed by DCL3 and AGO4 proteins; pol IVa may participate in a subunit exchange reaction with NRPD1b to form a functional pol IVb The presence of CTD at the NRPD1b subunit may provide a platform for the formation of a hypothetical NRPD1b-containg RISC complex that includes the AGO4 protein associated with siRNAs; NRPD1b-containg RISC delivers siRNA to a target site where pol IVb is formed by subunit exchange with pol IVa; pol IVb initiates RNA transcription at the siRNA-targeted site that exposes DNA for epigenetic modifications; NRPD1b possibly recruits de novo DNA methyltransferases MET1 and DRM2 that are aided by DRD1; together with DRD1, pol IVb reversibly controls promoters and LTRs in euchromatin DRD1 directs non-CpG DNA methylation in response to a RNA signal; DRD1 is required to facilitate the access of RNA signals to DNA; mediates full erasure of methylation when the signal is removed; together with pol IVb acts downstream of the siRNA biogenesis pathway, DRD possibly interacts with DNA methyltransferases and DNA glycosylases; preferentially targets the promoter and LTRs in euchromatin

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RNAi pathway RNA-DEPENDENT RNA POLYMERASE2 (RDR2)/ RNA-dependent RNA polymerase protein family

Synthesis of dsRNA from ssRNA templates; RDR2 provides substrates for DCL3 and AGO4 activity; may work on ssRNA templates produced by pol IVa

Loss of RDR2 results in decreased cytosine methylation at endogenous repeats and a complete loss of corresponding siRNAs

Local/ Repression

DICER-LIKE3 (DCL3)/ RNase III protein family

DCL3 processes dsRNA and produces RNA signals that guide siRNA-directed site-specific de novo DNA methylation

Loss of DCL3 results in decreased cytosine methylation at endogenous repeats and a complete loss of corresponding siRNAs; loss of DCL3 is partially compensated by DCL2 and DCL4 genes

Local/ Repression

ARGONAUT4 (AGO4)/ PPD protein (contains PAZ and PIWI domains)

AGO4 functions downstream of DCL3; initiates and maintains histone H3K9 methylation, initiates de novo DNA methylation; may be a functional part of NRPD1b-containing RISC

Loss of AGO4 results in decreased cytosine methylation and histone H3K9 methylation at endogenous repeats and leads to a complete loss of corresponding siRNAs

Local/ Repression

[95, 175, 196, 207] [101, 179, 198, 207] [95, 189, 190, 199, 207]

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Alex Boyko and Igor Kovalchuk

It is also necessary for a full loss of de novo DNA methylation after a silencing RNA trigger is withdrawn [160]. DRD1 interacts with two other factors, NRPD1b and NRPD2a, that represent the subunits of a novel plant-specific RNA polymerase, pol IVb [161]. DRD1 and the pol IVb complex act downstream of a small RNA (smRNA) biogenesis pathway (see below). Thus, they direct reversible silencing of euchromatic promoters in response to RNA signals; it is possibly done through recruitment of DNA methyltransferases to methylate homologous DNA sequences [162]. It is noteworthy that DNA glycosylases ROS1 and DME are putative DRD1 targets, and they both are involved in active DNA demethylation [108, 114]. Down-regulation of ROS1 in the drd1 and pol IVb mutants confirms the importance of the DRD1/pol IVb pathway for active loss of de novo DNA methylation [160].

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3.2. Small RNAs May Direct Epigenetic Modifications to a Specific Genomic Locus A significant advantage of epigenetic regulations is a fast stimulus-directed generation of new transcriptional states that are heritable and reversible. One of the key mechanisms involved in targeting chromatin structure and modifying a gene expression pattern in response to environmental stimuli is based on the activity of smRNAs. They were shown to guide sequence-specific gene regulation, mediate defense against viruses and change DNA methylation [79, 163, 164]. An initial DNA methylation imprint in response to a stress-induced RNA signal can be created by DRMs at asymmetric sites, then it can be perpetuated at symmetric CpG and CpNpG by MET1 and CMT3, respectively [100, 165]. In contrast to non-CpG methylation that is substantially reduced by removing a signal, CpG methylation can be maintained through several generations [79]. Consistently, virus-induced changes in methylation of the 35S promoter were inherited at CpG but not at non-CpG cytosines. [166]. The maintenance of CpG methylation is essential for stable inheritance of epigenetic memory, Therefore, plants deficient in maintenance of CpG methylation display the uncoordinated and aberrant formation of epigenetic patterns over successive generations [167].

3.2.1. The Complexity and Functional Redundancy of smRNA Biogenesis Pathways in Arabidopsis Many smRNA populations are usually present in plant cells. They derive from different types of precursors, and have different biogenesis pathways and functions [168, 169]. Small RNAs can be classified into two large groups. The first group includes micro RNAs or miRNAs, the 21 – 24-nt long gene-encoded smRNAs molecules that are processed from single-stranded imperfectly folded stem-loop like structures of a precursor miRNA (premiRNA) [170]. miRNAs exhinit a variety of regulatory functions in many cellular processes, specifically in plant development and stress response [171, 172]. In contrast to miRNAs, the second large group of smRNAs (that is called short/small interfering RNAs or siRNAs) derives from long perfect dsRNA duplex precursors that can be endogenous or exogenous by origin [169, 170]. Short interfering RNAs are usually 21 – 24-nt long, they are involved in the establishment of sequence-specific transcriptional and post-transcriptional gene silencing and can mediate cell defence against viruses [169, 170]. The group of siRNAs is very diverse and

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can be further divided into at least three subclasses, including trans-acting siRNAs (tasiRNAs), natural-antisense transcript-derived siRNA (nat-siRNAs), and repeat-associated siRNAs (ra-siRNAs) [169]. Trans-acting short-interfering RNAs are 21-nt long regulatory smRNAs that are found only in plants. They regulate the expression of targeted genes at the post-transcriptional level. The biogenesis of ta-siRNAs depends on miRNAs-directed cleavage of non-coding RNA precursors (TAS transcripts). This cleavage provides a substrate for an RNA dependent RNA polymerase (RDR6) that synthesizes dsRNA products which are cleaved to produce ta-siRNAs [169, 170]. Similar to miRNAs, ta-siRNAs guide the cleavage of complementary mRNAs molecules. The group of nat-siRNAs represents a recently identified class of siRNAs [173]. They are 24-nt long and derive from two overlapping and partially converging coding transcripts. Since the dsRNA transcript can be formed by two sense and antisense RNA transcripts derived from genes located at the same (cis-) or different locus (trans-), this group is further divided into cis-nat-siRNAs and trans-nat-siRNAs [170]. Similar to ta-siRNAs, nat-siRNAs regulated the targeted gene activity at the posttranscriptional level by guiding mRNA cleavage [169, 170]. Finally, the last group of siRNA (ra-siRNAs) plays a crucial role in maintaining gene expression via DNA methylation and histone modification, and it also controls the activity of retrotransposons and repetitive DNA sequences [174, 175]. Repeat-associated short-interfering RNAs are usually 24-nt long and their biogenesis involves the cleavage of dsRNA precursors [169]. Different classes of smRNAs display some similarities during their biogenesis. Their production usually involves the cleavage of a dsRNA precursor by a DICER protein. There are four DICER-LIKE proteins (DCL1-DCL4) known in Arabidopsis. They display some functional redundancy in biogenesis of different types of smRNAs via cleavage of dsRNA precursors [164, 169, 170]. DCL1 is involved in processing miRNAs; DCL2 processes natsiRNAs [173] and siRNAs required for viral resistance [175, 176]; DCL3 processes siRNAs that mediate transcriptional gene silencing and maintenance of DNA methylation [95, 175, 177]; DCL4 processes ta-siRNAs and some viral siRNAs [164, 178, 179]. Functional redundancy of Arabidopsis DCL proteins is also reported in mutational studies. Both DCL2 and DCL4 proteins are able to partially compensate for the loss of DCL3 in dcl3 mutants and maintain certain methylation marks [179]. Consistently, non-CpG methylation is not significantly affected by dcl3 mutation, but it is strongly altered in dcl2dcl3dcl4 triple mutant [180]. Similarly, inactivation of DCL4 and DCL2 genes in Arabidopsis dcl4 and dcl2dcl4 double mutants respectively, enhances miRNA production by DCL1 [178, 179]. Another example of functional redundancy can be found within the ARGONAUTE protein family which is comprised of ten members in Arabidopsis (AGO1-AGO10) [181]. ARGONAUTE proteins are involved in the formation of two different complexes. The first one, the RNA-induced silencing complex (RISC), cleaves the complementary mRNA in the centre of the region paired with smRNA [182]. The second one, the RNA-induced transcriptional silencing complex (RITS), mediates transcriptional inactivation of targeted genes, and it is well-studied in yeast [183]. AGO1 functions in miRNAs- and siRNAsmediated mRNA degradation [175, 184], and it shows some functional redundancy with AGO10 protein [185]. AGO7 functions in developmental timing and mediates the ta-siRNAsguided cleavage of targeted mRNAs in combination with an unknown AGO protein [186188]. Finally, AGO4 functions downstream of DCL3 and is involved in DNA and histone

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methylation, chromatin modification and epigenetic regulation at targeted sequences [189, 190]. Finally, there are six known members of the RDR protein family (RDR1-RDR6) in Arabidopsis [191]. RDR6 is necessary for viral resistance, ta-siRNAs production, and it is involved in sense-transgene-mediated silencing [192-194]. Similarly, RDR1 is important for resistance to virus infection [191, 195]. In contrast, RDR2 functions in silencing of repetitive DNA sequences and transposons via siRNA-directed DNA methylation and chromatin modification [175, 196]. Overall, smRNA biogenesis is represented by a network of complex and partially overlapping pathways. Since this chapter is focused on epigenetic regulation of the plant genome and not on mechanisms behind biogenesis of different types of smRNAs, we will restrict our consideration of smRNA biogenesis to DCL3, RDR2 and AGO4 proteins mediating smRNA-directed epigenetic modifications of DNA.

3.2.2. Mechanisms of smRNA-Directed Epigenetic Regulations Various endogenous and exogenous stimuli can initiate the production of dsRNAs. A list of these stimuli includes various types of viruses and viroids, transgenes, repetitive DNA sequences, and endogenous loci in particular [197]. These dsRNAs can operate at the genome level and initiate two important epigenetic processes such as RdDM and RNA interference (RNAi) [94]. The RdDM pathway involves DCL3, RDR2 and AGO4 proteins that mediate smRNAs biogenesis and function also in the RNAi pathway (Table 3). The RdDM pathway requires the activity of DRD1 and DRM2 proteins for chromatin remodelling and de novo DNA methylation, respectively. Finally, two forms of nuclear RNA polymerase IV (pol IVa and pol IVb) are needed for RNA synthesis as well as for directing the silencing machinery to its targets [95, 169]. In contrast to RNAi that is usually targeted to repetitive regions located in heterochromatin and spreads more than several thousands nucleotides away from the RNAtargeted nucleation site, RdDM is usually associated with non-spreading DNA methylation confined to short and frequently single-copy regions targeted by RNA-DNA homology [94]. A signal that initiates RdDM may arise from dsRNAs produced by the activity of RDR2 on ssRNA templates originated from various repetitive DNA sequences and transposons [175, 196]. Alternatively, the signal can be originated from mRNA transcripts produced by RNA polymerase II from genes containing inverted repeats. In fact, the first example of the endogenous promoter targeted by the RdDM pathway was the promoter of the FWA gene that contained direct repeats [136]. DCL3 protein processes dsRNA and produces RNA signals that target the site-specific DNA methyltransferases for de novo DNA methylation [101, 198]. Next, MET1 and DRM2 methyltransferases establish de novo CpG non-CpG methylation at targeted sequences, respectively. At this stage, the activity of a chromatin remodelling factor DRD1 is required to facilitate access of RNA signals to DNA [94, 95]. There are two current models that explain how RNA signals interact with a target locus in DNA. These are the DNA-recognition and RNA-recognition models, respectively [94]. The RNA-recognition model requires pairing of smRNA and nascent RNA transcribed from the original locus. In contrast, the DNA-recognition model suggests direct DNA-RNA pairing, and better experimental support has been provided for it. The initial DNA methylation imprint in response to an original RNA signal can be maintained at symmetric CpG and CpNpG sites by MET1 and CMT3, even in the absence of an original RNA trigger [100, 165]. Maintenance of CpG methylation also requires histone

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deacetylase HDA6 activity [121] and a SNF2-like chromatin remodeling protein DDM1 [110, 136]. Maintenance of CpNpG methylation involves KYP activity that reinforces DNA methylation by histone H3K9 methylation [119]. Moreover, AGO4 protein represents another important link between RNAi and CpNpG – a H3K9 methylation pathway. Together with DCL3 and RDR2, AGO4 is capable to initiate and maintain histone H3K9 methylation that may lead to de novo DNA methylation at a targeted locus [189, 190, 199]. Some experimental evidence indicates that AGO1 can play a role in mi/siRNAs post-transcriptional gene silencing [175, 184] and also can maintain chromatin modifications [200-202]. The activity of at least two DNA glycosylase-domain containing proteins, DME and ROS1 is required to remove RdDM and gene silencing [111, 112, 203, 204]. It is possible that this gene-activation step can also be guided by smRNAs, but direct experimental verification of this suggestion is still missing. However, this speculation is supported by data obtained from studies of vertebrate cells [205]. Active DNA demethylation by DNA glycosylases was shown to require the presence of an RNA strand complimentary to methylated DNA [205]. Another important player involved in siRNA-directed DNA methylation was recently described [95]. It is a new fourth class of nuclear RNA polymerase named RNA pol IV, or NRPD [95] (Table 3). This class is represented by two distinct forms named pol IVa and pol IVb that differ in a subunit composition. Pol IVa consists of NRPD1a and NRPD2a, whereas pol IVb consists of NRPD1b and NRPD2a subunits [161, 206]. Importantly, the presence of carboxy-terminal domain (CTD) at the NRPD1b subunit may provide an interface for proteinprotein interaction during RISC formation [95]. Consistent with their role in RdDM, loss of NRPD1a, NRPD2a, and NRPD1b genes results in loss of cytosine methylation [161, 177]. However, in contrast to nrpd1a and nrpd2a mutations, nrpd1b mutation has no effect on heterochromatic siRNA production. The foregoing suggests that pol IVa is involved in siRNAs production with pol IVb acting downstream to direct cytosine methylation [161]. A number of studies directed on co-localization of pol IV subunits with other factors participating in siRNA biogenesis and RdDM allowed the development of a hypothetical model explaining the spatial organization of the siRNA-directed DNA methylation pathway [95]. Subunits of pol IVa, DRD1 and a portion of the NRPD1b pool were found to be colocalized at siRNA source/target loci [207]. In contrast, NRPD1b, RDR2, DCL3, and AGO4 were co-localized within the nucleolus where they also interacted with the corresponding siRNAs [207]. The authors interpreted these results as direct evidences supporting the existence of a nucleolar siRNA processing center where dsRNAs produced by RDR2 are cleaved by DCL3 and loaded into AGO4- and NRPD1b-containing RISCs. According to Pontes et al. (2006), pol IVa transcripts produced at siRNA source/target loci move from the nucleoplasm to the nucleolus where RDR2 uses them as templates to produce dsRNAs [207]. In contrast, the functional pol IVb is formed during subunit exchange between the NRPD1bcontaining RISC complex and pol IVa localized at the siRNA source/target site. This is possible if the RISC complex leaves the nucleolus and arrives to the siRNA source/target site. Here, pol IVa donates its NRPD2a subunit to complete pol IVb [207]. Pol IVb could use siRNA delivered by AGO4 as primers to initiate RNA transcription from the siRNA source/target site with the help of the DRD1 protein [95]. Nucleosome displacement caused by RNA transcription or possibly by the invasion of the pol IVb into a DNA duplex may temporarily expose DNA to the activity of DRM2 and KYP proteins [95]. Next, DRM2 and KYP could produce de novo DNA methylation and histone H3K9 methylation, respectively.

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Overall, the ability of the DRD1/pol IVb complex to interact with DNA methyltransferases and DNA glycosylases suggests the involvement of this complex in the maintenance of reversible epigenetic states of euchromatic promoters in response to RNA signals [162].

3.2.3. Small RNA Biogenesis is a Sensitive Stress-Responsive System Many of smRNAs reported up-to-date were involved in the regulation of plant development [208-210]. They were tissues and organ specific [211, 212] and were regulated by a number of abiotic stresses including mechanical and oxidative stress, dehydration, salinity, cold, abscisic acid, and nutrient deprivation [170, 173, 211, 212]. This might allow directing methylation to specific loci by producing stress-specific smRNAs [213]. For example, oxidative stress down-regulates transcription of miR398 that normally guides cleavage of cytosolic (CSD1) and plastidic (CSD2) Cu-Zn superoxide dismutase gene transcripts [214]. This results in a rapid accumulation of CSD1 and CSD2 gene transcripts and increases plant resistance to stress. Interestingly, there is a large number of non-conservative miRNAs that are available in some species and absent in the others [212]. This might support a hypothesis that the development of a specialized miRNAs network was driven by physiological and stress conditions specific for each species [212]. The identification of 22 miRNAs from the developing secondary xylem of P. trichocarpa stems [212] further confirmed that species-specific miRNAs contributed to regulation of gene expression associated with specific growth/stress conditions. The expression of many ptrmiRNAs was induced in the developing xylem of stems in the presence of gravitropismmediated mechanical stress [212]. This stress triggers upregulation of ptr-miR408 expression and regulates the plastocyanin-like protein that mediates lignin polymerization. In addition, mechanical stress downregulates the expression of ptr-miR164 and ptr-miR171. These miRNAs target genes are involved in cell division and elongation in response to gravitropism. Among other non-conservative ptr-miRNAs that can be induced by abiotic stress are ptrmiR473, ptr-miR482, ptr-miR472, and ptr-miR159. ptr-miR473 and ptr-miR482 target the synthesis of cell wall polyphenolics via the UV-B resistant gene (UVR8) and three putative disease resistance genes, respectively. Similarly, ptr-miR472 and ptr-miR159 regulate the activity of putative disease resistance genes and cell wall polysaccharide synthesis [212]. Sunkar and Zhu (2004) demonstrated the existence of stress-inducible changes in the Arabidopsis miRNA pool [211]. The most interesting examples are miRNA402 and miRNA407 regulated by dehydration, salinity, cold, and abscisic acid. Whereas miRNA402 targets a ROS-like DNA glycosylase, miR407 targets a SET domain protein functioning in histone Lys methylation [211]. Some of miRNAs were shown to have multiple target sites within the same gene, which implies that different levels of gene repression might be achieved through a various number of miRNAs bound to the target [215]. Stress-induced miRNAs have a tissue-specific expression pattern. It defines requirements to organ-specific functional and metabolic differences in response to stress. Indeed, miR393 downregulates TIR1, a positive regulator of auxin signalling, and has its strongest expression in the inflorescence under physiological conditions. Hence, strong miR393 induction by stresses such as dehydration, salinity, cold and abscisic acid treatment is consistent with inhibition of plant growth under stress conditions [211]. Consistent with the role of miRNAs in the establishment of a stress-induced gene expression pattern, Arabidopsis mutants hen1-1 and dcl1-9 partially impaired in the production of miRNAs were shown to be hypersensitive to abiotic stresses [211].

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The known nat-RNAs were shown to be mainly produced under salt-stress conditions [169]. They are involved in stress-mediated regulation of genes located in antisense overlapping pairs that results in generation of complimentary transcripts. Borsani et al. (2005) showed that induction of one of these genes in an antisense pair by stress leads to the production of nat-siRNA which guides the cleavage of other gene transcripts followed by downregulation of the gene activity [173]. Similarly, studies of Katiyar-Agarwal et al. (2006) demonstrated induction of another specific nat-siRNA during Pseudomonas syringae infection that conferred resistance to pathogen [216]. This mechanism may play an important role since 4 – 20% of the all genes in eukaryotic genomes are grouped in antisense overlapping pairs [173].

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4. INDUCIBLE EPIGENETIC CHANGES MAY CHANGE GENOME STABILITY AND GUIDE GENOME EVOLUTION Modification of DNA sequence via selective cytosine methylation plays a crucial role in establishing a stable epigenetic mark in plants. Despite their reversibility, changes in DNA methylation are quite stable modifications that are not easily reset; and they are frequently transmitted for several generations. Indeed, backcrossing a ddm1 mutant to wild-type plants do not revert the mutant phenotype. This demonstrates that hypomethylation can be stably transmitted during meiosis, gametogenesis, and mitosis, irregardless of the presence of a functional DDM1 gene [137]. The progeny of MET1 antisense plants exhibit DNA hypomethylation independent of the presence of the transgene locus that previously triggered inhibition of the MET1 gene [91]. Perhaps, the best examples of methylation-mediated heritable changes are epialleles representing different forms of the same gene regulated epigenetically. Epialleles can be formed in response to a number of stimuli, and they may play an important role in acclimation. Good examples of such epialleles are methylated and demethylated forms of the FWA gene. Both of them are equally stable and can be inherited as a true Mendelian trait based on methylation rather than on the sequence difference [115]. Targeting DNA methylation to the FWA gene was triggered by positioning its promoter and transcription start site within two pairs of direct repeats [136]. Moreover, two Arabidopsis ecotypes, Ler and Da (1)-12, carry transposon insertions in the first intron of the FLC gene. This represents independent adaptive events that lead to the establishment of cold independent flowering initiation by preventing high expression of the FLC gene [217, 218]. Paramutations represent another class of heritable epigenetic traits. They are caused by interactions between two alleles of a single locus. Paramutation is essentially a heritable change of one allele (a target) that is induced by the other allele (a trigger). The silenced state of a target allele induced by a trigger remains stable, even if a trigger allele segregates out in the next generation [219]. Recent studies by Alleman et al. (2006) confirmed that siRNAs play a critical role in production and maintenance of chromatin states in maize [220]. They demonstrated that the MEDIATOR OF PARAMUTATION1 (MOP1) gene that encodes the RNA-dependent RNA polymerase is required for production of paramutations [220]. It can be hypothesized that the mi-/si-RNA-induced transgenerational response to stress indeed exists in plants.

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Genome rearrangements represent another epigenetic-sensitive mechanism affecting genome stability. The dual role of HR, as a DNA repair pathway and a putative evolutionary tool was intensively discussed over past several years [11, 221]. It has been suggested that HR can be involved in genome evolution through rearrangements of existing sequences, frequently resulting in gene duplication or deletion events. The degree of HR-mediated V(D)J rearrangements depends on DNA methylation [71]. It is possible that stress-directed changes in DNA methylation can stimulate or prevent rearrangements in different genomic loci [72]. Highly conserved gene families located in clusters could possibly increase their diversity using HR. It has been suggested that plant R gene evolution involved gene duplication and recombination events [67]. The fact that the frequency of meiotic and somatic HR can be altered by a variety of biotic and abiotic stresses could suggest that changes in the frequency of HR represent one of the mechanisms of stress adaptation [8, 52, 54, 55, 63-65, 222]. In several studies, the increased frequency of somatic HR was inherited by progeny of stressed plants as an epigenetic trait and persisted in a population for one or several non-stressed generations [55, 64, 222]. Interestingly, the increased spontaneous HR frequency observed in progeny of stressed plants correlated with increased tolerance to various stresses, including genotoxic ones. Progeny of salt-treated plants displayed an increased germination rate at high concentrations of NaCl, and was characterized by better growth on medium containing MMS (Boyko et al. unpublished data). Similarly, progeny of virus-challenged plants showed the delayed appearance of disease symptoms when it was infected with a virus (Kathiria and Kovalchuk, unpublished data). Our findings were consistent with the report by Blödner et al. (2007) that described an increased tolerance to low temperature conditions in progeny of cold-treated plants [223]. The foregoing studies suggest an intriguing hypothesis that stress can guide plant genome evolution using repair pathways, particularly HR, to trigger loci-specific genome rearrangements, thereby accelerating the evolution of targeted sequences and development of tolerance to stress [64, 78]. Our recent studies established a well-defined correlation between stress exposure, locispecific epigenetic changes and genome stability of exposed plants and their progeny using a well-studied model of TMV infection [64]. We have demonstrated that the progeny of tobacco plants treated with TMV inherited elevated rates of HR that correlated with an increased frequency of rearrangements in R-gene-like loci. Importantly, the progeny of stressed plants displayed increased levels of global genome methylation and exhibited locusspecific hypomethylation. Namely, the R-gene loci that carry homology to the N-gene conferring resistance to TMV were found to be hypomethylated and, as a consequence, rearranged more frequently [64]. Since plants used for infection (the SR1 cultivar) did not have the N-gene, we assumed that locus-specific changes in methylation and rearrangements could be a strategy for creating an active R-gene in plants. It is possible that such transgenerational changes in recombination and DNA methylation represent a general epigenetic control mechanism directed to selective relaxation of DNA sequences, thereby allowing their faster evolution under the influence of various environmental stimuli. This might somewhat resemble a phenomenon reported in flax where a number of heritable changes could be triggered by environment relatively fast [224, 225].

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CONCLUDING REMARKS Dynamic control of genome stability is an important prerequisite for the simultaneous occurrence of two distinctly different processes in plant genome: the preservation of the genome integrity and continuous generation of new sequence variants required to create novel traits carrying a selective advantage. DNA damage resulted from external and internal stress must be efficiently resolved via the activity of various DNA repair pathways. Repair of DNA damage is the primary mechanism that preserves the intactness of the plant genome and insures its stability. Many of DNA repair pathways lack high fidelity and their widespread activity may actually destabilize the genome, compromise its integrity, and even be lethal for the cell. A certain level of genome flexibility is absolutely required for the successful evolution of plant species. Mistakes produced during DNA repair may serve as a raw material for evolution. Similarly, rearrangement and duplication of existing DNA sequences may lead to the generation of new traits that confer a selective advantage under new growth conditions. A very delicate balance must exist between different DNA repair pathways to ensure a continuous production of new DNA sequence variants without affecting genome functions and cell vitality. It seems like different groups of organisms preferentially use specific DNA repair pathways depending on their genome content. Some organisms with a small genome size and a low content of repetitive sequences preferentially use HR that ensures that the production of a minimal number of mistakes. Increasing a genome size may increase the probability of a wrong template being chosen for damage repair via HR. The presence of a high number of repetitive DNA elements further complicates this problem as an improper recombination event may result in a larger size of deletions or duplications. Hence, a shift occurs toward using pathways that are more error-prone but less dangerous to the genome. For example, in plants, the NHEJ pathway is usually preferred to HR one. At the same time, HR is mainly used if reshaping of plant genome is needed. In the Arabidopsis genome, a substantial part of genetic material has been generated by duplication of existing DNA sequences possibly mediated by the HR pathway. Emerging evidence suggests that epigenetic marks and stress-induced sequence-specific signals like smRNAs may play an important role in the maintenance of genome stability and chromatin. Decreased DNA methylation and an open chromatin structure attract the activity of HR pathways and lead to activation of transposons, thus decreasing genome stability. At the same time, frequent rearrangements at unmethylated loci may help accelerate the evolution of targeted sequences. On the contrary, increasing genome methylation permits to preserve genome stability at the time of stress and prevents undesirable rearrangements and transpositions. The epigenetic landscape of the plant genome is rather complex, and it is a subject for continuous modifications in response to various stimuli. The forgoing leads to an interesting speculation that epigenetic marks are used not only to control the transcriptional activity of chromatin that helps adjust transcriptional profiles to new growth conditions but also to maintain genome stability and direct genome evolution. The fact that stress can lead to sequence-specific epigenetic changes permits further speculations about the involvement of epigenetic modifications in stress-directed genome evolution. Unfortunately, the direct evidence linking together stress, epigenetic modifications and mechanisms that control

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genome stability in one multifaceted system controls and directs genome evolution is still missing. It is the challenge for future studies to unravel these links and provide a better understanding of how stress-directed genome evolution and adaptation occur in nature.

ACKNOWLEDGMENTS We would like to acknowledge the help of Valentina Titova with proofreading the chapter. We acknowledge financial support of Alberta Agricultural Research Institute, Human Frontiers Science Program and NSERC.

REFERENCES [1] [2] [3]

[4] [5]

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

[6]

[7]

[8] [9] [10]

[11] [12] [13]

Madlung A, Comai L: The effect of stress on genome regulation and structure. Ann. Bot. (Lond) 2004, 94:481-495. Blokhina O, Virolainen E, Fagerstedt KV: Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. (Lond) 2003, 91 Spec No:179-194. Mittler R, Zilinskas BA: Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant J. 1994, 5:397-405. Breimer LH: Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Mol. Carcinog 1990, 3:188-197. Loeb LA, Preston BD: Mutagenesis by apurinic/apyrimidinic sites. Annu. Rev. Genet. 1986, 20:201-230. Babiychuk E, Kushnir S, Van Montagu M, Inze D: The Arabidopsis thaliana apurinic endonuclease Arp reduces human transcription factors Fos and Jun. Proc. Natl. Acad. Sci. U S A 1994, 91:3299-3303. Evans HH, Ricanati M, Horng MF, Jiang Q, Mencl J, Olive P: DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines. Radiat. Res. 1993, 134:307-315. Kovalchuk I, Abramov V, Pogribny I, Kovalchuk O: Molecular aspects of plant adaptation to life in the Chernobyl zone. Plant Physiol. 2004, 135:357-363. Gorbunova VV, Levy AA: How plants make ends meet: DNA double-strand break repair. Trends Plant Sci. 1999, 4:263-269. Orel N, Kyryk A, Puchta H: Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome. Plant J. 2003, 35:604-612. Puchta H: The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J. Exp. Bot. 2005, 56:1-14. Apel K, Hirt H: Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55:373-399. Mittler R: Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7:405-410.

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Genetic and Epigenetic Regulation of Genome Stability in Plants

57

[14] Vranova E, Inze D, Van Breusegem F: Signal transduction during oxidative stress. J. Exp. Bot. 2002, 53:1227-1236. [15] Allen RD: Dissection of Oxidative Stress Tolerance Using Transgenic Plants. Plant Physiol. 1995, 107:1049-1054. [16] Noctor G, Foyer CH: ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998, 49:249-279. [17] Desikan R, S AH-M, Hancock JT, Neill SJ: Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol. 2001, 127:159-172. [18] Knight H, Knight MR: Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci. 2001, 6:262-267. [19] Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, et al: Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J. 2002, 31:279-292. [20] Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K: Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr. Opin. Biotechnol. 2003, 14:194-199. [21] Critchlow SE, Jackson SP: DNA end-joining: from yeast to man. Trends Biochem. Sci. 1998, 23:394-398. [22] Shrivastav M, De Haro LP, Nickoloff JA: Regulation of DNA double-strand break repair pathway choice. Cell Res. 2008, 18:134-147. [23] Richardson C, Horikoshi N, Pandita TK: The role of the DNA double-strand break response network in meiosis. DNA Repair (Amst) 2004, 3:1149-1164. [24] Bleuyard JY, Gallego ME, White CI: Recent advances in understanding of the DNA double-strand break repair machinery of plants. DNA Repair (Amst) 2006, 5:1-12. [25] Dudas A, Chovanec M: DNA double-strand break repair by homologous recombination. Mutat. Res. 2004, 566:131-167. [26] Strathern JN, Shafer BK, McGill CB: DNA synthesis errors associated with doublestrand-break repair. Genetics 1995, 140:965-972. [27] Chen F, Nastasi A, Shen Z, Brenneman M, Crissman H, Chen DJ: Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52. Mutat. Res. 1997, 384:205-211. [28] Boyko A, Zemp F, Filkowski J, Kovalchuk I: Double-strand break repair in plants is developmentally regulated. Plant Physiol. 2006, 141:488-497. [29] Boyko A, Filkowski J, Hudson D, Kovalchuk I: Homologous recombination in plants is organ specific. Mutat. Res. 2006, 595:145-155. [30] Galbraith DW, Harkins KR, Knapp S: Systemic Endopolyploidy in Arabidopsis thaliana. Plant Physiol. 1991, 96:985-989. [31] Joubes J, Chevalier C: Endoreduplication in higher plants. Plant Mol. Biol. 2000, 43:735-745. [32] Swoboda P, Gal S, Hohn B, Puchta H: Intrachromosomal homologous recombination in whole plants. EMBO J. 1994, 13:484-489. [33] Paques F, Haber JE: Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 1999, 63:349-404.

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58

Alex Boyko and Igor Kovalchuk

[34] Aylon Y, Kupiec M: New insights into the mechanism of homologous recombination in yeast. Mutat. Res. 2004, 566:231-248. [35] Salomon S, Puchta H: Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J. 1998, 17:6086-6095. [36] Kirik A, Salomon S, Puchta H: Species-specific double-strand break repair and genome evolution in plants. EMBO J. 2000, 19:5562-5566. [37] Beaton MJ, Cavalier-Smitht T: Eukaryotic non-coding DNA is functional: evidence from the differential scaling of cryptomonad genomes. Proc. Biol. Sci. 1999, 266:20532059. [38] Gorbunova V, Levy AA: Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res. 1997, 25:4650-4657. [39] Sonoda E, Takata M, Yamashita YM, Morrison C, Takeda S: Homologous DNA recombination in vertebrate cells. Proc. Natl. Acad. Sci. U S A 2001, 98:8388-8394. [40] Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S: Differential usage of nonhomologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst) 2006, 5:1021-1029. [41] Chinnusamy V, Schumaker K, Zhu JK: Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J. Exp. Bot. 2004, 55:225-236. [42] Turunen M, Latola K: UV-B radiation and acclimation in timberline plants. Environ. Pollut. 2005, 137:390-403. [43] Zhu JK: Cell signaling under salt, water and cold stresses. Curr. Opin. Plant Biol. 2001, 4:401-406. [44] Thomashow MF: PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999, 50:571-599. [45] Zhu J, Dong CH, Zhu JK: Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation. Curr. Opin. Plant Biol. 2007, 10:290-295. [46] Larkindale J, Hall JD, Knight MR, Vierling E: Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 2005, 138:882-897. [47] Kotak S, Larkindale J, Lee U, von Koskull-Doring P, Vierling E, Scharf KD: Complexity of the heat stress response in plants. Curr. Opin. Plant Biol. 2007, 10:310316. [48] Chalker-Scott L, Scott JD: Elevated ultraviolet-B radiation induces cross-protection to cold in leaves of Rhododendron under field conditions. Photochem. Photobiol. 2004, 79:199-204. [49] Caldwell MM, Bornman JF, Ballare CL, Flint SD, Kulandaivelu G: Terrestrial ecosystems, increased solar ultraviolet radiation, and interactions with other climate change factors. Photochem. Photobiol. Sci. 2007, 6:252-266. [50] Arnholdt-Schmitt B: Stress-induced cell reprogramming. A role for global genome regulation? Plant Physiol. 2004, 136:2579-2586. [51] Ries G, Heller W, Puchta H, Sandermann H, Seidlitz HK, Hohn B: Elevated UV-B radiation reduces genome stability in plants. Nature 2000, 406:98-101. [52] Boyko A, Filkowski J, Kovalchuk I: Homologous recombination in plants is temperature and day-length dependent. Mutat. Res. 2005, 572:73-83.

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59

[53] Ries G, Buchholz G, Frohnmeyer H, Hohn B: UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation. Proc. Natl. Acad. Sci. U S A 2000, 97:13425-13429. [54] Boyko A, Greer M, Kovalchuk I: Acute exposure to UVB has a more profound effect on plant genome stability than chronic exposure. Mutat. Res. 2006, 602:100-109. [55] Molinier J, Ries G, Zipfel C, Hohn B: Transgeneration memory of stress in plants. Nature 2006, 442:1046-1049. [56] Puchta H, Swoboda P, Gal S, Blot M, Hohn B: Somatic intrachromosomal homologous recombination events in populations of plant siblings. Plant Mol. Biol. 1995, 28:281292. [57] Kovalchuk I, Kovalchuk O, Arkhipov A, Hohn B: Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident. Nat. Biotechnol. 1998, 16:1054-1059. [58] Brennan RJ, Schiestl RH: Free radicals generated in yeast by the Salmonella testnegative carcinogens benzene, urethane, thiourea and auramine O. Mutat. Res. 1998, 403:65-73. [59] Kovalchuk O, Titov V, Hohn B, Kovalchuk I: A sensitive transgenic plant system to detect toxic inorganic compounds in the environment. Nat. Biotechnol. 2001, 19:568572. [60] Filkowski J, Besplug J, Burke P, Kovalchuk I, Kovalchuk O: Genotoxicity of 2,4-D and dicamba revealed by transgenic Arabidopsis thaliana plants harboring recombination and point mutation markers. Mutat. Res. 2003, 542:23-32. [61] Lebel EG, Masson J, Bogucki A, Paszkowski J: Stress-induced intrachromosomal recombination in plant somatic cells. Proc. Natl. Acad. Sci. U S A 1993, 90:422-426. [62] Jiang N, Bao Z, Zhang X, Hirochika H, Eddy SR, McCouch SR, Wessler SR: An active DNA transposon family in rice. Nature 2003, 421:163-167. [63] Boyko A, Hudson D, Bhomkar P, Kathiria P, Kovalchuk I: Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress. Plant Cell Physiol. 2006, 47:736-742. [64] Boyko A, Kathiria P, Zemp FJ, Yao Y, Pogribny I, Kovalchuk I: Transgenerational changes in the genome stability and methylation in pathogen-infected plants: (virusinduced plant genome instability). Nucleic Acids Res. 2007, 35:1714-1725. [65] Kovalchuk I, Kovalchuk O, Kalck V, Boyko V, Filkowski J, Heinlein M, Hohn B: Pathogen-induced systemic plant signal triggers DNA rearrangements. Nature 2003, 423:760-762. [66] Lucht JM, Mauch-Mani B, Steiner HY, Metraux JP, Ryals J, Hohn B: Pathogen stress increases somatic recombination frequency in Arabidopsis. Nat. Genet. 2002, 30:311314. [67] Meyers BC, Kaushik S, Nandety RS: Evolving disease resistance genes. Curr. Opin. Plant Biol. 2005, 8:129-134. [68] Mauricio R, Stahl EA, Korves T, Tian D, Kreitman M, Bergelson J: Natural selection for polymorphism in the disease resistance gene Rps2 of Arabidopsis thaliana. Genetics 2003, 163:735-746.

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Alex Boyko and Igor Kovalchuk

[69] Tornero P, Chao RA, Luthin WN, Goff SA, Dangl JL: Large-scale structure-function analysis of the Arabidopsis RPM1 disease resistance protein. Plant Cell 2002, 14:435450. [70] Bender J: Cytosine methylation of repeated sequences in eukaryotes: the role of DNA pairing. Trends Biochem. Sci. 1998, 23:252-256. [71] Bassing CH, Swat W, Alt FW: The mechanism and regulation of chromosomal V(D)J recombination. Cell 2002, 109 Suppl:S45-55. [72] Rizwana R, Hahn PJ: CpG methylation reduces genomic instability. J. Cell Sci. 1999, 112 ( Pt 24):4513-4519. [73] Shinozaki K, Yamaguchi-Shinozaki K, Seki M: Regulatory network of gene expression in the drought and cold stress responses. Curr. Opin. Plant Biol. 2003, 6:410-417. [74] Sung S, Amasino RM: Vernalization and epigenetics: how plants remember winter. Curr. Opin. Plant Biol. 2004, 7:4-10. [75] Whitelaw NC, Whitelaw E: How lifetimes shape epigenotype within and across generations. Hum. Mol. Genet. 2006, 15 Spec No 2:R131-137. [76] Wagner D: Chromatin regulation of plant development. Curr. Opin. Plant Biol. 2003, 6:20-28. [77] Vanyushin BF: DNA methylation in plants. Curr. Top Microbiol. Immunol. 2006, 301:67-122. [78] Boyko A, Kovalchuk I: Epigenetic control of plant stress response. Environ. Mol. Mutagen. 2008, 49:61-72. [79] Bender J: DNA methylation and epigenetics. Annu. Rev. Plant Biol. 2004, 55:41-68. [80] Takeda S, Paszkowski J: DNA methylation and epigenetic inheritance during plant gametogenesis. Chromosoma 2006, 115:27-35. [81] Rassoulzadegan M, Grandjean V, Gounon P, Vincent S, Gillot I, Cuzin F: RNAmediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 2006, 441:469-474. [82] Ingelbrecht I, Van Houdt H, Van Montagu M, Depicker A: Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc. Natl. Acad. Sci. U S A 1994, 91:10502-10506. [83] Meyer P, Niedenhof I, ten Lohuis M: Evidence for cytosine methylation of nonsymmetrical sequences in transgenic Petunia hybrida. EMBO J. 1994, 13:2084-2088. [84] Finnegan EJ, Genger RK, Peacock WJ, Dennis ES: DNA Methylation in Plants. Annu Rev Plant Physiol Plant Mol. Biol. 1998, 49:223-247. [85] Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R: Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc. Natl. Acad. Sci. U S A 2000, 97:5237-5242. [86] Gowher H, Jeltsch A: Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites. J. Mol. Biol. 2001, 309:1201-1208. [87] Dieguez MJ, Vaucheret H, Paszkowski J, Mittelsten Scheid O: Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance. Mol. Gen. Genet. 1998, 259:207-215.

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61

[88] Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, Jacobsen SE: Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 2001, 292:2077-2080. [89] Kankel MW, Ramsey DE, Stokes TL, Flowers SK, Haag JR, Jeddeloh JA, Riddle NC, Verbsky ML, Richards EJ: Arabidopsis MET1 cytosine methyltransferase mutants. Genetics 2003, 163:1109-1122. [90] Ronemus MJ, Galbiati M, Ticknor C, Chen J, Dellaporta SL: Demethylation-induced developmental pleiotropy in Arabidopsis. Science 1996, 273:654-657. [91] Finnegan EJ, Peacock WJ, Dennis ES: Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc. Natl. Acad. Sci. U S A 1996, 93:8449-8454. [92] Jacobsen SE, Meyerowitz EM: Hypermethylated SUPERMAN epigenetic alleles in arabidopsis. Science 1997, 277:1100-1103. [93] Jacobsen SE, Sakai H, Finnegan EJ, Cao X, Meyerowitz EM: Ectopic hypermethylation of flower-specific genes in Arabidopsis. Curr. Biol. 2000, 10:179-186. [94] Matzke MA, Birchler JA: RNAi-mediated pathways in the nucleus. Nat. Rev. Genet. 2005, 6:24-35. [95] Pikaard CS: Cell biology of the Arabidopsis nuclear siRNA pathway for RNA-directed chromatin modification. Cold Spring Harb. Symp. Quant Biol. 2006, 71:473-480. [96] Tompa R, McCallum CM, Delrow J, Henikoff JG, van Steensel B, Henikoff S: Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3. Curr. Biol. 2002, 12:65-68. [97] Bartee L, Malagnac F, Bender J: Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes Dev. 2001, 15:17531758. [98] Kato M, Miura A, Bender J, Jacobsen SE, Kakutani T: Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis. Curr. Biol. 2003, 13:421426. [99] Cao X, Springer NM, Muszynski MG, Phillips RL, Kaeppler S, Jacobsen SE: Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. Proc. Natl. Acad. Sci. U S A 2000, 97:4979-4984. [100] Cao X, Jacobsen SE: Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr. Biol. 2002, 12:1138-1144. [101] Cao X, Aufsatz W, Zilberman D, Mette MF, Huang MS, Matzke M, Jacobsen SE: Role of the DRM and CMT3 methyltransferases in RNA-directed DNA methylation. Curr. Biol. 2003, 13:2212-2217. [102] Ramchandani S, Bhattacharya SK, Cervoni N, Szyf M: DNA methylation is a reversible biological signal. Proc. Natl. Acad. Sci. U S A 1999, 96:6107-6112. [103] Bhattacharya SK, Ramchandani S, Cervoni N, Szyf M: A mammalian protein with specific demethylase activity for mCpG DNA. Nature 1999, 397:579-583. [104] Weiss A, Keshet I, Razin A, Cedar H: DNA demethylation in vitro: Involvement of RNA. Cell 1996, 86:709-718. [105] Weiss A, Cedar H: The role of DNA demethylation during development. Genes to Cells 1997, 2:481-486.

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[106] Zhu J, Kapoor A, Sridhar VV, Agius F, Zhu JK: The DNA glycosylase/lyase ROS1 functions in pruning DNA methylation patterns in Arabidopsis. Curr. Biol. 2007, 17:5459. [107] Zhu B, Zheng Y, Angliker H, Schwarz S, Thiry S, Siegmann M, Jost JP: 5Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence. Nucleic Acids Res. 2000, 28:4157-4165. [108] Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marin MI, Martinez-Macias MI, Ariza RR, Roldan-Arjona T: DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases. Proc. Natl. Acad. Sci. U S A 2006, 103:68536858. [109] Penterman J, Zilberman D, Huh JH, Ballinger T, Henikoff S, Fischer RL: DNA demethylation in the Arabidopsis genome. Proc. Natl. Acad. Sci. U S A 2007, 104:6752-6757. [110] Kinoshita T, Miura A, Choi Y, Kinoshita Y, Cao X, Jacobsen SE, Fischer RL, Kakutani T: One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation. Science 2004, 303:521-523. [111] Choi Y, Gehring M, Johnson L, Hannon M, Harada JJ, Goldberg RB, Jacobsen SE, Fischer RL: DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in arabidopsis. Cell 2002, 110:33-42. [112] Gong Z, Morales-Ruiz T, Ariza RR, Roldan-Arjona T, David L, Zhu JK: ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 2002, 111:803-814. [113] Kapoor A, Agius F, Zhu JK: Preventing transcriptional gene silencing by active DNA demethylation. FEBS Lett. 2005, 579:5889-5898. [114] Penterman J, Uzawa R, Fischer RL: Genetic interactions between DNA demethylation and methylation in Arabidopsis. Plant Physiol. 2007, 145:1549-1557. [115] Zilberman D, Henikoff S: Epigenetic inheritance in Arabidopsis: selective silence. Curr. Opin. Genet. Dev. 2005, 15:557-562. [116] Soppe WJ, Jasencakova Z, Houben A, Kakutani T, Meister A, Huang MS, Jacobsen SE, Schubert I, Fransz PF: DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis. EMBO J. 2002, 21:6549-6559. [117] Tariq M, Saze H, Probst AV, Lichota J, Habu Y, Paszkowski J: Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc. Natl. Acad. Sci. U S A 2003, 100:8823-8827. [118] Jasencakova Z, Soppe WJ, Meister A, Gernand D, Turner BM, Schubert I: Histone modifications in Arabidopsis- high methylation of H3 lysine 9 is dispensable for constitutive heterochromatin. Plant J. 2003, 33:471-480. [119] Jackson JP, Lindroth AM, Cao X, Jacobsen SE: Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 2002, 416:556-560. [120] Johnson L, Cao X, Jacobsen S: Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation. Curr. Biol. 2002, 12:1360-1367. [121] Aufsatz W, Mette MF, van der Winden J, Matzke M, Matzke AJ: HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA. EMBO J. 2002, 21:6832-6841.

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[122] Lachner M, O'Carroll D, Rea S, Mechtler K, Jenuwein T: Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 2001, 410:116-120. [123] Grewal SI, Moazed D: Heterochromatin and epigenetic control of gene expression. Science 2003, 301:798-802. [124] Gaudin V, Libault M, Pouteau S, Juul T, Zhao G, Lefebvre D, Grandjean O: Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis. Development 2001, 128:4847-4858. [125] Mylne JS, Barrett L, Tessadori F, Mesnage S, Johnson L, Bernatavichute YV, Jacobsen SE, Fransz P, Dean C: LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC. Proc. Natl. Acad. Sci. U S A 2006, 103:5012-5017. [126] Ben-Porath I, Cedar H: Epigenetic crosstalk. Mol. Cell 2001, 8:933-935. [127] Zemach A, Grafi G: Methyl-CpG-binding domain proteins in plants: interpreters of DNA methylation. Trends Plant Sci. 2007, 12:80-85. [128] Havas K, Whitehouse I, Owen-Hughes T: ATP-dependent chromatin remodeling activities. Cell Mol. Life Sci. 2001, 58:673-682. [129] Geiman TM, Robertson KD: Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together? J. Cell Biochem. 2002, 87:117-125. [130] Zemach A, Li Y, Wayburn B, Ben-Meir H, Kiss V, Avivi Y, Kalchenko V, Jacobsen SE, Grafi G: DDM1 binds Arabidopsis methyl-CpG binding domain proteins and affects their subnuclear localization. Plant Cell 2005, 17:1549-1558. [131] Jeddeloh JA, Stokes TL, Richards EJ: Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nat. Genet. 1999, 22:94-97. [132] Miura A, Yonebayashi S, Watanabe K, Toyama T, Shimada H, Kakutani T: Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 2001, 411:212-214. [133] Kakutani T, Jeddeloh JA, Flowers SK, Munakata K, Richards EJ: Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proc. Natl. Acad. Sci. U S A 1996, 93:12406-12411. [134] Stokes TL, Kunkel BN, Richards EJ: Epigenetic variation in Arabidopsis disease resistance. Genes Dev. 2002, 16:171-182. [135] Kato M, Takashima K, Kakutani T: Epigenetic control of CACTA transposon mobility in Arabidopsis thaliana. Genetics 2004, 168:961-969. [136] Soppe WJ, Jacobsen SE, Alonso-Blanco C, Jackson JP, Kakutani T, Koornneef M, Peeters AJ: The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. Mol. Cell 2000, 6:791-802. [137] Kakutani T, Munakata K, Richards EJ, Hirochika H: Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana. Genetics 1999, 151:831-838. [138] Amedeo P, Habu Y, Afsar K, Mittelsten Scheid O, Paszkowski J: Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature 2000, 405:203-206. [139] Vaillant I, Schubert I, Tourmente S, Mathieu O: MOM1 mediates DNA-methylationindependent silencing of repetitive sequences in Arabidopsis. EMBO Rep. 2006, 7:1273-1278.

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[140] Henderson IR, Dean C: Control of Arabidopsis flowering: the chill before the bloom. Development 2004, 131:3829-3838. [141] Finnegan EJ, Genger RK, Kovac K, Peacock WJ, Dennis ES: DNA methylation and the promotion of flowering by vernalization. Proc. Natl. Acad. Sci. U S A 1998, 95:58245829. [142] Steward N, Kusano T, Sano H: Expression of ZmMET1, a gene encoding a DNA methyltransferase from maize, is associated not only with DNA replication in actively proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent cells. Nucleic Acids Res. 2000, 28:3250-3259. [143] Dyachenko OV, Zakharchenko NS, Shevchuk TV, Bohnert HJ, Cushman JC, Buryanov YI: Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress. Biochemistry (Mosc) 2006, 71:461465. [144] Sha AH, Lin XH, Huang JB, Zhang DP: Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis. Mol. Genet. Genomics 2005, 273:484-490. [145] Wada Y, Miyamoto K, Kusano T, Sano H: Association between up-regulation of stressresponsive genes and hypomethylation of genomic DNA in tobacco plants. Mol. Genet. Genomics 2004, 271:658-666. [146] Hashida SN, Kitamura K, Mikami T, Kishima Y: Temperature shift coordinately changes the activity and the methylation state of transposon Tam3 in Antirrhinum majus. Plant Physiol. 2003, 132:1207-1216. [147] Hashida SN, Uchiyama T, Martin C, Kishima Y, Sano Y, Mikami T: The temperaturedependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase. Plant Cell 2006, 18:104-118. [148] Beguiristain T, Grandbastien MA, Puigdomenech P, Casacuberta JM: Three Tnt1 subfamilies show different stress-associated patterns of expression in tobacco. Consequences for retrotransposon control and evolution in plants. Plant Physiol. 2001, 127:212-221. [149] Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M: Retrotransposons of rice involved in mutations induced by tissue culture. Proc. Natl. Acad. Sci. U S A 1996, 93:7783-7788. [150] Takeda S, Sugimoto K, Otsuki H, Hirochika H: A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors. Plant J. 1999, 18:383393. [151] Kalendar R, Tanskanen J, Immonen S, Nevo E, Schulman AH: Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proc. Natl. Acad. Sci. U S A 2000, 97:6603-6607. [152] Song WY, Pi LY, Wang GL, Gardner J, Holsten T, Ronald PC: Evolution of the rice Xa21 disease resistance gene family. Plant Cell 1997, 9:1279-1287. [153] McClintock B: The significance of responses of the genome to challenge. Science 1984, 226:792-801. [154] Chua YL, Watson LA, Gray JC: The transcriptional enhancer of the pea plastocyanin gene associates with the nuclear matrix and regulates gene expression through histone acetylation. Plant Cell 2003, 15:1468-1479.

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[155] Tsuji H, Saika H, Tsutsumi N, Hirai A, Nakazono M: Dynamic and reversible changes in histone H3-Lys4 methylation and H3 acetylation occurring at submergence-inducible genes in rice. Plant Cell Physiol. 2006, 47:995-1003. [156] Shaked H, Avivi-Ragolsky N, Levy AA: Involvement of the Arabidopsis SWI2/SNF2 chromatin remodeling gene family in DNA damage response and recombination. Genetics 2006, 173:985-994. [157] Takeda S, Tadele Z, Hofmann I, Probst AV, Angelis KJ, Kaya H, Araki T, Mengiste T, Mittelsten Scheid O, Shibahara K, et al: BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis. Genes Dev. 2004, 18:782-793. [158] Hanin M, Mengiste T, Bogucki A, Paszkowski J: Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene. Plant J. 2000, 24:183-189. [159] Kanno T, Mette MF, Kreil DP, Aufsatz W, Matzke M, Matzke AJ: Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation. Curr. Biol. 2004, 14:801-805. [160] Kanno T, Aufsatz W, Jaligot E, Mette MF, Matzke M, Matzke AJ: A SNF2-like protein facilitates dynamic control of DNA methylation. EMBO Rep. 2005, 6:649-655. [161] Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, Daxinger L, Kreil DP, Matzke M, Matzke AJ: Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet 2005, 37:761-765. [162] Matzke M, Kanno T, Huettel B, Daxinger L, Matzke AJ: RNA-directed DNA methylation and Pol IVb in Arabidopsis. Cold Spring Harb. Symp. Quant. Biol. 2006, 71:449-459. [163] Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette MF, Matzke AJ: Genetic analysis of RNA-mediated transcriptional gene silencing. Biochim. Biophys. Acta. 2004, 1677:129-141. [164] Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, Vazquez F, Robertson D, Meins F, Jr., Hohn T, Pooggin MM: Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res. 2006, 34:6233-6246. [165] Cao X, Jacobsen SE: Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc. Natl. Acad. Sci. U S A 2002, 99 Suppl 4:16491-16498. [166] Jones L, Ratcliff F, Baulcombe DC: RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Curr. Biol. 2001, 11:747-757. [167] Mathieu O, Reinders J, Caikovski M, Smathajitt C, Paszkowski J: Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation. Cell 2007, 130:851-862. [168] Vaucheret H: Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev. 2006, 20:759-771. [169] Vazquez F: Arabidopsis endogenous small RNAs: highways and byways. Trends Plant Sci 2006, 11:460-468. [170] Sunkar R, Chinnusamy V, Zhu J, Zhu JK: Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci. 2007, 12:301-309.

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[171] Jones-Rhoades MW, Bartel DP, Bartel B: MicroRNAS and their regulatory roles in plants. Annu. Rev. Plant Biol. 2006, 57:19-53. [172] Zhang B, Pan X, Cobb GP, Anderson TA: Plant microRNA: a small regulatory molecule with big impact. Dev. Biol. 2006, 289:3-16. [173] Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK: Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 2005, 123:1279-1291. [174] Hamilton A, Voinnet O, Chappell L, Baulcombe D: Two classes of short interfering RNA in RNA silencing. EMBO J. 2002, 21:4671-4679. [175] Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC: Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2004, 2:E104. [176] Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O: Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 2006, 313:68-71. [177] Onodera Y, Haag JR, Ream T, Nunes PC, Pontes O, Pikaard CS: Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 2005, 120:613-622. [178] Xie Z, Allen E, Wilken A, Carrington JC: DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U S A 2005, 102:12984-12989. [179] Gasciolli V, Mallory AC, Bartel DP, Vaucheret H: Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 2005, 15:1494-1500. [180] Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, Green PJ, Jacobsen SE: Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 2006, 38:721-725. [181] Carmell MA, Xuan Z, Zhang MQ, Hannon GJ: The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 2002, 16:2733-2742. [182] Llave C, Xie Z, Kasschau KD, Carrington JC: Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 2002, 297:2053-2056. [183] Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI, Moazed D: RNAi-mediated targeting of heterochromatin by the RITS complex. Science 2004, 303:672-676. [184] Baumberger N, Baulcombe DC: Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. U S A 2005, 102:11928-11933. [185] Lynn K, Fernandez A, Aida M, Sedbrook J, Tasaka M, Masson P, Barton MK: The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 1999, 126:469-481. [186] Hunter C, Sun H, Poethig RS: The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr. Biol. 2003, 13:1734-1739. [187] Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crete P: Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 2004, 16:69-79.

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[188] Yoshikawa M, Peragine A, Park MY, Poethig RS: A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 2005, 19:2164-2175. [189] Zilberman D, Cao X, Jacobsen SE: ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 2003, 299:716-719. [190] Zilberman D, Cao X, Johansen LK, Xie Z, Carrington JC, Jacobsen SE: Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr. Biol. 2004, 14:1214-1220. [191] Yu D, Fan B, MacFarlane SA, Chen Z: Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense. Mol. Plant Microbe Interact 2003, 16:206-216. [192] Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC: An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 2000, 101:543-553. [193] Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N, et al: Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 2000, 101:533-542. [194] Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS: SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of transacting siRNAs in Arabidopsis. Genes Dev. 2004, 18:2368-2379. [195] Xie Z, Fan B, Chen C, Chen Z: An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proc. Natl. Acad. Sci. U S A 2001, 98:65166521. [196] Chan SW, Zilberman D, Xie Z, Johansen LK, Carrington JC, Jacobsen SE: RNA silencing genes control de novo DNA methylation. Science 2004, 303:1336. [197] Bender J: Chromatin-based silencing mechanisms. Curr. Opin. Plant Biol. 2004, 7:521526. [198] Aufsatz W, Mette MF, Matzke AJ, Matzke M: The role of MET1 in RNA-directed de novo and maintenance methylation of CG dinucleotides. Plant Mol. Biol. 2004, 54:793804. [199] Vaucheret H, Vazquez F, Crete P, Bartel DP: The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 2004, 18:1187-1197. [200] Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, et al: Role of transposable elements in heterochromatin and epigenetic control. Nature 2004, 430:471-476. [201] Lippman Z, May B, Yordan C, Singer T, Martienssen R: Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification. PLoS Biol. 2003, 1:E67. [202] Morel JB, Godon C, Mourrain P, Beclin C, Boutet S, Feuerbach F, Proux F, Vaucheret H: Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 2002, 14:629-639. [203] Choi Y, Harada JJ, Goldberg RB, Fischer RL: An invariant aspartic acid in the DNA glycosylase domain of DEMETER is necessary for transcriptional activation of the imprinted MEDEA gene. Proc. Natl. Acad. Sci. U S A 2004, 101:7481-7486.

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[204] Xiao W, Gehring M, Choi Y, Margossian L, Pu H, Harada JJ, Goldberg RB, Pennell RI, Fischer RL: Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase. Dev. Cell 2003, 5:891-901. [205] Jost JP, Fremont M, Siegmann M, Hofsteenge J: The RNA moiety of chick embryo 5methylcytosine- DNA glycosylase targets DNA demethylation. Nucleic Acids Res. 1997, 25:4545-4550. [206] Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, Hakimi MA, Lerbs-Mache S, Colot V, Lagrange T: Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 2005, 19:2030-2040. [207] Pontes O, Li CF, Nunes PC, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS: The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 2006, 126:79-92. [208] Chen X: A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 2004, 303:2022-2025. [209] Juarez MT, Kui JS, Thomas J, Heller BA, Timmermans MC: microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature 2004, 428:84-88. [210] Kidner CA, Martienssen RA: Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 2004, 428:81-84. [211] Sunkar R, Zhu JK: Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 2004, 16:2001-2019. [212] Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL: Novel and mechanical stressresponsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 2005, 17:2186-2203. [213] Matzke M, Kanno T, Huettel B, Daxinger L, Matzke AJ: Targets of RNA-directed DNA methylation. Curr. Opin. Plant Biol. 2007, 10:512-519. [214] Sunkar R, Kapoor A, Zhu JK: Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 2006, 18:2051-2065. [215] Doench JG, Petersen CP, Sharp PA: siRNAs can function as miRNAs. Genes Dev. 2003, 17:438-442. [216] Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A, Jr., Zhu JK, Staskawicz BJ, Jin H: A pathogen-inducible endogenous siRNA in plant immunity. Proc. Natl. Acad. Sci. U S A 2006, 103:18002-18007. [217] Michaels SD, He Y, Scortecci KC, Amasino RM: Attenuation of FLOWERING LOCUS C activity as a mechanism for the evolution of summer-annual flowering behavior in Arabidopsis. Proc. Natl. Acad. Sci. U S A 2003, 100:10102-10107. [218] Liu J, He Y, Amasino R, Chen X: siRNAs targeting an intronic transposon in the regulation of natural flowering behavior in Arabidopsis. Genes Dev. 2004, 18:28732878. [219] Chandler VL, Eggleston WB, Dorweiler JE: Paramutation in maize. Plant Mol. Biol. 2000, 43:121-145. [220] Alleman M, Sidorenko L, McGinnis K, Seshadri V, Dorweiler JE, White J, Sikkink K, Chandler VL: An RNA-dependent RNA polymerase is required for paramutation in maize. Nature 2006, 442:295-298.

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[221] Schuermann D, Molinier J, Fritsch O, Hohn B: The dual nature of homologous recombination in plants. Trends Genet 2005, 21:172-181. [222] Kovalchuk O, Burke P, Arkhipov A, Kuchma N, James SJ, Kovalchuk I, Pogribny I: Genome hypermethylation in Pinus silvestris of Chernobyl--a mechanism for radiation adaptation? Mutat. Res. 2003, 529:13-20. [223] Blodner C, Goebel C, Feussner I, Gatz C, Polle A: Warm and cold parental reproductive environments affect seed properties, fitness, and cold responsiveness in Arabidopsis thaliana progenies. Plant Cell Environ. 2007, 30:165-175. [224] Chen Y, Schneeberger RG, Cullis CA: A site-specific insertion sequence in flax genotrophs induced by environment. New Phytol. 2005, 167:171-180. [225] Cullis CA: Mechanisms and control of rapid genomic changes in flax. Ann. Bot. (Lond) 2005, 95:201-206. [226] Naumann K, Fischer A, Hofmann I, Krauss V, Phalke S, Irmler K, Hause G, Aurich AC, Dorn R, Jenuwein T, Reuter G: Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis. EMBO J. 2005, 24:1418-1429. [227] Balogun MA, Ramsay ME, Hesketh LM, Andrews N, Osborne KP, Gay NJ, MorganCapner P: The prevalence of hepatitis C in England and Wales. J. Infect. 2002, 45:219226.

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In: Genome Instability and Transgenerational Effects ISBN: 978-1-60876-831-8 Editors: I. Kovalchuk, O. Kovalchuk, pp. 71-88 © 2010 Nova Science Publishers, Inc.

Chapter 4

EVOLUTION OF THE FPG/NEI FAMILY OF DNA GLYCOSYLASES Dorothy E. Pumo1,2, Ramiro Barrantes-Reynolds1, Scott Kathe1, Susan S. Wallace1 and Jeffrey P. Bond1 1

Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, Vermont 05405-0068 2 Department of Biology, Hofstra University, Hempstead, New York 11549-1140

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ABSTRACT Oxidative damage to DNA results in lesions such as thymine glycol, 5hydroxycytosine, formamidopyrimidine and 8-oxoguanine that have very different structures. Base excision repair is the main biological process for repairing DNA damage caused by oxidative stress. During the first step of base excision repair, an oxidative DNA lesion is recognized and excised by a DNA glycosylase. The Fpg/Nei family of DNA glycosylases is one of two families, each containing members that have distinct substrate specificities which collectively excise the oxidative lesions listed above. Events in the Fpg/Nei phylogeny include acquisition of novel substrate specificity, horizontal transfer, changes in a major structural motif (a zinc finger), and expansion within a bacterial clade. Importantly, the function of one clade, Fpg2, remains unknown.

ABBREVIATIONS AP site,

apurinic/apyrimidinic (abasic) site;

8-oxoG,

7,8-dihydro-8-oxo-2'-deoxyguanosine;

AthFpg,

Arabidopsis thaliana Fpg;

Fpg2,

Fpg-like protein clade apparently unique to Actinomycetes;

BER,

base excision repair;

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Dorothy E. Pumo, Ramiro Barrantes-Reynolds, Scott Kathe et al. CalFpg,

Candida albicans Fpg;

DHT,

5,6-dihydrothymine;

DHU,

5,6-dihydrouracil;

EcoFpg,

E. coli Fpg;

EcoNei,

E. coli Nei;

FapyAde,

4,6-diamino-5-formamidopyrimidine;

FapyGua,

2,6-diamino-4-hydroxy-5-formamidopyrimidine;

Fpg,

formamidopyrimidine-DNA glycosylase;

Fur,

ferric uptake regulator; Gh, guanidinohydantoin;

H2TH,

helix-two-turn-helix;

Nei,

endonuclease VIII;

Neil,

Nei-like;

Nth,

endonuclease III;

Ogg,

oxoguanine glycosylase;

OHC,

5-hydroxycytosine;

OHU,

5-hydroxyuracil;

PPMC,

Pearson product moment correlation;

Sp,

spiroiminodihydantoin;

Tg,

thymine glycol.

INTRODUCTION DNA in living cells is constantly damaged by light environmental chemicals and metabolic by-products that include superoxide and hydroxyl radicals. Normal metabolic processes such as respiration generate over 5,000 oxidative lesions per cell per day [1]. The oxidative lesions vary widely in structure (Figure 1). DNA integrity must be retained so that cells remain alive and accurate genetic information is passed to the next generation. When DNA damage is not corrected, mutations accumulate and in humans, they are associated with carcinogenesis and aging [2, 3]. Cells contain numerous repair enzymes that identify and correct specific types of DNA damage. The DNA glycosylases, members of the base excision repair (BER) pathway, remove damaged bases before they block replication or mispair and cause mutations. In the first steps of BER, members of two large unrelated families of DNA glycosylases recognize and remove damaged DNA bases including oxidized purines or pyrimidines; some members of these families act on alkylated bases (for reviews see [4-6]). With respect to oxidative damages and within each family, specific glycosylases act primarily on either oxidized purines or oxidized pyrimidines.

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Evolution of the Fpg/Nei Family of DNA Glycosylases

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Figure 1. Members of two different families of DNA glycosylases (Fpg/Nei, left; HhH-GPD, right) excise (arrows) a structurally diverse set of oxidative lesions (center). One family may provide for excision of different lesions, one lesion may be recognized by members of different families, and one enzyme may recognize different lesions.

The endonuclease III (Nth) or helix-hairpin-helix (HhH-GPD) superfamily members include Nth, Ogg I and II, MutY, Mig, MpgII, AlkA, and Tag. Proteins from the Nth superfamily are found in eubacteria, archaea, protists, plants, fungi and metazoans [7, 8]. The Fpg/Nei family members include formamidopyrimidine-DNA glycosylase (Fpg/MutM) and endonuclease VIII (Nei) proteins in several eubacterial prokaryotes, Fpg proteins in plants and fungi [9], and Nei-like (Neil) proteins found in metazoans [10, 11]. They are absent from protists and rare among Archaea. For the purposes of this review, the protein names Fpg, Nei, and Neil are used as they appear in the current literature and are based largely on primary sequence alignments. In practice, enzymes are named either for their activities or their ancestry which, as described below, presents a special problem for the Fpg/Nei family. Thus, names do not necessarily imply specific biochemical activities. Although Fpg/Nei proteins are found in all domains, they are not present in all taxa. The role of DNA glycosylases is to initiate base excision repair. Those that recognize oxidative lesions remove the damaged base and then use a lyase reaction to produce a single strand break (for reviews see [5, 12, 13]). General information concerning the biochemistry of Fpg/Nei proteins is compared in Table I. Differences in opposite base specificity between Fpg and Nei proteins are consistent with structural data. Fpg proteins have a strong preference for 8-oxoG:C but not 8-oxoG:A mispairs [14], whereas Nei proteins do not share a bias for opposite base recognition [15]. Note that neither protein names nor high sequence similarity

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is a reliable predictor of substrate specificity (for example, see enzyme activities for DHU in Table I). Table I. A comparison of preferred substrates for Fpg, Nei and Neil proteins in double stranded DNA. The information in this table represents general trends and not specific kinetic data. The relative activity rating can vary according to the conditions used in particular experiments. Because the biochemical activities of most Fpg/Nei proteins have not been empirically tested, and because there is variation among sequences, the activity of Fpg/Nei proteins not included in this table should be considered hypothetical. References for each column are in parentheses. Abbreviations: 8-oxoG, 7,8-dihydro-8oxo-2'-deoxyguanosine; AthFpg, Arabidopsis thaliana Fpg; CalFpg, Candida albicans Fpg; DHT, 5,6-dihydrothymine; DHU, 5,6-dihydrouracil; ds, double stranded; EcoFpg, E. coli Fpg; EcoNei, E. coli Nei; FapyAde, 4,6-diamino-5-formamidopyrimidine; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; Fpg, formamidopyrimidineDNA glycosylase; Gh, guanidinohydantoin; ND, not determined; Nei, endonuclease VIII; Neil, Nei-like; 5-OHC, 5-hydroxycytosine; 5-OHU, 5-hydroxyuracil; Tg, thymine glycol Fpg/Nei Proteins

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DNA Lesion

EcoFpg

EcoNei

Neil1

Neil2

AthFpg

CalFpg

[9, 14, 64]

[9, 15, 65]

[3, 9, 23, 66, 67]

[20, 24, 66]

[9]

[9]

Tg

No activity

Good

Good

Weak

No activity

Good

DHT

No activity

Good

Good

Good

No activity

Good

DHU

Weak

Good

Good

Weak

Good

Good

5-OHU

Weak

Good

Good

Weak

Weak

Good

5-OHC

Weak

Good

Good

Weak

Weak

Good

8-oxoG

Very good

No activity

Weak

Very weak

No activity

Weak

FapyGua

Very good

Weak

Good

ND

Good

Very good

FapyAde

Very good

Very good

Good

ND

Good

Good

Sp

Good

Weak

Very good

Very weak

Very good

Very good

Gh

Good

Weak

Very good

Good

Good

Good

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Evolution of the Fpg/Nei Family of DNA Glycosylases

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Key structural features of Fpg/Nei proteins have been studied in E. coli Fpg (EcoFpg) and Nei (EcoNei). The initial methionine is removed from translated proteins leaving an Nterminal proline which is the required nucleophile necessary for glycosylase and lyase activity [16, 17]. Glutamic acid that follows is required for glycosylase activity in both Fpg and Nei [18, 19]. A lysine at position 53 in EcoNei is used in catalysis [20], a helix-two-turns-helix (H2TH) domain (at positions 154-180 in EcoNei) and a zinc finger domain containing four cysteines and a requisite arginine (position 253 in EcoNei) are also signatures of bacterial proteins [21, 22]. The H2TH domain interacts with DNA by forming hydrogen bonds between phosphates in DNA. Hydrogen bonding involving the H2TH domain also helps maintain the active site. The zinc finger binds to DNA primarily in the loop between antiparallel β-strands. The K53 and H2TH and zinc finger motifs together create strong hydrogen bonding that stabilizes damaged DNA along the length of the enzyme [22]. Alignments that include Fpg/Nei members of other bacterial and eukaryotic phyla demonstrate that N-terminal sequences, K53 and the helix-two-turns-helix domain are generally common to all family members in prokaryotes as well as eukaryotes [20]. The zinc finger domain is common in the bacterial Fpg/Nei proteins. With the notable exception of insects and nematodes (based on the currently sequenced whole genomes), metazoans have one to three Fpg/Nei proteins named Neil1 (Nei-like 1), Neil2, and Neil3 [3, 11, 23-26]. Two Nei-like proteins are also found in Mimivirus [27]. The Neil3 protein and one of Mimivirus proteins differ from all other Fpg/Neis by having the Nterminal valine which, based on demonstrable catalytic activity, presumably replaces proline acting as a nucleophile required for glycosylase and lyase activities. The requisite glutamic acid and lysine residues are conserved along with the helix-two-turn-helix. Plant (Arabidopsis thaliana Fpg, AthFpg) and fungal (Candida albicans Fpg, CalFpg) homologs share about 50% sequence similarity. They have the same P2E3 and K53 (EcoNei positions) amino acids as well as the H2TH domain [9]. There is a putative zincless finger that is 50% similar between plant and fungal Fpgs [9]. AthFpg and CalFpg also have the Fpg insertion loop that determines the opposite base specificity of Fpg proteins [9]. The relationship between glycosylases and the oxidative lesions that they excise is complex. Members of a protein family may catalyze different reactions from one another[2831], one enzyme may catalyze multiple reactions[32], and proteins having different folds may catalyze the same reaction[33]; all these features can be seen among DNA glycosylases (Figure 1). Accounting for this complexity by identifying events in the co-evolution of these families presents a significant challenge. In particular, the sporadic occurrence of Fpg/Nei proteins within the tree of life and our current understanding of the relative importance of the identified structural elements of proteins leave many unanswered questions.

METHODS Fpg/Nei homologs were identified from genomic sequences deposited at the National Center for Biotechnology Information (NCBI) using known bacterial and eukaryotic Fpg, Nei, and Neil sequences as the query in BLAST [34]. Fpg/Nei homologs were identified using a PFAM domain profile (pfam06831) [35]. Proteins containing this domain were

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fetched using the Conserved Domain Database (CDD) [36]. CDTree 3.0 [37] was used to organize the data. Alignments were made using MUSCLE [38] and MAFFT [39], and some alignments were truncated using GBLOCKS [40]. For the eukaryotic sequences, alignments were refined by visual inspection using SEAVIEW [41]. Sequence logos were made with Weblogo v.3.0 [42, 43]. Maximum likelihood trees were built and tested with PhyML [44], and Baysian analysis was also used to evaluate trees with MrBayes [45].

EVENTS IN THE FPG/NEI PHYLOGENY

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Horizontal Transfer Fpg genes are found in conserved positions within operons in closely related γProteobacteria [46], and Fpg proteins are found in most eubacteria consistent with the Fpg genes being present early in eubacterial evolution. Nei proteins are missing in the majority of eubacterial genomes sequenced thus far, exceptions being the Actinobacteria and certain Proteobacteria. The phylogeny suggests that the Nei genes came to proteobacteria by horizontal transfer [20] that is they appear to be xenologous not paralogous. Although it is formally possible that vertebrates acquired the Fpg/Nei family members by horizontal transfer, there are several problems with this idea. Although horizontal gene transfer among bacteria is thought to be very common [47, 48], it is much harder to identify in vertebrates where the novel DNA must be inserted into the germ line in order to be passed to progeny [49]. Early reports of putative horizontal gene transfer from bacteria to vertebrates made as a result of BLAST searches were refuted [49]. Notably, some of gene transfers may be to single-celled or colonial eukaryotic ancestors, for which transfer to the germline does not present a problem. The possibility that plant, fungal and Neil1 clades arose from distinct horizontal transfers to ancestors without germline cells cannot be ruled out. Transposons allow limited horizontal transfer among metazoans [50, 51] and plants [52]. The Alphaproteobacteria of the genus Wolbachia are endosymbionts in the germ lines of invertebrates, and recently the Wolbachia genes were convincingly identified in chromosomes of insects and nematodes [53]. Because Wolbachia species inhabit germline cells of their insect and worm hosts, bacterial DNA could integrate into the hosts‘ genomes. Interestingly, Fpg/Nei proteins are absent from Drosophila (and all insects so far examined) and Caenorhabditis genomic databases. Hotopp and colleagues point out [51] that eukaryotic genome sequencing projects routinely remove bacterial genes as presumptive contaminants. It remains to be determined if insects and roundworms utilize Fpg proteins from genes that were transferred from Wolbachia but were routinely removed from eukaryotic genome databases. Except for Wolbachia infections of insects and nematodes, there are no other mechanisms known to allow incorporation of bacterial DNAs into the germlines of modern multicellular metazoans. The fully sequenced genomes from a variety of eukaryotic taxonomic groups allowed us to identify Fpg/Nei proteins in plants (Arabidopsis, grape, rice and algae), several fungi (but notably not important model organisms such as Saccharomyces cerevisiae and Schizosaccharomyces pombe), and metazoans including the primitive Tricoplax, sea anemone, and many vertebrates. At least one Fpg/Nei is present in an alga representing a

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basal plant lineage. This alga, Chlamydomonas, also shares traits, e.g., cilia, with metazoans and is considered to be an important link to ancestral eukaryotes [54]. The presence of the Fpg/Nei protein in the alga and modern plants suggests that the earliest plants likely had the Fpg protein. In fungi, sister taxonomic groups to Saccharomyces and Schizosaccharomyces as well as more distantly related taxa also have Fpg proteins. In both plants and fungi, Fpgs are similar, which thus suggests that the Fpg protein became a part of the eukaryotic genome early before the split of plants from the metazoan/fungi clade. The situation in metazoans is less clear as described in the following section.

Changes in Substrate Specificity Candida albicans Fpg (CalFpg) and Arabidopsis thaliana Fpg (AthFpg) are closely related DNA glycosylases that fall into a plant and fungal clade. These DNA glycosylases exhibit sequence features similar to bacterial Fpg such as the N-terminal proline, the insertion loop that determines opposite base specificity and the H2TH motif, and other key catalytic residues, suggesting the substrate specificity similar to bacterial Fpg. Unlike bacterial Fpg, however, they have a ‗zincless finger‘ similar to Neil1 sequences. The preferred substrates for CalFpg and AthFpg are formamidopyrimidines, Gh and Sp, but not 8-oxoG-like typical bacterial Fpg proteins. Like Neil1, these glycosylases have a zincless finger and can recognize oxidatively-induced damage in single-stranded DNA. The presence of Ogg in both Candida albicans and Arabidopsis thaliana may have allowed their Fpg to lose the activity against 8-oxoG and evolve the activity to recognize and remove Gh and Sp.

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Changes in the Structural Zinc-Finger Motif The majority of eubacterial Fpg/Nei proteins examined have a single common zinc-finger domain of the general pattern C-X2-C-X11-R-X4C-X2-C (Figure 2a) located near the Cterminus. The zinc finger structure has been confirmed by crystallography [22, 55]. The zinc finger works with the helix-two-turns-helix motif, the initial proline and other loops to contact DNA during excision repair. Mutation of the conserved arginine (EcoNei R252) to alanine disrupts the catalytic activity of the enzyme such that the mutant enzyme cannot cleave DHUcontaining DNA [22]. Metazoan Neil1 does not have a canonical zinc finger and does not appear to bind zinc. Nevertheless, Neil1 does recognize typical Fpg/Nei substrates and initiate the BER pathway (Table 1). No cysteines or histidines align with the bacterial zinc fingers, but an arginine (R277, human NEIL1) aligns with a conserved arginine in bacterial enzymes. Experiments have shown that the arginine is required for glycosylase activity [10]. Crystallographic analyses clearly demonstrated that the NEIL1 protein structure of the apo protein can be superimposed with the bacterial Nei (with DNA) and Fpg (without DNA) structures in the region of the bacterial zinc finger [10]. The arginine in the loop is conserved, as are the antiparallel strands. Two cysteine-containing loops that anchor zinc in the bacterial zinc finger are absent. The zincless finger may not be common, but it has also been found elsewhere [56, 57].

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Figure 2. Sequence logos illustrate the zinc-finger consensus patterns for bacterial (a) Nei and (b) Fpg. The conserved cysteines are in positions 2, 5, 22, and 25 along the x-axis. The conserved arginine is in position 17. Other conserved amino acids can also be found in one or both logos.

Both Neil2 and Neil3 have at least one zinc finger [3, 11, 20]. The Neil2 zinc finger is a CHCC-type, whereas Neil3 contains the CCCC-type finger (at amino acid positions 255- 282 in mouse) found in the bacterial proteins. The Neil2 zinc-finger also contains the conserved arginine (R310, human NEIL2) that is required for glycosylase activity [11]. Sequence alignments demonstrate that the arginine (R272 in mouse) is also conserved in Neil3 and therefore is probably required for activity. Although the Neil2 zinc finger has a histidine replacing one cysteine, the WCPxCQ consensus sequence is consistent with the evolutionary history shared by bacterial Nei proteins (Figure 2a). The Neil3 zinc finger sequence C-X2-CXn-I-Xn-R-X5-R-X4-CPXCQ is more similar to the bacterial Fpg proteins (Figure 2b). The Neil3 proteins are predicted to have 3 additional zinc fingers (initial cysteines at positions 324, 508, and 555 in mouse). One of these is a Ran-binding protein and the other two at the C-terminus are the presumptive GRF-family zinc fingers with sequence similarity to the zinc fingers predicted for DNA topoisomerase IIIα. The presence of the Ran-binding protein zinc finger family domain suggests that Neil3 proteins may depend on regulatory proteins for enhanced activity. Neil1-encoding DNA is found in the seemingly primitive metazoans, Trichoplax adhaerens and sea anemone (Nematostella vectensis) and none of them has an intact zinc finger. Sea urchins (Strongylocentrotus purpuratus) along with chordates are predicted to have Neil2. Interestingly, sea urchins appear to have two almost identical Neil2 proteins. The difference is that one contains a heat shock protein 70 (hsp70) domain, suggesting another potential target for regulatory protein binding. The Florida lancet (Branchiostoma floridae), like most vertebrates, appears to have genes encoding all three Neil proteins suggesting that these proteins arose before vertebrates diverged from other chordates. Representative plants, Arabidopsis thaliana, rice (Oryza sativa) and fungi that have the Fpg/Nei family proteins do not have a recognizable zinc finger domain. However, amino acid sequence alignments suggest a consensus sequence G-X2-(I/L)-X5-G-Xn-RT-Xn-P-X2-Q(K/R) where the second glycine, the arginine-threonine and proline align with the zincless finger in NEIL1 [9]. Parts of the consensus sequence can also be aligned with consensus regions of the zinc fingers present in other Fpg/Nei family members. When complete amino acid sequences are compared, plant and fungal Fpg proteins are most similar to each other and to the bacterial

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Fpg proteins compared with other Fpg/Nei sequences including the Neil proteins. However, when only zinc and zincless finger domains are compared, the data are consistent with an early eukaryotic conversion of a zinc finger to a zincless finger in at least one Fpg/Nei isoform followed by substantial additional diversification after the separation of metazoans from both plants and fungi. The levels of NEIL1 increase during the S-phase [23]. NEIL1 interacts with flap endonuclease 1 [58] and proliferating cell nuclear antigen [59], suggesting that its activity is increased during DNA replication. In contrast, NEIL2 activity is cell cycle-independent in fibroblasts [24]. The presence of the zincless finger does coincide with the occurrence of the canonical eukaryotic pattern of cell cycle regulation common in plants, metazoa and fungi. The functionality of the zincless finger could be related to the association with the other cell cycle or replication machinery. The flap endonuclease 1 binding site is in the disordered region of the structure of human NEIL1 [10], so the exact relationship between the zincless finger and endonuclease is still uncertain. However, only 14 amino acids separate two regions in human NEIL1 making the interaction between the zincless finger and the NEIL1-bound endonuclease a possibility. The endonuclease-binding domain is notably absent from the characterized bacterial Fpg/Nei proteins [58]. Plant and fungal Fpg amino acid sequence alignments are not inconsistent with homology of plant, fungal and Neil1 zincless fingers. The zincless finger motif common to plant/fungal Fpg proteins and NEIL1 and absent in prokaryotes along with other conserved residues argues for a common Fpg ancestry of all eukaryotes with apparent gene loss in the lineages leading to insects and roundworms (Neil1) and some modern fungi (loss of fungal Fpg).

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Expansion within the Actinobacteria Orthologs (i.e., Fpg versus Nei) differ by taxa and the number and type of paralogs (different proteins from the same family) within organisms are also highly variable. For example, there are four or more paralogs in the Actinomycetes and two or more in some proteobacteria (although they may be xenologs). Among eukaryotes, Fpg proteins are absent from Saccharomyces but are present in many closely related fungi such as Candida albicans as well as in monocot and dicot plants and in the green alga, Chlamydomonas. With the notable exception of the ecdysozoa which do not appear to have any Fpg/Nei proteins, the metazoa have one to three Nei-like (Neil) proteins. Most eubacteria contain at most one ortholog from the Fpg/Nei family (Table II), and a single member is usually the Fpg protein. The most sequenced Actinobacteria have multiple Fpg/Nei paralogs (Table II), with a few species having six or more genes. Even parasitic species of Mycobacterium have four paralogs, although at least one Fpg-like protein is most likely a pseudogene (Y. Guo, personal communication). Bayesian and maximum likelihood methods strongly suggest that the common ancestor of all actinomycetes had two Fpg and two Nei proteins (Figure 3). As actinomycetes evolved, so did their Fpg and Nei proteins; the phylogenetic analysis suggests that most (perhaps all) of the diversity of Fpg/Nei proteins in actinomycetes can be explained by vertical gene inheritance from a common actinomycete ancestor having four Fpg/Nei proteins. The phylogenetic analysis also suggests that actinomycete species with fewer paralogs probably lost them relative to the common ancestor.

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Figure 3. A maximum likelihood dendrogram of putative Fpg/Nei proteins found in Actinobacteria. All taxa shown, except Rubrobacter, are in the order Actinomycetales. All completed actinobacterial genomes (at the time of the analysis) containing one or more predicted Fpg/Nei proteins, including predicted pseudogenes, are included. Four distinct clades are resolved and labeled according to sequence similarity to the E. coli Fpg and Nei proteins. The Fpg2 proteins are found only in Actinomycetales, and their sequences are most similar to the Fpg proteins. The dendogram edges are colored according to the taxon key and represent either genera or families of closely related genera with few completely sequenced species. Among the Actinomycetales, the majority of proteins are most closely related to orthologous proteins in other closely related species rather than paralogs found within the same genome. This lends strong support to the hypothesis that the four clades evolved early in an ancestral Actinomycetales.

Table II. The number of the Fpg/Nei family members per bacterium found in sequenced genomes. Species with fully sequenced genomes that do not appear to have any members of the Fpg/Nei family are not included in this Table. Numbers in parentheses are the percentage of fully sequenced Actinobacteria or other genomes that are in each category The Number of Fpg/Nei paralogs/cell 1 (%)

2 (%)

≥ 3 (%)

The Number of sequenced genomes

Actinobacteria

5 (10%)

4 (8%)

41 (82%)

50

All others

386 (83%)

74 (16%)

5 (1%)

465

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The question remains: why would Actinobacteria maintain multiple paralogs within their genome? In general, the existence and maintenance of paralogs in bacteria may be a side effect of genome size, nucleotide composition bias, the environment including increases in environmental levels of superoxide radicals, horizontal gene transfer, and other factors. The sequenced genomes of Actinobacteria are biased towards species that have major impacts on humans, including agriculturally important plant and animal pathogens or species isolated from toxic environments. Additional DNA repair enzymes might make survival in contaminated environments more likely. One way to test for a relationship between Fpg/Nei paralogs and environmental effects is to look for correlations between Fpg/Nei proteins and other proteins that monitor, regulate, disable or otherwise interact with superoxide radicals or molecules that produce superoxide radicals. One such protein, the ferric uptake regulator (Fur) also has multiple paralogs in many Actinobacteria, and the number of paralogs is positively correlated with genome size within the phylum [60]. Several, but not all, ―metal related‖ proteins examined were positively correlated with Fpg/Nei proteins, but most of the PPMC values were less than 0.5. Fur and catalase, for example, had PPMC values of 0.36 and 0.29, respectively. Values for two protein families, siderophore-interacting proteins and FADH-binding protein 9, were slightly higher at 0.57 and 0.59, respectively. The positive correlations are consistent with the hypothesis that the superoxide radical environment may influence the maintenance of multiple Fpg/Nei paralogs in Actinobacteria. Although suggestive, PPMC do not provide strong evidence because multivariate observations are not independent, rather they are samples from a tree. The proper statistical evaluation of the correlation between the number of Fpg/Nei family members and the environment and genomic statistics remains an important outstanding task. Three types of evidence suggest that horizontal gene transfer is probably not a major contributor to the number of Fpg/Nei paralogs in Actinomycetes. The phylogenetic analysis (Figure 3) shows that Fpg/Nei protein clades can be explained by the evolution of four ancestral genes in evolving Actinomycete species. There is no evidence for G+C content variation among Fpg/Nei proteins and the remainder of the actinobacterial genomes (data not shown). In Mycobacterium tuberculosis where more proteins have been annotated, fpg is located close to and in the same orientation as hypothesized genes for a cell division protein ftsY and chromosome partition protein smc. Similarly, a nei gene is in the same orientation and close proximity to hypothesized genes for an ATP-dependent helicase and a transcriptional regulator. The neighboring genes suggest possible functional operons containing proteins that may be used in conjunction with DNA repair. Horizontal gene transfer is expected to insert proteins randomly in the new genome without regard to associated gene functions. Drake [61] demonstrated that in microbes genome size was inversely related to the number of mutations per genome. We hypothesized that larger genomes in Actinobacteria may be better at repairing mutations (and so have a lower mutation rate) because additional copies of oxidative repair enzymes are present. We compared genome-related characteristics with the number of Fpg/Nei paralogs within Actinobacteria (Table III). Although there is a positive correlation (PPMC = 0.45), genome size is probably not the only contributing factor. Massey [62] suggested the concept of "proteomic constraint" to explain data showing that a mutation rate is inversely proportional to proteome size in prokaryotes. He reasoned that smaller genomes have fewer errors because there are fewer bases to begin with.

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Table III. Genome data compared to the number of Fpg/Nei paralogs per organism. The Table contains Pearson product moment correlations (PPMC) comparing genome size, proteome size and the percentage of G+C to with the number of Fpg/Nei paralogs for all fully sequenced Actinobacteria currently available. The analysis was repeated after separating the free-living species from the usually eukaryotic host-associated species. Possible or suspected pseudogenes were included in the paralog count Total Actinobacteria

Free-living

Host-associated

Genome size

0.45

0.33

0.48

Proteome size

0.51

0.37

0.56

Percent G + C

0.41

0.03

0.5

As genome size increases, the need for improved mechanisms for genomic fidelity increases. Table III demonstrates a slightly higher correlation between the number of total proteins and the number of Fpg/Nei DNA repair proteins when compared to genome size; it is therefore consistent with the proteomic constraint hypothesis. When all Actinobacteria are considered, nucleotide composition bias is also positively correlated with the number of paralogs; however, when free-living Actinobacteria are tested separately from host-associated species, the data shift notably (Table III). The number of Fpg/Nei proteins in free-living Actinobacteria is random with respect to the G+C content of the genome, whereas hostassociated species are positively correlated. Host-associated organisms typically lose proteins compared to their free-living relatives [63], so the higher correlations listed in Table III for host-associated species may initially appear surprising. However, free-living species are likely to have more unusual or unique proteins compared to host-associated species because free-living species have a more variable environment. Increased overall protein diversity dilutes the correlation of proteome size with Fpg/Nei paralogs. The proteomic constraint hypothesis also offers an explanation as to why mimivirus maintains two Fpg/Nei proteins. Relative to other viruses, the mimivirus genome is huge. BER enzymes such as the Fpg/Nei proteins help sustain a necessary lower mutation rate compared to other viruses with much smaller genomes and therefore less DNA to mutate.

THE ENZYMATIC FUNCTION OF THE FPG2 GLADE REMAINS UNKNOWN There are four major clades of Fpg/Nei proteins in the Actinobacteria (Figure 3). Functions of these individual clades are still being determined; however sequence alignments and biochemical data (Y. Guo, pers. comm.) suggest that the sequence most similar to EcoFpg has a similar biochemical profile, and the sequence most similar to EcoNei also has a similar biochemical profile. A Nei2 clade and another Fpg-like clade, named Fpg2, are less understood.

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In some parasitic Mycobacterium species, the predicted Fpg2 protein is missing between 120-140 amino acids from the amino terminus. The amino terminal proline is the requisite nucleophile in other Fpg proteins for glycolsylase and lyase activities, suggesting that the predicted protein without the terminal proline and other conserved amino acids has lost activity and is a pseudogene. Enzyme and Schiff base assays confirm the absence of glycosylase and lyase activities (Y. Guo personal communication). Corynebacterium and Nocardiaceae species are in the same suborder as the Mycobacterium. None of the six fully sequenced genomes of Corynebacterium species have detectable fpg2 genes; but the two Nocardiaceae have fpg2 genes that appear to be able to encode ―normal,‖ (i.e. nonpseudogene) Fpg-type proteins. Translations of the fpg2 genes and putative pseudogenes predict a highly conserved zinc finger typical of Fpg/Nei proteins, except for the conserved arginine (Figure 4). Replacement of the arginine with lysine is the most common substitution, however in Streptomyces and Salinispora, the arginine is replaced by serine. Previous studies on other Fpg/Nei proteins reported that replacement of the arginine with alanine [10] or glutamine [11] caused marked reductions in glycosylase activity. Amino acid alignments show that most Fpg2 proteins differ from other bacterial Fpg/Neis in the zinc finger domain. In Mycobacterium, a lysine replaces the conserved arginine, and in Streptomyces and Salinispora Fpg2, the arginine is replaced by serine. Arthrobacter species also have fpg2 genes. Arthrobacter are free-living soil bacteria. A. sp. FB24 was isolated from heavy metal and aromatic solvent containing soil, and A. aurescens TC1 was found in atrazine-contaminated soil, and it can metabolize many 3triazine compounds contained in pesticides, dyes and plastic waste. Given the unfriendly environment of Arthrobacter species, it is difficult to argue that BER enzymes are likely candidates for pseudogenation. If the Fpg2 protein is unnecessary, it is surprising that Fpg2 remains in most genera of Actinobacteria that have been sequenced. The most parsimonious assumption for the Fpg2 protein function is that it is similar to Fpg proteins because of their overall sequence similarity. Nevertheless, the presence of both Fpg1 and Fpg2 proteins in the same organisms raises the possibility that the protein has assumed a new function or mode of operation or interactions with other proteins in at least a few species. This function may be quite different from that of other proteins in the Fpg/Nei family, and in fact it may not even be a BER enzyme or have glycosylase or lyase activity.

Figure 4. A sequence logo showing the conserved amino acid residues for the Fpg2 protein zinc fingers. Residue 25 on the x-axis corresponds to the conserved arginine found in other Fpg/Nei proteins (residue 17 in Figure 2). The conserved cysteines that stabilize zinc are found in positions 10, 13, 30, and 33. Only positions 25 and 27 are highly variable compared to the consensus sequence.

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CONCLUSION

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The common domain architecture, including the N-terminus, conserved lysine, helix-twoturns-helix and zinc/zincless finger found among bacterial and eukaryotic Fpg/Neis, is evidence of homology. We propose the events consistent with the phylogenetic distribution of this family described above. An early Fpg/Nei gene was transferred to eukaryotes by one or more horizontal gene transfers to early, likely single-celled or colonial, ancestors. This transfer may have occurred due to a close association in a soil or marine environment or perhaps a symbiosis. Some combinations of gene duplication and transfer (Figure 5) involving early actinomycetes produced four clades of Fpg/Nei proteins. Figure 3 convincingly demonstrates the placement of the origin of Nei among Actinobacteria before the most recent common ancestor of actinomycetes. An early duplication occurred in eukaryotes or two genes were transferred in the case of symbiosis. In one replicate, the zinc-binding site but not the DNA-binding ability was lost shortly after the transfer. Plant and fungal lineages did not markedly change the protein after the loss of the zincbinding motif and apparently had an early loss of the replicate with the intact zinc finger. Mutations in early metazoans were more extreme than in plants and fungi resulting in proteins (Neils) that had the least overall sequence similarity compared to other Fpg/Nei proteins. Neil1 retains the zincless finger motif as in the plant and fungal Fpgs. A relatively recent Neil2 duplication in sea urchins was followed by the addition of a heat shock 70 domain. In a chordate ancestor, additional mutations (or domain addition) resulted in new zinc-fingers in Neil3.

Figure 5. Broad substrate specificity may facilitate the origin of new protein functions by gene transfer. The red and blue bars quantify the specificity of an enzyme for different substrates, A and B. Cell division is followed by the change, through adaptation or drift, of the substrate specificity. Selection pressure for maintaining enzymes is present throughout the process. Gene transfer, therefore, may result in a cell having two xenologous enzymes.

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REFERENCES [1] [2]

[3]

[4]

[5] [6] [7] [8]

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

[9]

[10]

[11]

[12] [13] [14]

[15] [16]

Friedberg EC: DNA repair and mutagenesis. 2nd edn. Washington, D.C.: ASM Press; 2006. Maynard S, Schurman SH, Harboe C, de Souza-Pinto NC, Bohr VA: Base excision repair of oxidative DNA damage and association with cancer and aging. Carcinogenesis 2008:bgn250. Bandaru V, Sunkara S, Wallace SS, Bond JP: A novel human DNA glycosylase that removes oxidative DNA damage and is homologous to Escherichia coli endonuclease VIII. DNA Repair (Amst) 2002, 1:517-529. Mol CD, Parikh SS, Putnam CD, Lo TP, Tainer JA: DNA repair mechanisms for the recognition and removal of damaged DNA bases. Annu. Rev. Biophys. Biomol. Struct. 1999, 28:101-128. Wallace SS: Enzymatic processing of radiation-induced free radical damage in DNA. Radiat. Res. 1998, 150:S60-79. Zharkov DO: Base excision DNA repair. Cell Mol. Life Sci. 2008, 65:1544-1565. Denver DR, Swenson SL, Lynch M: An Evolutionary Analysis of the Helix-HairpinHelix Superfamily of DNA Repair Glycosylases. Mol. Biol. Evol. 2003, 20:1603-1611. Robey-Bond SM, Barrantes-Reynolds R, Bond JP, Wallace SS, Bandaru V: Clostridium acetobutylicum 8-Oxoguanine DNA Glycosylase (Ogg) Differs from Eukaryotic Oggs with Respect to Opposite Base Discrimination. Biochemistry 2008, 47:7626-7636. Kathe SD, Barrantes-Reynolds R, Jaruga P, Newton MR, Burrows CJ, Bandaru V, Dizdaroglu M, Bond JP, Wallace SS: Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases. DNA Repair (Amst) 2009. Doublie S, Bandaru V, Bond JP, Wallace SS: The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc. Natl. Acad. Sci. U S A 2004, 101:10284-10289. Das A, Rajagopalan L, Mathura VS, Rigby SJ, Mitra S, Hazra TK: Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein. J. Biol. Chem. 2004, 279:47132-47138. David SS, Williams SD: Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair. Chem. Rev. 1998, 98:1221-1262. McCullough AK, Dodson ML, Lloyd RS: Initiation of base excision repair: glycosylase mechanisms and structures. Annu. Rev. Biochem. 1999, 68:255-285. Tchou J, Bodepudi V, Shibutani S, Antoshechkin I, Miller J, Grollman AP, Johnson F: Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA. J. Biol. Chem. 1994, 269:15318-15324. Jiang D, Hatahet Z, Melamede RJ, Kow YW, Wallace SS: Characterization of Escherichia coli Endonuclease VIII. J. Biol. Chem. 1997, 272:32230-32239. Zharkov DO, Rieger RA, Iden CR, Grollman AP: NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/ AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein. J. Biol. Chem. 1997, 272:5335-5341.

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Dorothy E. Pumo, Ramiro Barrantes-Reynolds, Scott Kathe et al.

[17] Sidorkina OM, Laval J: Role of the N-terminal Proline Residue in the Catalytic Activities of the Escherichia coli Fpg Protein. J. Biol. Chem. 2000, 275:9924-9929. [18] Lavrukhin OV, Lloyd RS: Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase. Biochemistry 2000, 39:15266-15271. [19] Burgess S, Jaruga P, Dodson ML, Dizdaroglu M, Lloyd RS: Determination of active site residues in Escherichia coli endonuclease VIII. J Biol Chem 2002, 277:2938-2944. [20] Wallace SS, Bandaru V, Kathe SD, Bond JP: The enigma of endonuclease VIII. DNA Repair (Amst) 2003, 2:441-453. [21] Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP, Shoham G: Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J. Biol. Chem. 2002, 277:19811-19816. [22] Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G: Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 2002, 21:789-800. [23] Hazra TK, Izumi T, Boldogh I, Imhoff B, Kow YW, Jaruga P, Dizdaroglu M, Mitra S: Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA. Proc. Natl. Acad. Sci. U S A 2002, 99:3523-3528. [24] Hazra TK, Kow YW, Hatahet Z, Imhoff B, Boldogh I, Mokkapati SK, Mitra S, Izumi T: Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions. J. Biol. Chem. 2002, 277:30417-30420. [25] Rosenquist TA, Zaika E, Fernandes AS, Zharkov DO, Miller H, Grollman AP: The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death. DNA Repair (Amst) 2003, 2:581-591. [26] Takao M, Kanno S, Kobayashi K, Zhang QM, Yonei S, van der Horst GT, Yasui A: A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue. J. Biol. Chem. 2002, 277:42205-42213. [27] Bandaru V, Zhao X, Newton MR, Burrows CJ, Wallace SS: Human endonuclease VIIIlike (NEIL) proteins in the giant DNA Mimivirus. DNA Repair 2007, 6:1629-1641. [28] Jensen RA: Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol. 1976, 30:409-425. [29] Francino MP: An adaptive radiation model for the origin of new gene functions. Nat. Genet. 2005, 37:573-577. [30] Tocchini-Valentini GD, Fruscoloni P, Tocchini-Valentini GP: Structure, function, and evolution of the tRNA endonucleases of Archaea: an example of subfunctionalization. Proc. Natl. Acad. Sci. U S A 2005, 102:8933-8938. [31] Hughes AL: Gene duplication and the origin of novel proteins. Proc. Natl. Acad. Sci. U S A 2005, 102:8791-8792. [32] Piatigorsky J: Gene sharing and evolution : the diversity of protein functions. Cambridge, Mass.: Harvard University Press; 2007. [33] Steitz TA: DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 1999, 274:17395-17398. [34] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J. Mol. Biol. 1990, 215:403-410.

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[35] Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL, Bateman A: The Pfam protein families database. Nucleic Acids Res. 2008, 36:D281-288. [36] Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, et al: CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res. 2009, 37:D205210. [37] Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, et al: CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res. 2007, 35:D237-240. [38] Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32:1792-1797. [39] Katoh K, Toh H: Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinformatics 2008, 9:212. [40] Talavera G, Castresana J: Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 2007, 56:564-577. [41] Galtier N, Gouy M, Gautier C: SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci. 1996, 12:543-548. [42] Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res. 2004, 14:1188-1190. [43] Schneider TD, Stephens RM: Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 1990, 18:6097-6100. [44] Guindon S, Lethiec F, Duroux P, Gascuel O: PHYML Online--a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 2005, 33:W557559. [45] Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19:1572-1574. [46] Gifford CM, Wallace SS: The Genes Encoding Formamidopyrimidine and MutY DNA Glycosylases in Escherichia coli Are Transcribed as Part of Complex Operons. J. Bacteriol. 1999, 181:4223-4236. [47] Doolittle WF: Phylogenetic Classification and the Universal Tree. Science 1999, 284:2124-2128. [48] Koonin EV, Wolf YI: Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world. Nucl. Acids Res. 2008, 36:6688-6719. [49] Stanhope MJ, Lupas A, Italia MJ, Koretke KK, Volker C, Brown JR: Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates. Nature 2001, 411:940-944. [50] Loreto ELS, Carareto CMA, Capy P: Revisiting horizontal transfer of transposable elements in Drosophila. Heredity 2008, 100:545-554. [51] Pace JK, Gilbert Cm, Clark MS, Feschotte Cd: Repeated horizontal transfer of a DNA transposon in mammals and other tetrapods. Proceedings of the National Academy of Sciences 2008, 105:17023-17028. [52] Diao X, Freeling M, Lisch D: Horizontal Transfer of a Plant Transposon. PLoS Biology 2006, 4:e5.

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Dorothy E. Pumo, Ramiro Barrantes-Reynolds, Scott Kathe et al.

[53] Hotopp JCD, Clark ME, Oliveira DCSG, Foster JM, Fischer P, Torres MCM, Giebel JD, Kumar N, Ishmael N, Wang S, et al: Widespread Lateral Gene Transfer from Intracellular Bacteria to Multicellular Eukaryotes. Science 2007, 317:1753-1756. [54] Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, et al: The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 2007, 318:245-250. [55] Sugahara M, Mikawa T, Kumasaka T, Yamamoto M, Kato R, Fukuyama K, Inoue Y, Kuramitsu S: Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8. EMBO J. 2000, 19:3857-3869. [56] Struthers MD, Cheng RP, Imperiali B: Design of a monomeric 23-residue polypeptide with defined tertiary structure. Science 1996, 271:342-345. [57] Kumar RA, Koc A, Cerny RL, Gladyshev VN: Reaction Mechanism, Evolutionary Analysis, and Role of Zinc in Drosophila Methionine-R-sulfoxide Reductase. J. Biol. Chem. 2002, 277:37527-37535. [58] Hegde ML, Theriot CA, Das A, Hegde PM, Guo Z, Gary RK, Hazra TK, Shen B, Mitra S: Physical and Functional Interaction between Human Oxidized Base-specific DNA Glycosylase NEIL1 and Flap Endonuclease 1. J. Biol. Chem. 2008, 283:27028-27037. [59] Dou H, Theriot CA, Das A, Hegde ML, Matsumoto Y, Boldogh I, Hazra TK, Bhakat KK, Mitra S: Interaction of the Human DNA Glycosylase NEIL1 with Proliferating Cell Nuclear Antigen: The Potential for Replication-Associated Repair of Oxidized Bases in Mammalian Genomes J. Biol. Chem. 2008, 283:3130-3140. [60] Santos C, Vieira J, Tavares F, Benson D, Tisa L, Berry A, Moradas-Ferreira P, Normand P: On the nature of fur evolution: A phylogenetic approach in Actinobacteria. BMC Evolutionary Biology 2008, 8:185. [61] Drake JW: A constant rate of spontaneous mutation in DNA-based microbes. Proc. Natl. Acad. Sci. U S A 1991, 88:7160-7164. [62] Massey SE: The Proteomic Constraint and Its Role in Molecular Evolution. Mol. Biol. Evol. 2008, 25:2557-2565. [63] Wernegreen JJ: For better or worse: genomic consequences of intracellular mutualism and parasitism. Current Opinion in Genetics and Development 2005, 15:572-583. [64] Hatahet Z, Kow YW, Purmal AA, Cunningham RP, Wallace SS: New substrates for old enzymes. 5-Hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2'-deoxyuridine is a substrate for uracil DNA N-glycosylase. J. Biol. Chem. 1994, 269:18814-18820. [65] Melamede RJ, Hatahet Z, Kow YW, Ide H, Wallace SS: Isolation and characterization of endonuclease VIII from Escherichia coli. Biochemistry 1994, 33:1255-1264. [66] Hailer MK, Slade PG, Martin BD, Rosenquist TA, Sugden KD: Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2. DNA Repair (Amst) 2005, 4:41-50. [67] Krishnamurthy N, Zhao X, Burrows CJ, David SS: Superior removal of hydantoin lesions relative to other oxidized bases by the human DNA glycosylase hNEIL1. Biochemistry 2008, 47:7137-7146.

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Chapter 5

STRESS-INDUCED MUTAGENESIS IN BACTERIA Andrey Golubov Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, CANADA

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ABSTRACT Stress-induced mutagenesis in bacteria has been puzzling researchers for decades. Bacteria regularly encounter a variety of chemical, physical and biological stressors. Some bacteria manage to survive and proliferate, very often giving rise to cell populations with new phenotypes. Numerous attempts have been made to study mutagenic factors and their influence on microbial physiology, genome stability and evolution. These attempts have raised more questions than answers. A classical example is the fact that until now there is no unambiguous theory regarding mechanisms of adaptive mutation in a Lac- strain of Escherichia coli. In this chapter, we will attempt to outline the influence of main mutagenic factors on bacterial physiology and the bacteria‘s ability to adapt to these factors.

INTRODUCTION The term ―stress‖ was introduced by a famous Canadian researcher of Hungarian origin Hans Selye in 1936. Initially, he founded a concept of the General Adaptation Syndrome (GAS) which was the response of an organism to negative stress (distress) or positive stress (eustress) [1]. Later, the term ―stress‖ coined by him was introduced into many languages and accepted now worldwide. Bacteria living in nature always experience different types of stress. In order to survive in the harmful environment, they have developed an efficient system of the stress-induced response that enhances their adaptability and finally speeds-up evolution. Such stress response can be carried out by a few genetic processes which direct genetic changes and finally result in creating bacteria with a new phenotype. Therefore, we can consider the stress response as a mutagenic process induced by various stressful (mutagenic) factors. Historically, ―adaptive mutagenesis‖ and ―classical‖ SOS mutagenesis were considered as

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separate processes [2]. However nowadays, there is a tendency to group these two processes together based on the fact that they both reflect the selective environment and produce better adapted variants [3,4]. In this chapter, we will discuss various mutagenic factors and their influence on bacterial genome stability. We will also describe mutagen-induced stress-response mechanisms that help bacteria survive in a new environment.

STRESSFUL (MUTAGENIC) FACTORS The term ―mutagen‖ refers to any factor that damages DNA. In this chapter, any severe factor that induces the stress-response system, damages DNA or puts bacterial development at risk will be considered as mutagenic. They include, but are not limited to, changes in temperature and oxygen concentration, starvation, exposure to antibiotics, UV light and ionizing radiation, etc. We will discuss various mechanisms of bacterial response leading to elimination or suppression of negative consequences triggered by exposure to these stresses.

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STRESS RESPONSE TO ENDOGENOUS AND EXOGENOUS DNADAMAGING AGENTS Endogenous DNA-damaging agents are primarily represented by ROS - reactive oxygen species that are the products of normal metabolic processes - H2O2 and O2-. The source of ROS in E. coli has been described by Gonzalez-Flecha and Demple [5]. It was shown that the majority of H2O2 molecules are produced by the respiratory chain. Ubiquinone and NADP give away electrons that form O2-. Afterwards, superoxide dismutases convert O2- to H2O2. ROS can cause various types of DNA damage, for example, double- and single-strand breaks, base modifications (oxidation, alkylation and hydrolysis of bases), DNA-protein crosslinks formation, etc. [6]. Exogenous DNA-damaging agents include artificial mutagenic chemicals (for example, aromatic compounds that act as DNA intercalating agents), high temperature, ionizing radiation (gamma rays and x-rays), ultraviolet (λ = 280 - 400nm) radiation, etc. Types of DNA damage can vary according to a DNA-damaging agent: UVA light creates mostly free radicals; UVB light creates pyrimidine dimmers due to crosslinking between adjacent cytosine and thymine bases; ionizing radiation causes breaks in DNA strands (radiationinduced water radiolysis generates .OH radicals inducing the formation of more than 100 distinct DNA adducts [7]); high temperature increases the rate of hydrolytic depurination (base loss), deamination and backbone scission [8]; DNA intercalating agents such as ethidium bromide intercalate into double-stranded DNA between bases. Thus, all above-mentioned factors change DNA structure, impeding protein-DNA interaction and jeopardizing the most important cell processes (DNA synthesis, replication and transcription). These mutagenic factors also activate the SOS DNA-damage response (see review by Friedberg et al., 2006 [9]). The formal concept of the SOS response has been formed in 1974 by M. Radman [10]. The SOS response is a complex series of changes, including DNA repair. There are two main

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regulators of the SOS response in bacteria: LexA and RecA proteins. The LexA protein represses transcription of a few dozen genes in E. coli [11] by binding to an SOS box in their operator regions. SOS boxes consist of 20 bp (8 of them are absolutely conserved) and overlap with the respective promoters. Following DNA damage, the amount of single-stranded DNA in bacterial cells increases drastically due to unsuccessful attempts of bacteria to replicate damaged DNA. The very first step in the SOS response is coating of single-stranded DNA with the RecA protein and forming a nucleoprotein filament (Figure 1). The RecA protein in this complex is ―activated‖ and promotes the autoproteolytic cleavage of the LexA repressor. It opens SOS boxes in the corresponding promoters and induces transcription of SOS genes. The level and duration of their expression depends mostly on the amount of single-stranded DNA in cells. The order of their expression depends on the affinity of LexA protein to the corresponding SOS box. Once damaged DNA has been repaired and replication restored, the LexA protein represses expression of SOS genes (Figure 1) [12]. The SOS response can help bacteria survive by using two mechanisms: nucleotide excision repair and/or translesion synthesis. Their main functions are either restoration of the original genetic information or generation of genetic variability. The choice of the repair pathway depends on the intensity and duration of a damaging factor. Both pathways are biochemically complex processes, even in bacteria. Their brief outline is given below.

Figure 1. Schematic representation of the first step of the SOS response: The major steps are: coating of single-stranded DNA with RecA protein, formation of nucleoprotein filaments, the autoproteolytic cleavage of the LexA repressor and induction of SOS gene transcription. Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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Nucleotide excision repair is an ATP-dependent process in which damaged bases such as pyrimidine dimers and (6-4) photoproducts are excised from DNA as part of oligonucleotide fragments rather than free bases [9]. Logically, nucleotide excision repair genes should be induced at the beginning of SOS response [13]. For example, RecA is induced in less than 1 min after exposure to a damaging agent (for instance, UV), becomes activated and promotes autocleavage of LexA, triggering expression of the nucleotide excision repair genes [14]. There are four formal steps in the excision repair pathway: incision, excision, synthesis and ligation (Figure 2). The incision step requires a combination of UvrA, UvrB and UvrC proteins. First of all, damaged DNA is recognized by the UvrAB complex, then UvrA dissociates and UvrC binds to UvrB. The formed UvrBC complex makes two incisions: 7 nucleotides from the 5‘ side of the damaged site and 3-4 nucleotides away from the 3‘ side. During the excision step UvrD (DNA helicase II) unwinds DNA allowing release of the oligonucleotide between the two cuts and UvrC protein from the postincision complex [15]. It is possible that this step also requires DNA polymerase I (PolA). The DNA synthesis reaction is catalyzed by DNA polymerase I which fills the resulting gap and displaces the UvrB protein bound to DNA. DNA ligase finalizes the process: it ligates the newly incorporated nucleotides with a polynucleotide chain [9]. If the effect of damage is persistent or nucleotide excision repair cannot fix damaged DNA bases, RecA de-represses translesion DNA synthesis gene expression.

Figure 2. Four formal steps of the excision repair pathway: incision, excision, synthesis and ligation.

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TRANSLESION DNA SYNTHESIS Translesion DNA synthesis is a true basis for SOS mutagenesis. It allows the replication machinery presented by three DNA polymerases to bypass DNA lesions which block polymerization by E. coli DNA polymerase III. Normally, cells ‗pay‘ for the ability to continue replication by decreased fidelity of synthesis and generated genetic variability. There are three characterized SOS DNA polymerases: DNA Pol II, DNA Pol IV and DNA Pol V. DNA Pol II belongs to the B-family encoded by the polB gene and is a usual polymerase that possesses reasonably good fidelity and processivity in normal conditions. It can bypass some DNA lesions and may help restart a stalled replication fork [16,17]. DNA Pol IV belongs to the Y-family and is encoded by the dinB gene [18]. It is an errorprone polymerase, but the type and number of lesions it can fix are restricted. Similarly to DNA Pol II, DNA Pol IV may restart stalled replication forks and extend polynucleotide chains started by another polymerase [16,19]. The last polymerase in our list – DNA Pol V – seems to be a major player in this game; it is induced ~50 min after the initial activation of RecA. It is encoded by the umuDC operon which was identified by screening for ultravioletresistant E. coli mutants [20]. DNA Pol V is a heterotrimer composed of one UmuC protein and two truncated variants of UmuD protein (=Umu D2‘C) [21]. It is working together interacts with RecA, the single-stranded DNA (ssDNA)-binding protein (SSB), the β sliding clamp, and the γ clamp loading complex. Being error-prone, DNA Pol V can pass through many DNA lesions (see details in [19,21,22]). Logically, we can expect that the nucleotide excision repair process will either fix mutated bases and restore the status quo of bacterial genome or promote the activity of the translesion DNA synthesis machinery. It is apparent that translesion DNA synthesis will introduce many mutations into the genome and thus will lead to genetic variability. Some of those mutations will be lethal or will have no effect on the ability of cells to survive in new environmental conditions. But there are a few heritable mutations that will be advantageous and have long-term consequences for bacteria evolution.

THE GENERAL STRESS RESPONSE The general stress response is induced by transition of growing bacterial cells into stationary phase. It can also be triggered by various environmental stress conditions such as starvation, high and low temperatures, high osmolarity, and low pH. Despite a different nature of all these stress factors, they have one common outcome: bacterial cells stop their growth. The master regulator of the general stress response is RpoS (also known as ζS, ζ38, Nuv, KatF, XthA, AppR, and Csi-2) [23-28]. RpoS is an RNA polymerase sigma factor in E. coli that can replace a vegetative sigma factor (RpoD) under stress conditions [29-31]. The rpoS gene has been identified in many other proteobacteria and seems to be very conservative. Regulation of RpoS is a very complex and multi-level process. Depending on the type of stress, it can involve transcriptional regulation (including various trans-acting factors such as inorganic polyphosphates, ppGpp, etc.) and translational regulation (mRNA secondary

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structure and trans-acting factors such as Hfq, HU, H-NS, small regulatory RNAs, etc.) (see review by Hengge-Aronis [32]). Since cells experience a specific stressful condition, the amount of RpoS increases drastically, resulting in the formation of the RNAP-RpoS complex. This complex directly or indirectly regulates expression of 481 positively controlled and 95 negatively controlled genes in E. coli (up to 10% of the E. coli genes) which are required for survival during stress [33]. Layton and Foster have shown that Pol IV is induced in the late stationary cell phase under positive control of RpoS and is independent of LexA inactivation [34]. In another work, Yeiser and colleagues have shown that the dinB (Pol IV) gene has a higher expression level in a 5-day- old culture [35]. It suggests that the RpoS regulon can overlap with other stress response networks, particularly with the SOS system. The RpoS-dependent regulatory pathway can increase the mutation rate of stressed cells either through up-regulation of DNA Pol IV expression or down-regulation of mismatch repair protein (MMR) expression [16]. In the first case, the error-prone DNA Pol IV increases the amount of mutations in newly synthesized DNA chains. In the second case, RpoS downregulates the expression level of two of the MMR proteins: MutS and MutH [36]. Thus, their low activity can promote preservation of mutations generated by DNA Pol IV. Also, in cells with the low amount of MMR proteins, interspecies recombination can be enhanced, presenting another way of genetic variation [37].

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THE HEAT-SHOCK RESPONSE The heat-shock response can be induced not only by a rapid increase in environmental temperature but also by other conditions that result in protein unfolding, for example, oxidative stress, exposure to antibiotics and heavy metals, phage infection, and carbonsource/ amino-acid starvation. Thus, the heat-shock response can also be considered as the general stress response. E. coli and other bacteria contain two main types of chaperones [38] which regulate the heat-shock response: the sigma factors RpoH (ζ32) and RpoE (ζ24) [39-41]. It has been shown that heat-shock results in high-level synthesis of two heat shock chaperons GroE (GroES and GroEL) and DnaK (hsp70 homolog) which act to aid proper protein folding and protection from degradation. GroEL and its cofactor GroES can promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Interestingly, GroE plays a key protective role in supporting growth at normal physiological temperatures (20-40° C), whereas high levels of DnaK are required primarily at higher temperatures [42]. Kerner and co-authors have shown that approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence was limited to approximately 85 substrates, including 13 essential proteins [43]. It has been shown that GroE interacts with UmuC, the polymerase subunit of Pol V, protecting it from degradation [44-46]. Also GroE is likely to interact with Pol IV and is required for Pol IV-dependent adaptive mutation [47].

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Thanks to chaperones of the RpoH regulon that can be expressed under various stress conditions, DNA polymerases Pol IV and Pol V will be protected from degradation and will be able to perform their mutagenic (and possibly adaptogenic) functions.

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THE STRINGENT RESPONSE The bacterial stringent response is typically triggered by amino acid starvation. It causes accumulation of the signaling nucleotides pppGpp and ppGpp [48]. These nucleotides are produced not only in response to an amino acid shortage but also in response to many different kinds of nutrient limitations that cause growth arrest. (p)ppGpp, guanosine pentaphosphate or tetraphosphate is an alarmone which causes inhibition of rRNA and tRNA synthesis, when there is a shortage of amino acids. It inhibits translation and thus ‗saves‘ amino acids. There are two different pathways for ppGpp production in E. coli: the RelA- and SpoT-dependent pathways. RelA is associated with ribosomes and produces ppGpp during amino acid starvation. SpoT is very likely responsible for ppGpp accumulation of in response to most stresses and nutrient limitations apart from amino acid starvation. In addition, SpoT exhibits dual functions and is also responsible for hydrolyzing ppGpp [49]. The most important feature of (p)ppGpp is its ability to positively regulate expression of RpoS and RpoS-dependent genes [50]. It has been shown that (p)ppGpp increases the ability of RpoS and RpoH to compete with the vegetative sigma RpoD factor for RNA polymerase [51]. Thus, (p)ppGpp-, RpoS- and RpoH-mediated stress response mechanisms are co-induced and overlapping. It seems that the (p)ppGpp-mediated stringent response can serve as an enhancer of the RpoS-dependent general stress response and the RpoH-dependent heat-shock response. The mutagenic outcome of both of them is based on the activity of DNA Pol IV, Pol V and mismatch repair proteins. As a result of (p)ppGpp-mediated regulation, the levels of DNA Pol IV and/or Pol V should increase, and the levels of the mismatch repair proteins should decrease, resulting in a general mutagenic state.

NUTRITIONAL STRESS AND ADAPTIVE MUTATION Adaptive mutation is a phenomenon previously called directed mutation that appears when specific nutritional selection is applied, for instance, Lac- cells are incubated on lactose medium [16]. During this process, the stress-response apparatus of bacteria produces advantageous mutations that relieve selective pressure [52]. Interestingly, mutations do not occur and accumulate when cells merely experience starvation [52-55]. The starving population may be dynamic, with some cells dying and slowly proliferating. Even during very long incubation in nutritionally limited medium, mutant bacteria can survive on waste products of their neighbors and of course on their dead remnants) [56,57]. The classical study of adaptive mutation in the E. coli strain FC40 was done by Cairns and Foster [53]. Below, we will try to give just a brief description of models explaining adaptive mutagenesis. The detailed characteristics of FC40 and an excellent comprehensive picture of the current situation in this subject area as well as polemics between three major

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research groups on the subject in this area is are presented elsewhere [16,37,58-68]. The description of FC40 was adapted from reviews by Foster, 2005 and 2007 [16,37]. The E. coli strain FC40 is unable to catabolize lactose (Lac−) but reverts to lactose utilization (Lac+) when lactose is its sole carbon and energy. FC40 has a +1 frameshift mutation at the 320th codon of lacI, changing CCC to CCCC that affects the lacZ gene which makes the strain Lac− [69]. This strain has a conjugal plasmid, F′128, which carries the lac region as well as proAB deleted from the chromosome. The FC40 Lac- strain has different growth abilities on solid and in liquid medium supplemented with lactose. When incubated in liquid minimum lactose medium, the Lac- population is completely static for at least 4 days [70]. However, when plated on minimal lactose medium, FC40 can double several times over the course of a few days (Figure 3). This growth may be due to either the impurities in the agar or, possibly, breakdown products of lactose. Because of the low amount of βgalactosidase made by Lac+ revertants of FC40, Lac+ colonies take 2 days to become visible. Therefore, most of the colonies that appear on day 2 are due to mutations that occurred during the growth of colonies prior to plating. Thereafter, from day 3 to day 5, new Lac+ colonies appear at a constant rate. These late-arriving revertants are due to mutations that occurred after the cells were on lactose, and are called adaptive mutations [16,37]. There are two types of stress-induced mutants: Lac+ ―point mutants‖ that carry a compensatory frameshift mutation in the lac gene [71,72], and lac-amplified cells that carry a tandem array of 20-50 or more repeats of a region of 7-134 kb including the lac gene [73,74]. Currently, three main models have been suggested to explain adaptive mutation: hypermutation, recombination-dependent mutation (RDM), and amplification-dependent mutation (ADM) [16,64,65].

Figure 3. Different growth abilities of E. coli strain FC40 grown on solid and in liquid media supplemented with lactose. When incubated in liquid minimum lactose medium, the Lac- population is completely static for at least 4 days. When plated on minimal lactose medium, FC40 can double several times over the course of a few days.

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Hypermutations It has been proven that about 1% of Lac+ mutants which arose on lactose-supplemented medium have a second mutation in their genome [16]. These isolates, however, did not show an increase in the mutation rate upon subsequent testing [75,76]. It suggests that there was a cell population with a transient increase in mutation rate during the process of lactose selection. In E. coli strain FC40, the hypermutator state requires an increased level of DNA Pol IV activity and a simultaneous decrease in mismatch repair protein activity [76,77]. Rosche and Foster [76] have shown that about 0.1% of cells of the initial population are hypermutators with elevated mutation rates (approximately 200-fold). They can produce up to 10% of the Lac+ adaptive mutations. Two alternative pathways – RDM or ADM – might account for the rest 90% of adaptive mutations.

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RDM and ADM The RDM model requires a special mutagenic mechanism at work in cells during selection. RDM can be triggered by production of a double-strand break in the DNA, initiating double-strand break repair. This process is RecA- and RecBCD-dependent and occurs via strand invasion. DNA synthesis involves either DNA Pol II or DNA Pol IV (the last one has a high frequency of errors). Thus, any DNA region of any replicon can be mutated, including the lac region on the episom. The ADM model proposes that mutations occur by an ordinary mechanism [16]. When selective pressure is removed, a duplication process in cells occurs at a frequency of approximately one new event in every 1000 cells [16]. It can also involve duplication of the lac region. On lactose medium, cells with the duplicated lac region can slowly proliferate and amplify the lac regions via homologous recombination (a RecA- and RecBCD-dependent strand invasion process). Sooner or later, a ―true‖ Lac+ revertant will appear, allowing further proliferation of a selected clone. Within this clone, amplified arrays can be de-amplified, leaving the clone with a single Lac+ copy which will overgrow its slow- growing neighbors [16]. Obviously, bacterial populations that have minor subpopulations of hypermutated cells have a better chance to survive in adverse circumstances. Under nutritional stress, hypermutated cells can proliferate and multiply mutations which will help bacterial populations survive. If the environmental conditions quickly return to normal, the cells with normal mutation rates will survive and carry no extra mutations. Under normal conditions, the hypermutated cells that carry advantageous mutations will survive as well. The genetic burden carried by their progeny will be minimal due to the transient nature of their hypermutable state.

MUTAGENESIS IN AGING COLONIES (MAC) The first experiments showing that bacteria living in liquid medium and on solid surfaces can have different phenotypes were done a decade ago [78-80]. It was also shown that E. coli

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cells living on solid rich medium experience some stress and respond to it with an activated SOS system [78,79]. These studies, however, were mostly carried out on laboratory strains of bacteria, which in fact may have different physiology compared to their free-living ancestors. For instance, the majority of laboratory strains have acquired mutations, such as proAB, that allow selection on minimal medium. It was difficult to assess the evolutionary significance, physiology and genetic determinants of any of these phenomena without knowing its frequency and distribution in natural populations. In 2003, Bjedov et al. published an excellent work on stress-induced mutagenesis in aging colonies (MAC) of Escherichia coli natural isolates [81]. They analyzed 787 E. coli isolates from various environments around the world: commensal and pathogenic isolates from a variety of hosts and isolates from air, water, and sediments. To mimic stress conditions that bacteria can experience in natural environments, they used progressive starvation following an exponential growth phase occurring in colonies. For the estimation of MAC in natural E. coli isolates, the frequency of mutations conferring resistance to rifampicin in 1day- and 7-day-old colonies was measured. Rifampicin resistance is conferred by mutations only in one gene, rpoB. In order to show that mutations are not restricted to rpoB, they monitored mutations in several other genomic targets by using a lacI papillation assay [82] and looked at the mutation rate in a subset of strains growing on media supplemented with other antibiotics. Their results clearly indicated that genome-wide mutagenesis increased significantly in the majority of strains tested as a consequence of stresses cells encounter in aging colonies. It was shown that MAC was quite strong, and the high MAC variability reflected the diversity of selective pressures in ecological niches. The authors showed that MAC arose due to carbon source starvation and oxidative metabolism. MAC was controlled by the RpoS and adenosine 3',5'-monophosphate regulons. Genetic analysis showed that MAC was also dependent on the two SOS genes, recA and polB, and on the activity of the methyl-directed mismatch-repair system. It was suggested that MAC might be a by-product of genetic strategies for improving survival under stress. Using computer modeling and analysis of stress-inducible and constitutive mutagenesis patterns, it was shown that MAC could also be selected through beneficial mutations it generated. Based on the analyzed data, the authors suggested that stress-induced mutations participated in adaptive evolution regardless of the origin of their appearance [79, 80].

CONCLUSION As we can see from all above-mentioned examples, there are many responses in bacteria that can be artificially defined as stress responses. But we still believe that there is just one common stress-response mechanism which triggers various pathways depending on the type of stress stimulus. Indeed, each stress response employs various regulatory elements, proteins and low-molecular weight molecules that help bacteria survive. Nevertheless, almost all stress responses activate just a few key players, such as RecA, LexA, sigma factors, and Y-family DNA polymerases, which are responsible for long-term survival and evolution of bacteria in new environmental conditions. Some of the questions researchers are trying to answer are:

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Are these mutations induced by stress? Are they just byproducts of cell reactions? Are these mutations directed to useful changes or not? Numerous data confirm the existence of stress-induced mutagenesis in bacteria. Bjedov with colleagues [83] have shown that of all tested natural bacterial isolates of E. coli grown on agar plates approximately 40% had a 10-fold increase and 13% had a 100-fold increase in stress-induced mutant frequency. At the same time, they observed that 3% and 1% of the same isolates tested exhibited equivalently increased constitutive mutant frequencies. Computer simulations have shown that stress-induced mutations have a greater probability to be fixed than constitutive mutations, and thus they speed up adaptive evolution under particular stress conditions [83]. Also, these findings strongly suggest that stress-induced mutagenesis is widespread in nature and has a big impact on evolvability. The question that still remains unanswered is whether stress-induced mutations are directed (a ―natural genetic engineer‖ hypothesis [52]) or random. For many years various research groups have been struggling for the right model of stress-induced mutagenesis, and eventually they have agreed that mutations are not directed [84,85]. Indeed, in the case of directed mutagenesis, we may expect that the stress-response apparatus of bacteria will mostly generate useful mutations and fix them in the genome. However, it has been shown that mutations may occur at any location in the genome and can be accumulated in the population. This was confirmed in at least two experiments: Foster (1997) [84] has shown that Lac+ adaptive mutants accumulate unselected mutations in a tet gene on the F` episome, and using the lacI papillation assay, Bjedov with colleagues (2003) have shown that mutations are distributed throughout the genome [81,86]. The issue of whether mutations are directed or random is still debatable. The bacterial population consists of millions of living cells. The size of bacterial genomes varies widely, in general it is approximately 2-6 Mb. Ideally, mutations will be introduced randomly under stress conditions (according to probability theory). For instance, if one mutation is introduced during each round of replication of bacterial genomes of 6 Mb, the bacterial population of 106 cells will generate at least 106 variants. Of course, there is a chance that some cells will acquire two or more mutations, while other cells will be mutation-free. In any case, there will be plenty of cells living under selective pressure with randomly distributed mutations in their genomes. Obviously, there is quite a high probability that only those bacterial cells that acquired useful mutations will survive and multiply. Moreover, one mutated cell in the bacterial population is very likely to be enough to raise a new population.

REFERENCES [1] [2] [3] [4] [5]

Hans Selye. A Syndrome Produced by Diverse Nocuous Agents. Nature 138, 32. 7-41936. Galhardo RS, Hastings PJ, Rosenberg SM: Mutation as a stress response and the regulation of evolvability. Crit. Rev. Biochem. Mol. Biol. 2007, 42: 399-435. George J, Devoret R, Radman M: Indirect ultraviolet-reactivation of phage lambda. Proc. Natl. Acad. Sci. U S A 1974, 71: 144-147. Echols H: SOS functions, cancer and inducible evolution. Cell 1981, 25: 1-2. Gonzalez-Flecha B, Demple B: Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli. J. Biol. Chem. 1995, 270: 13681-13687.

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

100 [6] [7] [8] [9] [10]

[11]

[12]

[13]

[14]

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

[15]

[16] [17]

[18] [19]

[20]

[21]

[22]

Andrey Golubov Breimer LH: Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Mol. Carcinog 1990, 3: 188-197. Hutchinson F: Chemical changes induced in DNA by ionizing radiation. Prog. Nucleic. Acid. Res. Mol. Biol. 1985, 32: 115-154. Lindahl T: Instability and decay of the primary structure of DNA. Nature 1993, 362: 709-715. Errol C. Friedberg. Edited by Graham C. Walker and Wolfram Siede. p. 192. ASM Press, 2006. Radman M: Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis. In: L. Prakash, F. Sherman. M. W Miller, C. Lawrence, and H W Tabor (eds.). Molecular and Environmental Aspects of Mutagenesis. pp. 128-142 Springfield. III.: Charles C Thomas. Publisher, 1974. Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC: Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 2001, 158: 41-64. Tang M, Shen X, Frank EG, O'Donnell M, Woodgate R, Goodman MF: UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V. Proc. Natl. Acad. Sci. U S A 1999, 96: 8919-8924. Sanchez H, Kidane D, Reed P, Curtis FA, Cozar MC, Graumann PL et al.: The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in Bacillus subtilis. Genetics 2005, 171: 873-883. Goodman MF: Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 2002, 71: 17-50. Caron PR, Kushner SR, Grossman L: Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex. Proc. Natl. Acad. Sci. U S A 1985, 82: 4925-4929. Foster PL: Stress-induced mutagenesis in bacteria. Crit. Rev. Biochem. Mol. Biol. 2007, 42: 373-397. Bonner CA, Hays S, McEntee K, Goodman MF: DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli. Proc. Natl. Acad. Sci. U S A 1990, 87: 7663-7667. Ohmori H, Friedberg EC, Fuchs RP, Goodman MF, Hanaoka F, Hinkle D et al.: The Yfamily of DNA polymerases. Mol. Cell 2001, 8: 7-8. Tang M, Pham P, Shen X, Taylor JS, O'Donnell M, Woodgate R et al.: Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis. Nature 2000, 404: 1014-1018. Kato T, Shinoura Y: Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol. Gen. Genet. 1977, 156: 121-131. Pham P, Rangarajan S, Woodgate R, Goodman MF: Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli. Proc. Natl. Acad. Sci. U S A 2001, 98: 8350-8354. Sutton MD, Smith BT, Godoy VG, Walker GC: The SOS response: recent insights into umuDC-dependent mutagenesis and DNA damage tolerance. Annu. Rev. Genet. 2000, 34: 479-497.

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Stress-Induced Mutagenesis in Bacteria

101

[23] Lange R, Hengge-Aronis R: Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol. Microbiol. 1991, 5: 49-59. [24] Tuveson RW, Jonas RB: Genetic control of near-UV (300-400 NM) sensitivity independent of the recA gene in strains of Escherichia coli K12. Photochem. Photobiol. 1979, 30: 667-676. [25] Loewen PC, Triggs BL: Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli. J. Bacteriol. 1984, 160: 668675. [26] Sak BD, Eisenstark A, Touati D: Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product. Proc. Natl. Acad. Sci. U S A 1989, 86: 3271-3275. [27] Touati E, Dassa E, Boquet PL: Pleiotropic mutations in appR reduce pH 2.5 acid phosphatase expression and restore succinate utilisation in CRP-deficient strains of Escherichia coli. Mol. Gen. Genet. 1986, 202: 257-264. [28] Touati E, Dassa E, Dassa J, Boquet PL, Touati D: Are appR and katF the same Escherichia coli gene encoding a new sigma transcription initiation factor? Res. Microbiol. 1991, 142: 29-36. [29] Mulvey MR, Loewen PC: Nucleotide sequence of katF of Escherichia coli suggests KatF protein is a novel sigma transcription factor. Nucleic Acids Res. 1989, 17: 99799991. [30] Nguyen LH, Jensen DB, Thompson NE, Gentry DR, Burgess RR: In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product. Biochemistry 1993, 32: 11112-11117. [31] Tanaka K, Takayanagi Y, Fujita N, Ishihama A, Takahashi H: Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli. Proc. Natl. Acad. Sci. U S A 1993, 90: 3511-3515. [32] Hengge-Aronis R: Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase. Microbiol. Mol. Biol. Rev. 2002, 66: 373-95, table. [33] Weber H, Polen T, Heuveling J, Wendisch VF, Hengge R: Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J. Bacteriol. 2005, 187: 1591-1603. [34] Layton JC, Foster PL: Error-prone DNA polymerase IV is controlled by the stressresponse sigma factor, RpoS, in Escherichia coli. Mol. Microbiol. 2003, 50: 549-561. [35] Yeiser B, Pepper ED, Goodman MF, Finkel SE: SOS-induced DNA polymerases enhance long-term survival and evolutionary fitness. Proc. Natl. Acad. Sci. U S A 2002, 99: 8737-8741. [36] Tsui HC, Feng G, Winkler ME: Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12. J. Bacteriol. 1997, 179: 7476-7487. [37] Foster PL: Stress responses and genetic variation in bacteria. Mutat. Res. 2005, 569: 311. [38] Lund PA: Microbial molecular chaperones. Adv. Microb. Physiol. 2001, 44: 93-140.

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[39] Hiratsu K, Amemura M, Nashimoto H, Shinagawa H, Makino K: The rpoE gene of Escherichia coli, which encodes sigma E, is essential for bacterial growth at high temperature. J. Bacteriol. 1995, 177: 2918-2922. [40] Grossman AD, Erickson JW, Gross CA: The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell 1984, 38: 383-390. [41] Grossman AD, Straus DB, Walter WA, Gross CA: Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes. Dev. 1987, 1: 179-184. [42] Kusukawa N, Yura T: Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress. Genes. Dev. 1988, 2: 874-882. [43] Kerner MJ, Naylor DJ, Ishihama Y, Maier T, Chang HC, Stines AP et al.: Proteomewide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 2005, 122: 209-220. [44] Donnelly CE, Walker GC: groE mutants of Escherichia coli are defective in umuDCdependent UV mutagenesis. J. Bacteriol. 1989, 171: 6117-6125. [45] Liu SK, Tessman I: Error-prone SOS repair can be error-free. J. Mol. Biol. 1990, 216: 803-807. [46] Liu SK, Tessman I: groE genes affect SOS repair in Escherichia coli. J. Bacteriol. 1990, 172: 6135-6138. [47] Layton JC, Foster PL: Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli. J. Bacteriol. 2005, 187: 449-457. [48] Magnusson LU, Farewell A, Nystrom T: ppGpp: a global regulator in Escherichia coli. Trends Microbiol. 2005, 13: 236-242. [49] M.Cashel. The stringent response. 1, 1458-1496. 1996. Escherichia coli and Salmonella: Cellular and molecular biology. F.C.Neidhardt. [50] Kvint K, Farewell A, Nystrom T: RpoS-dependent promoters require guanosine tetraphosphate for induction even in the presence of high levels of sigma(s). J. Biol. Chem. 2000, 275: 14795-14798. [51] Jishage M, Kvint K, Shingler V, Nystrom T: Regulation of sigma factor competition by the alarmone ppGpp. Genes. Dev. 2002, 16: 1260-1270. [52] Cairns J, Overbaugh J, Miller S: The origin of mutants. Nature 1988, 335: 142-145. [53] Cairns J, Foster PL: Adaptive reversion of a frameshift mutation in Escherichia coli. Genetics 1991, 128: 695-701. [54] Foster PL: Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli. J. Bacteriol. 1997, 179: 1550-1554. [55] Hughes D, Andersson DI: Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates. J. Bacteriol. 1997, 179: 6688-6691. [56] Saumaa S, Tarassova K, Tark M, Tover A, Tegova R, Kivisaar M: Involvement of DNA mismatch repair in stationary-phase mutagenesis during prolonged starvation of Pseudomonas putida. DNA Repair (Amst) 2006, 5: 505-514. [57] Finkel SE: Long-term survival during stationary phase: evolution and the GASP phenotype. Nat. Rev. Microbiol. 2006, 4: 113-120. [58] Foster PL: Adaptive mutation in Escherichia coli. J. Bacteriol. 2004, 186: 4846-4852. [59] Foster PL: Rebuttal: growth under selection stimulates Lac(+) reversion (Roth and Andersson). J. Bacteriol. 2004, 186: 4861. [60] Foster PL: Rebuttal: adaptive point mutation (Rosenberg and Hastings). J. Bacteriol. 2004, 186: 4845.

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[61] Rosenberg SM, Hastings PJ: Genomes: worming into genetic instability. Nature 2004, 430: 625-626. [62] Rosenberg SM, Hastings PJ: Rebuttal: growth under selection stimulates Lac(+) reversion (Roth and Andersson). J. Bacteriol. 2004, 186: 4862-4863. [63] Rosenberg SM, Hastings PJ: Rebuttal: adaptive mutation in Escherichia coli (Foster). J. Bacteriol. 2004, 186: 4853. [64] Rosenberg SM, Hastings PJ: Adaptive point mutation and adaptive amplification pathways in the Escherichia coli Lac system: stress responses producing genetic change. J. Bacteriol. 2004, 186: 4838-4843. [65] Roth JR, Andersson DI: Adaptive mutation: how growth under selection stimulates Lac(+) reversion by increasing target copy number. J. Bacteriol. 2004, 186: 4855-4860. [66] Roth JR, Andersson DI: Rebuttal: adaptive mutation in Escherichia coli (Foster). J. Bacteriol. 2004, 186: 4854. [67] Roth JR, Andersson DI: Rebuttal: adaptive point mutation (Rosenberg and Hastings). J. Bacteriol. 2004, 186: 4844. [68] Roth JR, Andersson DI: Amplification-mutagenesis--how growth under selection contributes to the origin of genetic diversity and explains the phenomenon of adaptive mutation. Res. Microbiol. 2004, 155: 342-351. [69] Calos MP, Miller JH: Genetic and sequence analysis of frameshift mutations induced by ICR-191. J. Mol. Biol. 1981, 153: 39-64. [70] Foster PL: Population dynamics of a Lac- strain of Escherichia coli during selection for lactose utilization. Genetics 1994, 138: 253-261. [71] Foster PL, Trimarchi JM: Adaptive reversion of a frameshift mutation in Escherichia coli by simple base deletions in homopolymeric runs. Science 1994, 265: 407-409. [72] Harris RS, Longerich S, Rosenberg SM: Recombination in adaptive mutation. Science 1994, 264: 258-260. [73] Hastings PJ, Bull HJ, Klump JR, Rosenberg SM: Adaptive amplification: an inducible chromosomal instability mechanism. Cell 2000, 103: 723-731. [74] Kugelberg E, Kofoid E, Reams AB, Andersson DI, Roth JR: Multiple pathways of selected gene amplification during adaptive mutation. Proc. Natl. Acad. Sci. U S A 2006, 103: 17319-17324. [75] Torkelson J, Harris RS, Lombardo MJ, Nagendran J, Thulin C, Rosenberg SM: Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation. EMBO J. 1997, 16: 3303-3311. [76] Rosche WA, Foster PL: The role of transient hypermutators in adaptive mutation in Escherichia coli. Proc. Natl. Acad. Sci. U S A 1999, 96: 6862-6867. [77] Tompkins JD, Nelson JL, Hazel JC, Leugers SL, Stumpf JD, Foster PL: Error-prone polymerase, DNA polymerase IV, is responsible for transient hypermutation during adaptive mutation in Escherichia coli. J. Bacteriol. 2003, 185: 3469-3472. [78] Taddei F, Matic I, Radman M: cAMP-dependent SOS induction and mutagenesis in resting bacterial populations. Proc. Natl. Acad. Sci. U S A 1995, 92: 11736-11740. [79] Taddei F, Halliday JA, Matic I, Radman M: Genetic analysis of mutagenesis in aging Escherichia coli colonies. Mol. Gen. Genet. 1997, 256: 277-281. [80] Shapiro JA: The significances of bacterial colony patterns. Bioessays 1995, 17: 597607.

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[81] Bjedov I, Tenaillon O, Gerard B, Souza V, Denamur E, Radman M et al.: Stressinduced mutagenesis in bacteria. Science 2003, 300: 1404-1409. [82] Nghiem Y, Cabrera M, Cupples CG, Miller JH: The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions. Proc. Natl. Acad. Sci. U S A 1988, 85: 2709-2713. [83] Bjedov I, Tenaillon O, Gerard B, Souza V, Denamur E, Radman M et al.: Stressinduced mutagenesis in bacteria. Science 2003, 300: 1404-1409. [84] Hersh MN, Ponder RG, Hastings PJ, Rosenberg SM: Adaptive mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress. Res. Microbiol. 2004, 155: 352-359. [85] Matic I, Taddei F, Radman M: Survival versus maintenance of genetic stability: a conflict of priorities during stress. Res. Microbiol. 2004, 155: 337-341. [86] Foster PL: Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli. J. Bacteriol. 1997, 179: 1550-1554.

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Chapter 6

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) LOCI AS AN EXAMPLE OF BACTERIAL ADAPTIVE IMMUNITY AGAINST PHAGES Igor Kovalchuk and Andrey Golubov Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, CANADA

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ABSTRACT Bacteria and archaea lacking sexual reproduction need to use a variety of mechanisms for gene exchange/gene acquisition. These mechanisms of horizontal gene transfer (HGT) in bacteria and archaea include phage transduction, transformation, and conjugation. At the same time, bacteria require protection against their ancient enemies, phages. One of the most interesting mechanisms of protection is based on the function of regulatory RNAs encoded by clustered regularly interspaced short palindromic repeat (CRISPR) loci to confer sequence-directed immunity against phages. According to recent publications, this is an active protection mechanism by which prokaryotes integrate short fragments of phage/viral nucleic acids into clusters of CRISPRs. As such, this process represents an ancient adaptive mechanism of protection against pathogens.

VARIOUS GROUPS OF REGULATORY RNAS IN PROKARYOTES Bacteria contain a number of regulatory RNAs, often called small RNA (sRNA) or noncoding RNA since the vast majority of them do not encode any protein [1, 2]. They can be broadly classified into those that act in cis, being part of mRNAs they regulate, and those that act in trans, having their own dedicated promoters and terminators. Regulatory RNAs can influence transcription, translation, mRNA stability and can be involved in silencing [1]. Riboswitches represent the simplest class of bacterial regulatory elements that consists of the sequence at the 5‘ end of mRNAs. Riboswitches function by

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changing the conformation that leads to changes in gene expression. These conformational changes often depend on the presence of various ligand molecules (including various metabolites), stalled ribosomes, and shifts in temperature [1, 3]. Another group of sRNAs consists of sRNAs that modulates protein activity. Three protein-binding sRNAs antagonize the activity of their cognate proteins. CsrB, 6S and GlmY sRNAs are able to mimic the structure of other nucleic acids and compete for protein binding [1]. These processes are often activated by poor nutrition conditions, they are also known to influence quorum sensing and secondary metabolism [4, 5]. Two groups of sRNAs that bind mRNA consist of cis- and trans-acting small RNAs. The first group of cis-encoded sRNAs represents small RNAs located on the strand opposite the target RNA which have perfect or nearly perfect complemantarity [6, 7]. The majority of these sRNAs are expressed from plasmid, bacteriophages, and transposons and are believed to regulate the copy number of these elements in the bacterial cell [7]. The second group consists of trans-encoded sRNAs in bacteria that are similar in function to eukaryotic microRNAs (miRNAs); these sRNAs have partial complemantarity to the target mRNA and function through either inhibition of translation or cleavage of their target mRNAs [8]. Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs are perhaps the most unique class of regulatory sRNAs. CRISPR loci consist of an array of short direct repeats that are interrupted by non-repetitive space sequences. Typically, upstream of these loci are the CRISPR-associated (CAS) genes that can also be located at substantial genomic distance. Repeats can vary in length from 24 to 47 nt and can be repeated up to 250 times [9]. It is believed that CRISPR function is associated with bacterial immunity, namely protection against invading phages. In this respect, CRISPR RNAs share certain functional similarities with eukaryotic small interfering RNAs (siRNAs) in silencing foreign RNAs. Recent data show that CRISPR loci are highly unstable and acquire new genetic material upon exposure to phages. This phenomenon is particularly relevant to stress adaptation and genome evolution, and thus it is the topic of this review.

THE ROLE OF CRISPR LOCI IN PROTECTION AGAINST PHAGES CRISPR arrays were first reported by Ishino et al. (1987); they found a group of 14 29bp-long repeats separated by 32-33 bp of non-repetitive elements in E. coli. Similar arrays were later on found in Thermotoga maritima [10], Methanocaldococcus jannaschii [11], Haloferax mediterranei [12], Mycobacterium tuberculosis, [13] and other bacteria. It is believed that 40% of bacteria and 90% of archaea contain these elements [9]. As it has been mentioned above, CRISPR loci contain 24-47 bp repeat units, alternated by unique 27-72 bp spacer sequences, and they are almost always accompanied by CAS genes reported originally by Jansen et al. (2002) [14]. Further analysis identified 25-45 additional CAS genes in proximity to the CRISPR arrays [15-17]. Several CAS genes are typically associated with CRISPR loci, and those of them that do not contain these arrays normally do not contain CAS genes. CRISPR loci are classified into 12 major groups, CRISPR-1 to CRISPR-12, based on sequence similarities [18]. The arrays of the same repeats are sometimes followed by a ATrich sequence, referred to as a leader [14, 19]. In the arrays containing degenerated terminal

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repeats, the leader is often located upstream of the CRISPR locus. The role of leaders is not clear, but it is possible that they promote transcription through repeats [20, 21]. Organization and processing CRISPR loci is depicted in Figure 1.

Figure 1. Organization and processing of the CRISPR elements. CRYSPR loci consist of inverted repeats, ―R‖ and spacers, ―S‖ preceeded by the leader sequence. CRYSPR loci contain genes of the cascade complex, Cas genes. CRISPR-Cas system presumably functions through RNAi mechanism. Transcribed pre-crRNAs are processed via cascade complex. Processed crRNAs are presumably blocking the bacteriphage translation process.

The function of CRISPRs remained unknown for a long time. Shortly after being discovered, CRISPRs were suggested to participate in replicon partitioning [12], thermal adaptation [22], DNA repair [15] and transposition [14]. In 2005, several groups discovered that spacer sequences consisted of phage- or plasmid-derived DNA, and thus it was suggested that CRISPRs were involved in bacterial immunity against invading foreign DNA molecules [23-25]. An indirect confirmation of this hypothesis was the discovery of correlation between the number of these repeats and bacteria tolerance to phage infection [25].

THE PROPOSED FUNCTION OF CRISPR LOCI CRISPR limits horizontal gene transfer by targeting DNA (discussed in [26]). Based on computational analysis, Makarova and colleagues proposed that the CRISPRCas system (CASS) is a prokaryotic system of defence against phages and plasmids that function via the RNAi mechanism [16]. They further suggested that the function of this system involves integration of fragments of foreign genes into bacterial and archael genomes resulting in heritable immunity to the respective agents [16].

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Brouns and colleagues (2008) showed how virus-derived sequences contained in CRISPRs are used by Cas proteins from the host to mediate an antiviral response that counteracts infection [20]. It was shown that a complex of five Cas proteins, CasA, CasB, CasC, CasD and CasE, collectively termed Cascade cleaves a CRISPR RNA pre-crRNA precursor in each repeat and retains cleavage products containing virus-derived sequences (Figure 1). These mature crRNAs consist of the antiviral spacer unit flanked by short RNA sequences derived from the repeat on either side termed the 5‘ and 3‘ handle. It is possible that these ―handles‖ serve as conserved binding sites for Cascade proteins [18, 20]. The helicase Cas3 assists these mature crRNAs to serve as guide RNAs that direct the complex to viral RNA and interfere with virus proliferation (Figure 1). This hypothesis comes from the observation that anti- CRISPRs of both polarities lead to a reduction of bacteria sensitivity to phages. This model is supported by previous reports showing that virus-derived sequences integrate into CRISPR loci in either orientation [16, 2325, 27]. CRISPR-CAS have several analogies with the eukaryotic RNAi systems, particularly with piwi-interacting RNAs (piRNAs) [28]. Like prokaryotic repeats, piRNAs are arranged into a limited number of loci in the genome. Precursors of piRNAs are long single-stranded RNA molecules that are cleaved to produce piRNAs involved in silencing [29]. Similarly, it has been shown that CRISPR loci are transcribed into a single molecule which is further processed into smaller discrete RNAs, each of which is the size of a repeat-spacer unit [20, 30].

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INCORPORATION OF NEW SEQUENCES INTO CRISPR LOCI The incorporation of new sequences into CRISPR loci was suggested by several groups. Pourcel et al. (2005) analyzed CRISPR sequences of various Yersinia pestis strains and hypothesized that these bacteria acquired new spacers very recently [24]. Mojica and colleagues suggested that CRISPR spacers derive from pre-existing sequences such as chromosomal sequences or those within transmissible genetic elements (bacteriophages or conjugative plasmids) [23]. At the same time, the authors suggested that bacteriophages fail to infect strains carrying specific spacers, suggesting the relationship between CRISPR and immunity against targeted DNA [23]. In 2007, it was shown that Streptococcus thermophilus bacterial strains carrying CRISPR became resistant to phage infection after acquiring new spacers derived from the virus [31]. Barrangou and colleagues used the S. thermophilus strain DGCC7710 and the virulent pac-type phages 2972 and 858 to show that CRISPRs play a role in the development of bacteriophage-insensitive mutants (BIMs) [31]. Specifically, they found that in response to infection with phages 858 or 2972, the aforementioned bacterial strain integrates new spacers derived from the phage genome generating a phage-resistant phenotype. The specificity of the resistance was determined by the identity between newly acquired spacers and phage sequences [31]. Curiously, the small population of phages was able to infect BIM cells, thus suggesting that both CRISPR loci and phage genomic regions are subject to rapid evolutionary changes [31]. More recent data revealed the decreased sensitivity to  phage of E. coli strains carrying artificial CRISPR systems against essential viral genes [20].

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Acquisition of phage sequences can be either a random or a directed process. It could be hypothesized that the CRISPR-leader system is capable to recognize the ‗to be spacer sequence‘. This sequence was in past referred to as the proto-spacer [27]. Proto-spacers might have been derived only from dsDNA phages which have no RNA stage (for example, phages 858 or 2972). Also, we cannot exclude the probability of phage integration into bacterial chromosomes (lysogeny) as an initial step of CRISPR formation. In this case, even RNA phages can be a source of CRIPSR. It would be very interesting to investigate in detail the dependence of CRIPSR formation on different types of bacteriophages (DNA or RNA phages). Mojica et al. (2009) explored the possibility that proto-spacers contain special motifs, termed PAMs [32]. They analyzed several groups of phage sequences containing homology to CRISPR and found the conservation of di- and trinucleotides, starting immediately or one position after the proto-spacer. Computational analysis performed by Mojica et al. (2009) revealed an interesting relationship between CRISPR and spacer/PAM orientation. Spacer ends equivalent to the proto-spacer edges adjacent to PAM (referred to as PAME, proto-spacer adjacent motif end) are oriented towards the leader irrespective of the location of the CAS genes. The conservation of the orientation of spacers defined by the PAME, with respect to both repeats and the leader, together with the preferential incorporation of new spacers at the leader‘s end of CRISPR arrays [27, 31], suggests that CRISPR and the leader could participate in PAM recognition. The proto-spacers are then base-paired with CRISPR and inserted through the recombination process [32]. This process results in acquisition of new spacers and potentially new phage-resistant bacterial strains (Figure 2).

Figure 2. Acquisition of new spacer into array of the CRISPR elements. Acquisition of new spacer elements into CRISPR may occur though recombination between the areas of homology in bacteriophage and CRISPR loci.

CONCLUSION Incorporation of new sequences into the bacterial genome is not a rare process, as horizontal gene transfer in bacteria occurs frequently during transformation, transduction and conjugation. The process of acquisition of new spacer elements when bacteria choose the

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sequence to pick and integrate is quite an amazing biological phenomenon. It suggests that organisms as simple as bacteria are able to adapt to phage infection by modifying their genome in a directed manner. These new sequences are taken directly from the phage genome and are used for sRNA-mediated inhibition of translation of phage RNAs during the process of infection. This is sufficient to substantially slow down the infection process so that bacteria become tolerant to phage progression. A certain percentage of phages can quickly become infectious, suggesting that phages can modify their genomes very efficiently to avoid recognition. The fact that phages are able to adjust almost immediately suggests a constant adaptive arms race between bacteria and phages.

REFERENCES [1] [2] [3] [4]

[5] [6]

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[7] [8] [9] [10]

[11]

[12]

[13]

Waters LS, Storz G: Regulatory RNAs in bacteria. Cell 2009, 136:615-628. Novick RP, Geisinger E: Quorum sensing in staphylococci. Annu. Rev. Genet. 2008, 42:541-564. Montange RK, Batey RT: Riboswitches: emerging themes in RNA structure and function. Annu. Rev. Biophys. 2008, 37:117-133. Lapouge K, Schubert M, Allain FH, Haas D: Gac/Rsm signal transduction pathway of gamma-proteobacteria: from RNA recognition to regulation of social behaviour. Mol. Microbiol. 2008, 67:241-253. Babitzke P, Romeo T: CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr. Opin. Microbiol. 2007, 10:156-163. Brantl S: Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr. Opin. Microbiol. 2007, 10:102-109. Wagner EG, Altuvia S, Romby P: Antisense RNAs in bacteria and their genetic elements. Adv. Genet. 2002, 46:361-398. Aiba H: Mechanism of RNA silencing by Hfq-binding small RNAs. Curr. Opin. Microbiol. 2007, 10:134-139. Sorek R, Kunin V, Hugenholtz P: CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat. Rev. Microbiol. 2008, 6:181-186. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, et al: Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 1999, 399:323-329. Bult CJ, White O, Olsen GJ, Zhou L, Fleischmann RD, Sutton GG, Blake JA, FitzGerald LM, Clayton RA, Gocayne JD, et al: Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 1996, 273:1058-1073. Mojica FJ, Ferrer C, Juez G, Rodriguez-Valera F: Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol. Microbiol. 1995, 17:85-93. Hermans PW, van Soolingen D, Bik EM, de Haas PE, Dale JW, van Embden JD: Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Loci…

[14] [15]

[16]

[17]

[18] [19]

[20]

[21]

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[22] [23]

[24]

[25]

[26] [27]

[28] [29]

111

integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect. Immun. 1991, 59:2695-2705. Jansen R, Embden JD, Gaastra W, Schouls LM: Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 2002, 43:1565-1575. Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV: A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res. 2002, 30:482-496. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV: A putative RNAinterference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 2006, 1:7. Haft DH, Selengut J, Mongodin EF, Nelson KE: A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 2005, 1:e60. Kunin V, Sorek R, Hugenholtz P: Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 2007, 8:R61. Mojica FJ, Diez-Villasenor C, Soria E, Juez G: Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. 2000, 36:244-246. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J: Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008, 321:960-964. Willkomm DK, Minnerup J, Huttenhofer A, Hartmann RK: Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog. Nucleic Acids Res. 2005, 33:1949-1960. Riehle MM, Bennett AF, Long AD: Genetic architecture of thermal adaptation in Escherichia coli. Proc. Natl. Acad. Sci. U S A 2001, 98:525-530. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E: Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 2005, 60:174-182. Pourcel C, Salvignol G, Vergnaud G: CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005, 151:653-663. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD: Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 2005, 151:2551-2561. Marraffini LA, Sontheimer EJ: CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008, 322:1843-1845. Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S: Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 2008, 190:1390-1400. Kawaji H, Hayashizaki Y: Exploration of small RNAs. PLoS Genet. 2008, 4:e22. Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Hannon GJ: Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 2007, 128:1089-1103.

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[30] Tang TH, Polacek N, Zywicki M, Huber H, Brugger K, Garrett R, Bachellerie JP, Huttenhofer A: Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus. Mol. Microbiol. 2005, 55:469-481. [31] Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P: CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007, 315:1709-1712. [32] Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C: Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 2009, 155:733-740.

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Chapter 7

WHITE TAIL TIP INHERITANCE IN MICE: FROM THE “KIT LOCUS” TO A NEW MODE OF HEREDITY Minoo Rassoulzadegan Inserm, U636, F-06108 Nice, France. Université de Nice-Sophia Antipolis, Laboratoire de Génétique du Développement Normal et Pathologique, F-06108 Nice, France

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ABSTRACT In contrast with a wide definition of ‗epigenetic variation‘, including all changes in gene expression that do not result from the alteration of gene structure, a more restricted class has been defined, initially in plants, under the name ‗paramutation‘. It corresponds to epigenetic modifications distinct from regulatory interactions of cell differentiation pathways, which are meiotically stable and sexually transmitted by non-Mendelian ratios. This class of epigenetic changes appeared for some time restricted to the plant world, but examples of epigenetic inheritance progressively accumulate in organisms ranging from mice to humans. Occurrence and possible mechanisms of paramutation in the mouse were first established with a tail color alteration in the paradigmatic case of a mutant phenotype maintained and hereditarily transmitted by genotypically wild-type homozygotes. Studies in the mouse point to a new role of RNA as an inducer and hereditary determinant of epigenetic variation. Given the known presence of a wide range of RNAs in human spermatozoa as well as a number of unexplained cases of familial disease predisposition and transgenerational maintenance, one may consider a role of RNA-mediated inheritance in mammals. A possible involvement in evolutionary processes makes for interesting speculations.

Keywords: Hereditary RNA, epigenetic modification, paramutation, mice, sperm RNA, c-kit, tail color.

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INTRODUCTION: AN OVERVIEW The main rules of genetic inheritance have been established since Mendel. Because these modes of transmission from one generation to the next have been largely confirmed, we are all very much aware of their importance. Knowledge of these robust rules is fundamental in order to follow the inheritance of any particular trait but also in order to be able to explain any deviation from the rule involving such a trait. The rules of non-Mendelian heredity are not yet known, and this makes investigators uncomfortable and sometimes suspicious of its very existence, leading them to wonder how these rules operate and what mechanisms are involved. There are, in fact, endless possibilities. It is still a difficult matter to know how and where to start. Variation is an intrinsic property of the living, from the egg to the embryo and the adult. This is not new; it was already largely discussed by biologists in the past. Variations in gene expression and in phenotypes of parents and progeny have also been reported; however key experiments that convince the public of the reality of epigenetic inheritance are missing. Now it is becoming more and more clear that Mendelian genetics does not provide an explanation for all variations and changes in the living world. It is necessary to construct, in parallel with conventional genetics, the basis for non-Mendelian variations. The term ‗epigenetic‘ is currently used to cover a wide range of modifications at different cellular levels, including differentiation processes from the egg to somatic and germ cells. One could ask whether mechanisms of cellular differentiation [1] are to be considered as ‗epigenetic‘ and whether they overlap with epigenetic inheritance [2]. At this stage, we do not know. In addition, adult phenotype can also be affected by epigenetic events in the early embryo [3]. Until now, epigenetic inheritance was studied mostly in plants, in which ‗paramutation‘ was discovered by R. A Brink [4]. He referred to paramutation as an interaction between two alleles leading to heritable changes of one allele influenced by another allele. The basic observation in plants is of non-Mendelian transmission of color, with important variations in a visible phenotype. Fifty years later, epigenetic inheritance in an animal model, similar in some respects to paramutation in plants [5], was made possible by the observation of nonMendelian segregation of a visible ‗coat color‘ marker. In both cases in plants and in mice, these phenomena were referred to as paramutations, but these processes differ at the molecular level, and they do not necessarily follow the same rules in transmission of traits. Any extrapolation from one model (plant) to other (mice) still leaves many matters to be considered. Brink [6] used the term paramutation instead of epigenetic inheritance to describe the case of BI locus inheritance in maize. Why was a new term necessary? Since Brink first used it, the term has been continuously used when referring to non-Mendelian inheritance rather than epigenetic inheritance. It allows us to identify properly this biological phenomenon and to distinguish it from any epigenetic modification limited to somatic cells. Paramutation thus is a consequence of a ―cross-talk‖ and exchange of information between chromosomes using still unknown mechanisms [7] [8]. The result of these exchanges is a modification of an allele at a given locus. This is normally initiated by interaction in heterozygotes of a ‗paramutable‘ allele with a ‗paramutagenic‘ form of the allele. It is absolutely necessary to identify the ‗paramutable‘ allele when a heritable epigenetic change of phenotype is observed. Some degree of variation in the progenies of heterozygote

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organisms constitutes an exception to the first law of Mendel, which states that genetic factors segregate unchanged from heterozygotes. Paramutation, first discovered in maize as a case of inheritance from a heterozygote and observed by Brink, has been extended to additional loci in plants (for review see [9]). It is now established that it occurs without a change in the primary DNA sequence of the affected locus [10]. In the case of the BI locus, it is a consequence of methylation of a repeated motif far upstream of the B locus [7]. Genetic proofs indicate that this motif is playing a central role in setting DNA methylation. In mice, white tail color of mutant heterozygotes of the Kit locus was maintained in the genetically wild-type progeny in clear contradiction with the first law of Mendel which states that alleles are inherited unchanged from heterozygotes. Following the tail tip color phenotype, we found that wild-type progenies born in non-Mendelian ratios from a heterozygote had changed by expressing the white tail tip mutant phenotype [5]. Reduced pigmentation of tail tip was shown to correlate with a defect in expression of the Kit protein during development [11, 12]. Modification of Kit gene expression observed in our work makes it a reference locus for epigenetic inheritance. We have not found any modification of the primary DNA sequence of the Kit locus so far. This is in contrast to paramutation in plants where changes in DNA methylation have been observed [13]. However, we have found a determinant role of RNA molecules. Our observations have led us to propose that hereditary transmission of epigenetic modifications is also active in the animal kingdom. Inheritance of epigenetic variations often involves subtle changes which may only be detected by duly alerted observers. These variations could appear unpredictably and to varying degrees. An additional degree of complexity of paramutation phenomena is due to the fact that the phenotypes often show quantitative variations between individuals in the same families, even in the same litters. These quantitative variations made authors in the plant field describe paramutation as a ‗rheostat‘ mechanism [14] rather than an ‗on-off switch‘ model. Importantly, our ignorance of modes of inducing the variation and rules of its transmission creates even more mystery surrounding epigenetic inheritance. The activity of the Kit gene provided us with an easy and powerful test for inheritance just by looking at tail color variations [15, 16]. The main aim of this assay is to focus on the Kit locus and also to attract the attention of readers and breeders to the possible importance of epigenetic heredity. Other examples have been identified [17], and it is clear that a number of genes can be targets of epigenetic modifications. Fortunately, the ‗Kit* paramutation‘ generates a visible phenotype amplified in certain heterozygote configurations. Given the expected variety of such changes, it may be in some cases difficult to identify the modified gene(s). These modifications are broad, and there is constant ongoing pressure to induce epigenetic variances. Vast molecular changes should be involved, and it is not yet clear what determinant modifications and original target molecules are. Accordingly, it is only in a genetically well-defined model that it is possible to conclude that this is an epigenetic cause. We have recently reported the case of hereditary transmission of an epigenetic change in the expression of the Kit gene of the mouse [5]. In fact, when a mutant with the white tail tip phenotype characteristic of the heterozygote for Kit tm1Alf/+ crosses with a wild-type partner, the homozygous wild-type progenies obtained inherit and

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express to some extent the phenotype of heterozygous mutants. Thus the wild-type progenies obtained from a heterozygote become a variance of wild-type parent animals. In the mouse, the initial observation was based on a dramatic departure from the Mendelian distribution in the progeny of crosses between heterozygotes carrying the wild type Kit allele together with a null allele (Kit tm1Alf). Most of the progeny genotyped as wild type Kit +/+ homozygotes maintained (with some degree of quantitative variation) and transmitted to their progenies the mutant phenotype of a heterozygous parent, namely, the characteristic patches of white fur on the tail and feet of the mouse. We know that a variable phenotypic extent depends on the level of Kit expression, making it a useful tool for the geneticist. In Kit tm1Alf/+ intercrosses, the Kit +/+ genotypes were generated with the expected Mendelian frequency. A majority of them, however, maintained and, most importantly, transmitted to their progeny the mutant phenotype. The same observation was made in the progeny of crosses of either male or female heterozygotes with wild-type partners. Whenever the Kit + allele had been confronted with the tm1Alf mutant, its expression was modified in a manner stable enough to persist in the following generations. It returned to normal only after more than two generations of out-bred crosses, a reversibility that would not be compatible with a classical mutational event. Following the nomenclature of plant genetics, a heritable epigenetic modification was referred to as paramutation and designated by the Kit* symbol. A reduced expression of Kit mRNA in Kit* animals was apparent due to post-transcriptional RNA cleavage. At the same time, however, transcriptional activity was increased especially during the terminal stages of spermatogenic differentiation when the gene is normally silent. In this chapter we will discuss our experiments focusing on the previously reported c-kit locus.

PHENOTYPIC DESCRIPTION OF EPIGENETIC INHERITANCE FROM KIT PARAMUTANTS Coat color is one of the oldest phenotypic traits used by both farmers and researchers in domestic selection of a variety from a given species. In mice, mutations at the W locus in the heterozygote leads to a visible phenotype known as the ―White spotting‖ phenotype [15, 16]. This murine white spotting locus is allelic with the proto-oncogene c-kit which encodes the transmembrane tyrosine protein kinase receptor for the Kit ligand. However, in addition to the coat color defect, the W locus on chromosome 5 leads to multiple developmental defects, including sterility and anemia. The defects in all these lineages are cell autonomous and intrinsic, leading to varying degrees of severity during development. The size and area of the ―White spotting‖ expression coat color phenotype is truly variable from one W mutant to another and depends on the strain of mice. However, a defect in pigmentation visible in the tail tip is a common character trait of W mutants. It is inherited and is visible on the tail in each generation as early as one week old. A spontaneous short white tail tip occurs infrequently in laboratory mice, on average in only 1 to 2% of mice. However, it is not systematically transmitted to the next generation, and the white portion is light and generally short. In the present study, we will discuss cases of transmission of a white tail from W

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mutants that contain deletion in the first exon of the c-kit gene. The genotype of this mutant is Kit tm1Alf/+ [18]. Figure 1 shows the picture of a two month old Kit tm1Alf/+ mutant mouse of the C57BL/6 background. Here, we are interested in the expression of the adult tail color ‗white tail tip‘ characteristic caused by a defect in the migration of melanocyte progenitors responsible for pigmentation. The white tail tip is well delimitated and visible in contrast to the black full tail color tip of the wild-type mouse. Three independent strains of heterozygote Kit tm1Alf/+ are routinely maintained by crosses with a wild-type partner C57Black/6, B6/D2 or 129/sv. Heterozygote progenies obtained from these crosses are currently transmitting with the Mendelian ratio the white tail tip phenotype (Table 1). In contrast, we start to observe a deviation from Mendelian laws if the cross occurs with heterozygotes obtained from the Kit tm1Alf/+ mutant inter-crosses as shown in Table 2. As expected, homozygous Kit tm1Alf/tm1Alf mice are dead at birth and are not integrated into these studies. However, surprisingly, a phenotypic analysis of 3- to 6weeks-old mice shows that the majority of progenies with wild-type genotype mimic to certain degree the phenotype of the heterozygote parents with a short but bright white tail tip (Table 2; Figure 2). These mice with the wild-type genotype at the c-kit locus are referred to as Kit* (Figure 2).

Figure 1. Tail tip pigmentation phenotype of wild type and heterozygote Kit mutant Comparison of tail phenotypes: genetically Kit+/+ wild type laboratory mouse (full tail color) and heterozygote KittmlAlf1/+ paramutant / paramutant KittmlAlf1/+ heterozygote / KittmlAlf1/+ heterozygote paramutant (white tail), both mice are of 2 months old.

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Table 1. Mendelian segregation of genotypes and white tail tip phenotypes in the progeny of Kit heterozygotes crossed with wild type partners Heterozygote KittmlAlf1/+ animals that are maintained in crosses with wild type partners transmit, as expected, Mendelian ratios of the white tail tip phenotype. Kit* paramutants are observed rarely. § “het”: Kit tm1Alf/+; “wsp”: white- spotted; “full”: full color. $ Genotype: lacZ determination by genomic PCR determination of tail DNA and expression by the X-gal assay on tail Crosses§

Progeny Tail Number of Male Female phenotype LacZ$ mice Class Kit het Kit +/+ wsp + 31 Kit het wsp 1 Kit* full 33 Kit +/+ Kit +/+ Kit het 9 wsp + 43 Kit het wsp 2 Kit* full 51 Kit +/+ § tm1Alf/+ ―het‖: Kit ; ―wsp‖: white-spotted; ―full‖: full color $ Genotype: lacZ determination by genomic PCR on tail DNA and by analysis of lacZ activity using the X-gal assay on tail tissues. B6D2 genetic background. Number of litters 7

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Table 2. Mendelian segregation of genotypes and non-Mendelian segregation of white tail tip phenotypes in the progeny of paramutated Kit heterozygotes Only one intercross between KittmlAlf1/+ heterozygote is sufficient to produce Kit* paramutants even in crosses with wild type partners Crosses§ Number of litters 13

Male Kit het (1)

Female Kit het (1)

Kit het Progeny from breeding (1)

Kit het Progeny from breeding (1)

6

Kit het

Kit +/+

10

Progeny Tail phenotype wsp wsp full wsp wsp

LacZ$ + + -

Number of mice 43 54 11 23 19

Class Kit het Kit* Kit +/+ Kit het Kit*

full

-

5

Kit +/+

wsp + 38 Kit het wsp 32 Kit* full 9 Kit +/+ Kit +/+ Kit het 11 wsp + 49 Kit het wsp 36 Kit* full 11 Kit +/+ § ―het‖: Kit tm1Alf/+; ―wsp‖: white-spotted; ―full‖: full color $ Genotype: lacZ determination by genomic PCR on tail DNA and by analysis of lacZ activity using the X-gal assay on tail tissues. B6D2 genetic background.

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Figure 2. Tail tip pigmentation phenotype of heterozygote Kit and Kit* paramutant Comparison of the length of the white tail tip: heterozygote KittmlAlf1/+ and the first generation of Kit* paramutant (both mice are of the same age).

The white tail tip in Kit* is shorter than in the Kit tm1Alf/+ mutant, but the length of the white tail could increase in the progeny; an increase in the length of the tail depends on the number of inter-crosses between Kit*and Kit tm1Alf/+. The frequency of occurrence of the Kit* genotype in the progeny is very high (close to 40%) (see Table 2). This was not expected. The reduced number of heterozygote Kit tm1Alf/+ mutant is not apparently due to a given stage of development. A large variability in tail tip colors and in defects at other stages of development was noticed. Interestingly, with regard to the phenotype and genotype of each cross, the remaining ~12% of animals are perfect wild types with a full black tail color.

FLEXIBLE PHENOTYPIC EPIGENETIC INHERITANCE Kit* paramutants are obtained from crosses of Kit tm1Alf/+; they have quantitative variation in tail length and a strong white tail tip phenotype. These Kit* paramutant mice transmit efficiently the same phenotype to their progenies at an average frequency of 80% to 90%. The white tail phenotype is then transmitted again to the next generation with variable frequencies, from one individual to another and with a variable level of expression. Subsequently, in the following generation, these phenotypic changes eventually disappear. In contrast, an intercross of Kit* x Kit* results in the maintenance of the white tail tip phenotype through a number of generations with 90% efficiency (three years currently).

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Kit tm1Alf/+ mutant mice back-crossed to C57BL/6 parents maintain and transmit the white tail phenotype according to Mendelian rules as expected (see Table 1), with incidences of Kit* parmutants being very low. By contrast, only one cross with a heterozygote Kit tm1Alf/+ mutant is enough to induce and obtain in the progeny the wild-type genotype with phenotypic changes of tail tip color (paramutants designated by the Kit* symbol) (Table 2 and Figure 3). Furthermore, each intercross of the heterozygote Kit tm1Alf/+ mutants increases the frequency, efficiency and extent of paramutation (see Table 2 and Figure 4). Finally, beyond six intercrosses, the whiteness of coat color extends to the rest of the body of mutants (not shown).

Figure 3. Scheme for generation of Kit paramutants. An expected Mendelian ratio of white tail tip phenotype is observed by maintaining crosses of female or male KittmlAlf1/+ heterozygote mutants with a wild type partner (two first lines). However, one round intercross between heterozygote KittmlAlf1/+ mutants (third line) is sufficient to produce Kit* paramutants. Then, all progenies from these crosses are able in their turn to transmit a paramutated phenotype (white tail tip) to the next generation, even in crosses with the wild type partner (line four). More crosses (at least three) between KittmlAlf1/+ mutants increase the length of white color of the tail. The last line shows the white tail phenotype of progeny obtained after three rounds of intercrosses between KittmlAlf1/+ mutants.

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Figure 4. Paramutation at the Kit locus. Heterozygote KittmlAlf1/+ mutants KittmlAlf1/+ heterozygote mutants obtained from intercrosses between heterozygote KittmlAlf1/+ mutants transmit the white tail phenotype to most of their progenies even in crosses with wild type partners.

On the other hand, we observe a rapid reversibility of the white tail tip phenotype in two or three generations by backcrossing to wild-type animals in contrast to an additive increase of the white tail tip phenotype with successive crosses between Kit tm1Alf/+ mutants. These differences in phenotypic appearance underline epigenetic determinism of inheritance. Flexible epigenetic inheritance, in contrast to ‗rigid‘ genetic inheritance, opens up a new field of research that allows the analysis of phenotypic variability. The so-called ―determinant‖ is apparently sensitive to the passage from one generation to another, as if it undergoes dilution, attenuation and amplification, depending on genotypes used for crosses.

RNAS ARE INVOLVED IN PHENOTYPIC EPIGENETIC INHERITANCE Female and male Kit tm1Alf/+ mutants transmit the paramutated white spotted phenotype with indistinguishable frequency. So the ‗determinant‘ is acting in the male and in the female

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germ line. Thus in order to identify the molecular basis of this phenomenon, we could use either sperm or ovum cells. Since sperm cells are more abundant and less complex than oocytes, we decided to use sperm cells for further experiments. The main question was the nature of a signal inducing phenotypic modifications after fertilization of a normal egg. Sperm has one haploid copy of the genome required for fertilization and formation of the zygote. Searching for a distinct basis of epigenetic information, we considered RNA as a possible candidate. We hypothesized that sperm RNA could act as a signal for the transgenerational maintenance of epigenetic changes. Our studies analyzed RNA content in the sperm and searched for the white tail tip phenotype induced by a specific RNA after microinjection into fertilized mouse eggs as a key molecule in epigenetic inheritance or ‗paramutation‘. RNA extraction from a purified sperm population was performed from heterozygotes Kit tmlAlf1/+, Kit* paramutants and wild types to initiate key molecular events. Independent RNA preparation from sperm tested by PCR for c-kit transcripts systematically revealed higher amounts of transcripts from and covering the whole c-kit locus mRNA in the sperm of male paramutants. Analysis of mRNA showed that the c-kit transcripts consist of only fully spliced RNA (not shown). In addition to the analysis of c-kit mRNA accumulation, we have performed staining of total RNA of sperm nuclei. This latter technique known as the EDTA regressive staining method [19] and coupled with electron microscopy (EM) allowed us to perform a visualization of total RNA in the sections of cells. The staining protocol of EM samples starts first with uranyl acetate and is followed by several washing steps with EDTA. The EDTA washing process removes all signals from the organelles, the cytoplasm and nuclei, leaving RNA molecules intact. Cytological analysis of the sections of spermatozoa heads washed with EDTA for RNA content appeared to be very informative. In fact, it demonstrated (Figure 6) that there were greater amounts of RNA staining in the sperm of Kit tmlAlf1/+ heterozygotes and Kit* paramutants than in the spermatozoa of normal mice (‗normal‘ referring to congenic strains maintained in the laboratory). This strong RNA staining signal observed in the sperm of Kit tmlAlf1/+ mutants versus the wild-type laboratory mouse strain indicates that the sperm head, in addition to the c-kit mRNA, is filled with RNA. This allowed us to suggest that an accumulation of RNA in the head of sperm could act as a signal for the transgenerational maintenance of epigenetic changes. Further studies on germ cell development and accumulation of RNA in the sperm are required in order to learn more about sequence specificity of these transcripts. Now deep sequencing technologies make it feasible to compare the RNA population of paramutants and RNA from wild-type animals. To confirm the impact of a specific RNA on the modification of a given trait and subsequently on its transmission to the next generation, we have established a biological test based on microinjection of RNA into normal fertilized mouse zygotes. Microinjection of total RNA prepared from various tissues (brain, testes, sperm) of Kit tmlAlf1/+ mutants, Kit*, wildtype mice into fertilized zygotes did not interfere with a normal birth and did not produce any gross signs of developmental arrest. On average, a 50% birth rate is obtained after RNA microinjection. About one-half of the mice born from the manipulated eggs with RNA from heterozygotes demonstrated the white tail phenotype and efficiently transmitted it to their progeny. Microinjection of RNA samples extracted either from sperm or somatic tissues (brain) was efficient at inducing paramutation. Furthermore, differences observed between RNA from both male and female somatic tissues (brain) were not significant. In contrast, upon microinjection of RNA, samples from wild-type tissues and/or irrelevant oligo RNA

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were significantly different and inefficient in generating the white tail phenotype in comparison with the RNA samples from the mutants. Founder paramutant animals with a white tail tip born after RNA microinjection transmitted efficiently to their progeny. Paramutants maintain the white tail tip phenotype during their whole life time, and subsequently the F1 generation transmits the phenotype efficiently to the next generation independently of gender. After two-to-three generations of back cross to a wild-type parent, expression of the white tail tip phenotype is lost, it drops to the level of control animals. However, an inter-cross between F1 paramutants maintains and transmits the phenotype over three years (as followed in our laboratory). After microinjection of RNA samples derived from wild type tissues and/or irrelevant oligo RNAs, white tail tip mice are sporadically born at low frequencies. These animals transmit the phenotype to the next generation at the same low sporadic level. Concomitantly we have checked whether Kit-related RNA would also act as an inducer of paramutation because we have observed an accumulation of short fragments of c-kit mRNA in the spermatozoa of Kit tmlAlf1/+ paramutants. To induce degradation of endogenous c-kit mRNA, we microinjected fertilized eggs of wild-type animals with two microRNAs, miR-221 and miR-222, that harbored homology to c-kit mRNA and thus down-regulating Kit RNA. In this way, we efficiently (in 40% of mice) generated a paramutagenic effect at the Kit locus. In the resulting miR-221* and miR-222* paramutants, we observed the same result of epigenetic maintenance and transmission of a down-regulation of expression as in the progeny of tm1Alf heterozygote mice. Down-regulation occurs with high variations from one individual to another. These large variations provide a strong reminder of what had been noted as a characteristic of plant paramutation. A ‗rheostat‘ model for epigenetic variations describes variability as a main reason of these paramutagenic events.

CONCLUDING REMARKS AND PERSPECTIVES It is therefore apparent that paramutation that is defined as a hereditary epigenetic modification of gene expression occurs in the mouse, and it is inherited by a direct flexible heredity process involving RNA molecules. The notion of an essential role of RNA is also supported by recent results generated in parallel in plant systems (reviewed by Chandler, [9]). In heterozygotes, two models have been proposed to explain the occurrence of paramutation at the molecular level. In the pairing model [20], a region of one chromosome with an altered chromatin structure interacts with a homologous region of another and transfers altered chromatin complexes from one sequence to the other. In the ―trans-RNA‖ model, either long RNAs (silencing of the X chromosome [21, 22]) expressed in cells interfere with the function of the paramutable locus or small interfering RNAs (siRNA) lead to degradation of RNAs transcribed from the locus [23], with or without associated chromatin changes [24]. The interfering RNAs could originate from either the paramutagenic or paramutable locus. Both the pairing and the trans-RNA model accommodate the fact that the paramutated locus becomes itself paramutagenic because altered chromatin structures can be transferred perpetually and the paramutated locus can provide interfering RNAs. The aforementioned example describing the Kit locus and establishment of paramitation via RNAs microinjection into zygotes in mice is unique in paramutation studies; in contrast to the known phenomenon

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of allelic inactivation, our experiments describe RNA-induced hereditary modifications leading to the activation of transcription [25]. This represents a new way of transmission of epigenetic information, that is different from known cases of epimutations [26]. In addition, it is still not clear how modified structure is maintained and transmitted in the absence of effectors after microinjection of RNA molecules into the fertilized mouse eggs and transmission from the Kit*. The denomination ―histone code‖ has been proposed for transcription control by chromatine structure [27, 28]. RNAs are possible playing an important role in these complex chromatin structures. Specificity of RNA sequences makes them attractive candidates for structural modification of the gene machinery. In this case, small RNA molecules could potentially recruit other snRNAs/mRNAs, histones and/or other chromatin modifiers and regulatory proteins for the control of gene activity. Does an RNA-based ―rheostat code‖ exist, can it be stably maintained and transferred to the next generation? In fact, our results document the possibility of RNA transfer to zygotes and transmission of the paramutated state in a non-Mendelian fashion. A ―molecular rheostat‖ has already been proposed for L1 gene expression [29]. RNA ‗transmission‘ from one cell to another is in part explained by the RNA-dependant RNA polymerase (RDRP) RNA amplification machinery in plants [30] or C. Elegans [31]. The absence of RDRP in mammals suggests the existence of an alternative mechanism of RNA amplification and RNA-mediated DNA methylation and chromatin modification. Other processes such as mechanisms of RNA surveillance or some unknown mechanisms of maintenance and amplification of RNA may exist in mammalian cells [32, 33]. Our results obviously raise a number of questions. The first is whether this process is unique to the Kit locus of the mouse. Our results [34] (manuscript in preparation) indicate that this is not the case, as a variety of phenotypes can be observed in mouse families generated after RNA microinjection in fertilized eggs. The observed phenotype is dependent on a particular RNA or microRNA to which the embryonic genome was exposed. This raises questions such as: ‗Which phenotype is triggered by the presence of which RNA sequences?‘ and ‗Could any locus be paramutable and could any RNA sequences be active?‘ The internal structure and natural stability of microRNAs [35] make them powerful but not exclusive candidates involved in modification of gene expression [36]. A number of microRNAs are expressed in a tissue-specific manner [37]. What is the significance of the presence of large amounts of RNA and small RNAs in the spermatozoa? Will exposure of genomes of early stage embryos to tissue-specific microRNAs modify the phenotype of target tissues in an adult organism? In our search for the white tail tip phenotype induced by specific RNAs, we have observed the specificity of the RNA sequence for the sensitive c-kit locus. MicroRNAs are more active in the generation of white tail tips as compared to randomly chosen c-kit oligo RNA. Indeed, microinjection of oligo RNA chosen from the 5‘ or 3‘ ends of the c-kit transcript induced the white tail phenotype in around 15% of cases. These mice carry the specificity of Kit* paramutants described before, with the white tail tip phenotype, nonMendelian transmission and reversibility of the phenotype in back-cross with wild-type animals. A key role of small RNAs in shaping the genome has been recently reported [38]. It would be interesting to identify some other phenotypes influenced by small RNA molecules. The effect of RNA could be tested using a systematic search, for example, it could be started with RNA of an important gene responsible for a defined phenotype. Microinjection of small

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RNAs targeting this specific locus into the fertilized egg should produce a variety of expected phenotypes. It will be challenging however to dissect the ‗behavior‘ of a complex phenotype in normal physiological aspects and pathological deviations. A second important question is how any RNA accumulates in the sperm of the mouse. Do all lifestyle variables, such as nutrition, climate, stress, etc., induce variations in gene expression via small RNAs? Do RNAs induce DNA methylation of IAP elements [39] or DNA methylation of others loci of interest? How do they influence these changes and become active in inducing variations? Do they induce the accumulation of a specific class of RNA in the sperm? Is it a consequence of modification of proliferating stem germ cells or modification of the gamete [40]? In this case, one can ask how it is regulated and under which conditions? Does RNA-mediated modification lead to systemic DNA methylation? If yes, what determines the maintenance and stability of this response? A third series of important questions is obviously raised when considering a possible extension to other species of mammals, including humans. One obviously important element is the presence of relatively high amounts of RNA in human sperm [41], which suggests the possibility of RNA-mediated inheritance. EDTA reverse staining of normal human spermatozoa confirms a strong signal for RNA in a human sperm head. Comparison of the RNA content of spermatozoa in different mammals (domestic or commercially used) by deep sequencing will allow identifying RNAs of interest in normal and/or species- specific development. Epigenetics teaches us that RNA is important and is involved in complex pathways of gene expression. It does not yet teach us, however, how RNA becomes accumulated in the sperm to be paramutagenic. An interesting hypothesis is derived from our mice studies: the presence of sperm RNA is strongly correlated with the inheritance of epigenetic modifications, which themselves result from structural differences between alleles and chromosome mispairing as illustrated in Figure 5. This model drawn from studies on the locus-specific gene silencing processes and known as co-suppression in plants, meiotic silencing in Neurospora [42, 43] and the regulation of the activity of transposable elements [44] was tentatively extended to Kit* paramutation. We noted that paramutation was induced with identical frequencies and properties in the progeny of Kit tmlAlf1/+ heterozygotes and another mutants that carry insertion into the Kit gene, but not in the Kit mutants that carry point mutation (Kit W-v) [5, 16] and (Figure 6). Localized chromosomal mispairing is likely to be relatively infrequent in the extensively homozygous congenic strains of laboratory mice. However, it is predicted to occur more frequently in the extensively outbred human species. It may also be predicted that sperm RNA content can depend on time of day, age of an animal and environmental influences, and it can also be influenced by distinct pathology. The determination of sperm RNAs will provide an important support for learning more about variation. This hypothesis is currently under investigation; we are comparing the RNA content in mouse strains with different degrees of heterozygocity at various loci. The most important question for the future is whether this type of effect could create psychopathological conditions or disease-prone states that would be inherited for at least a number of generations in a non-Mendelian way. This would mean that it is possible to induce immense variation within the same pool of genes but with a distinct combination and order of expression. In fact, such quantitative ‗rheostat-type‘ effects [14] are well recognized and usually described as ‗variable penetrance‘ with no further explanation in molecular terms.

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Figure 5. Staining of RNA on in the EM sections of mouse sperm. EDTA reverse staining in the EM sections of mouse sperm maintains dark signal characteristics of dense RNA in KittmlAlf1/+ heterozygote compared to light staining of wild type mouse sperm.

Figure 6. The absence of paramutation in the progeny of heterozygote Kitwv/+ Kitwv/+heterozygote. In all types of crosses, heterozygote Kitwv/+ produces only wild type (full color) and heterozygote (white tail) animals.

On the other hand, several instances of pathological phenotypes in humans have been reported as being possibly triggered by epigenetic marks. Unfortunately, in the absence of well-defined genetic backgrounds, it is not yet possible to conclude that epigenetic inheritance plays a role [45]. In addition, Bennett et al. have described transmission of information on occurrence of type 1 diabetes from untransmitted paternal chromosome [46].

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If established in model animal systems, inheritance of a disease-prone state maintained over several generations in a non-Mendelian manner and associated with the zygotic transfer of RNA would help us to understand some instances of familial risk with no Mendelian factor identified. Our preliminary results indicate that epigenetic pathological changes can be induced in the mouse by exposure of the embryonic genome to defined RNAs and microRNAs. At some point in the future, one might hope to find RNA for transcripts of oncogenes, tumor suppressors and related microRNAs in sperm. That could then help identify potential responsible RNAs. At present, general tentative and more extensive investigations of the aforementioned phenomena, using experimental epigenetic animal models, are required [47]. One can also ask for a experimental proof for the molecular mechanism of hereditary epigenetic variation in plant [48]. Is it mediated by RNA? In practice, one of the challenges in the near future is to understand the ‗raison d‘être‘ of RNA molecules in epigenetic function: in terms of sequence specificity in shaping the gene expression profile; in terms of the identity and modes of transfer through germ cells; in terms of their influence on heredity; and in terms of normal and pathological development.

ACKNOWLEDGMENTS

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I thank François Cuzin and Igor Kovalchuk for discussion and C. Vannetti, L. Martin, M. Radjkhumar, F. Millot, F. Paput, M. Cutajar, T. Amine for skilled technical assistance. The work was made possible by grants to MR from Ligue Nationale Contre le Cancer (France) as ―Equipe Labellisée‖ and from ―Agence Nationale de la Recherche‖ (grant ANR-06-BLAN0226 PARAMIR).

REFERENCES [1] [2] [3]

[4] [5]

[6] [7] [8]

Barlow DP: Gametic imprinting in mammals. Science 1995, 270:1610-1613. Lyon MF: Epigenetic inheritance in mammals. Trends Genet. 1993, 9:123-128. Reik W, Romer I, Barton SC, Surani MA, Howlett SK, Klose J: Adult phenotype in the mouse can be affected by epigenetic events in the early embryo. Development 1993, 119:933-942. Brink RA: A genetic change associated with the R locus in maize which is directed and potentially reversible. Genetics 1956, 41:872-879. Rassoulzadegan M, Grandjean V, Gounon P, Vincent S, Gillot I, Cuzin F: RNAmediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 2006, 441:469-474. Brink RA: Paramutation. Annu. Rev. Genet. 1973, 7:129-152. Hollick JB, Dorweiler JE, Chandler VL: Paramutation and related allelic interactions. Trends Genet. 1997, 13:302-308. Kennison JA, Southworth JW: Transvection in Drosophila. Adv. Genet. 2002, 46:399420.

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

128 [9] [10] [11]

[12]

[13]

[14] [15] [16]

[17]

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

[18]

[19] [20] [21] [22]

[23]

[24] [25]

Minoo Rassoulzadegan Chandler VL: Paramutation: RNA-mediated instructions passed across generations. Cell 2007, 23:641-645. Walker EL: Paramutation of the r1 locus of maize is associated with increased cytosine methylation. Genetics 1998, 148:1973-1981. Keshet E, Lyman SD, Williams DE, Anderson DM, Jenkins NA, Copeland NG, Parada LF: Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development. EMBO J. 1991, 10:2425-2435. Nocka K, Majumder S, Chabot B, Ray P, Cervone M, Bernstein A, Besmer P: Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice. Genes Dev. 1989, 3:816-826. Stam M, Belele C, Dorweiler JE, Chandler VL: Differential chromatin structure within a tandem array 100 kb upstream of the maize b1 locus is associated with paramutation. Genes Dev. 2002, 16:1906-1918. Jiang ALBaY-h: A Rheostat Model for a Rapid and Revrsible Form of ImprintingDependent Evolution. Am. J. Hum. Genet. 2002, 70:1389-1397. Little C, Cloudman A: The occurence of a dominant spotting mutation in the house mouse. Proc. Natl. Acad. Sci. USA 1937, 23:535-537. Motro B, van der Kooy D, Rossant J, Reith A, Bernstein A: Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci. Development 1991, 113:1207-1221. Wagner KD, Wagner N, Ghanbarian H, Grandjean V, Gounon P, Cuzin F, Rassoulzadegan M: RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Dev. Cell 2008, 14:962-969. Bernex F, De Sepulveda P, Kress C, Elbaz C, Delouis C, Panthier JJ: Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos. Development 1996, 122:3023-3033. Bernhard W: A new staining procedure for electron microscopical cytology. J. Ultrastruct Res. 1969, 27:250-265. Grant-Downton RT, Dickinson HG: Plants, pairing and phenotypes - two's company? Trends Genet. 2004, 20:188-195. Sleutels F, Zwart R, Barlow DP: The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 2002, 415:810-813. Ciaudo C, Bourdet A, Cohen-Tannoudji M, Dietz HC, Rougeulle C, Avner P: Nuclear mRNA degradation pathway(s) are implicated in Xist regulation and X chromosome inactivation. PLoS Genet. 2006, 2:e94. Yuan YR, Pei Y, Chen HY, Tuschl T, Patel DJ: A Potential Protein-RNA Recognition Event along the RISC-Loading Pathway from the Structure of A. aeolicus Argonaute with Externally Bound siRNA. Structure 2006, 14:1557-1565. Bernstein E, Allis CD: RNA meets chromatin. Genes. Dev. 2005, 19:1635-1655. Chess A, Simon I, Cedar H, Axel R: Allelic inactivation regulates olfactory receptor gene expression. Cell 1994, 78:823-834.

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[26] Chong S, Yougson NA, Whitelaw E: Heritable germline epimutation is not the same as transgenerational epigenetic inheritance. Nat. Genet. 2007, 39:574-575. [27] Alleman M, Sidorenko L, McGinnis K, Seshadri V, Dorweiler JE, White J, Sikkink K, Chandler VL: An RNA-dependent RNA polymerase is required for paramutation in maize. Nature 2006, 442:295-298. [28] Jenuwein T, Allis CD: Translating the histone code. Science 2001, 293:1074-1080. [29] Han JS, Szak ST, Boeke JD: Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature 2004, 429:268-274. [30] Schiebel W, Pelissier T, Riedel L, Thalmeir S, Schiebel R, Kempe D, Lottspeich F, Sanger HL, Wassenegger M: Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato. Plant Cell 1998, 10:2087-2101. [31] Smardon A, Spoerke JM, Stacey SC, Klein ME, Mackin N, Maine EM: EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans. Curr. Biol. 2000, 10:169-178. [32] Isken O, Maquat LE: Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev. 2007, 21:1833-1856. [33] Behm-Ansmant I, Kashima I, Rehwinkel J, Sauliere J, Wittkopp N, Izaurralde E: mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett. 2007, 581:2845-2853. [34] Wagner KD, Wagner N, Ghanbarian H, Grandjean V, Gounon P, Cuzin F, Rassoulzadegan M: RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Dev. Cell 2008, 14:962-969. [35] Berezikov E, Cuppen E, Plasterk RH: Approaches to microRNA discovery. Nat. Genet. 2006, 38 Suppl:S2-7. [36] Meister G, Tuschl T: Mechanisms of gene silencing by double-stranded RNA. Nature 2004, 431:343-349. [37] Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T: Identification of tissue-specific microRNAs from mouse. Curr. Biol. 2002, 12:735-739. [38] Nowacki M, Vijayan V, Zhou Y, Schotanus K, Doak TG, Landweber LF: RNAmediated epigenetic programming of a genome-rearrangement pathway. Nature 2008, 451:153-158. [39] Blewitt ME, Vickaryous NK, Paldi A, Koseki H, Whitelaw E: Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice. PLoS Genet. 2006, 2:e49. [40] Ariel M, Cedar H, McCarrey J: Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis. Nat. Genet. 1994, 7:59-63. [41] Miller D, Ostermeier GC, Krawetz SA: The controversy, potential and roles of spermatozoal RNA. Trends Mol. Med. 2005, 11:156-163. [42] Aramayo R, Metzenberg RL: Meiotic transvection in fungi. Cell 1996, 86:103-113. [43] Shiu PK, Raju NB, Zickler D, Metzenberg RL: Meiotic silencing by unpaired DNA. Cell 2001, 107:905-916.

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[44] Slotkin RK, Martienssen R: Transposable elements and the epigenetic regulation of the genome. Nat. Rev. Genet. 2007, 8:272-285. [45] Chong S, Vickaryous N, Ashe A, Zamudio N, Youngson N, Hemley S, Stopka T, Skoultchi A, Matthews J, Scott HS, et al: Modifiers of epigenetic reprogramming show paternal effects in the mouse. Nat. Genet. 2007, 39:614-622. [46] Bennett ST, Wilson AJ, Esposito L, Bouzekri N, Undlien DE, Cucca F, Nistico L, Buzzetti R, Bosi E, Pociot F, et al: Insulin VNTR allele-specific effect in type 1 diabetes depends on identity of untransmitted paternal allele. The IMDIAB Group. Nat. Genet. 1997, 17:350-352. [47] Pembrey ME: Time to take epigenetic inheritance seriously. Eur. J. Hum. Genet. 2002, 10:669-671. [48] Vaughn MW, Tanurd Ic M, Lippman Z, Jiang H, Carrasquillo R, Rabinowicz PD, Dedhia N, McCombie WR, Agier N, Bulski A, et al: Epigenetic Natural Variation in Arabidopsis thaliana. PLoS Biol. 2007, 5:e174.

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Chapter 8

ROLE OF EPIGENETIC CHANGES IN THE NONTARGETED RADIATION-INDUCED EFFECTS: UNDERSTANDING THE EPIGENETICS OF BYSTANDER AND TRANSGENERATIONAL EFFECTS Jody Filkowski, Yaroslav Ilnytsky and Olga Kovalchuk Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4, CANADA

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ABSTRACT Ionizing radiation, in addition to being an important treatment modality, is a potent tumor causing agent. The resultant risk of secondary radiation treatment-related cancers is a growing clinical problem. Some studies link secondary radiation-induced cancers to an enigmatic phenomenon of bystander effects whereby exposed cells signal damage and distress to their naïve neighbors resulting in genome instability. There is also welldocumented evidence that radiation exposure leads to transgenerational genome instability in the offspring of exposed parents. The exact molecular mechanisms of these indirect/non-targeted radiation effects have yet to be defined; however, recent evidence suggests that they may be epigenetic in nature. Epigenetic phenomena seem to be mediators of indirect radiation effects, including radiation-induced genome instability, bystander and transgenerational effects. DNA methylation and histone modification changes directly impact chromatin packaging and therefore influence gene expression and susceptibility of DNA to rearrangements. Short RNAs (such as microRNAs and piRNAs) may potentially be acting as key mediators of these non-targeted effects due to their small size, relative stability, their roles in maintenance of gene expression, stability of transposable elements, and the genome as a whole. This chapter will summarize the current knowledge of the existence of non-targeted radiation-induced bystander and transgenerational effects and the roles of epigenetic changes in their initiation and maintenance.

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INTRODUCTION Radiation refers to a broad spectrum of omnipresent energy quanta; some examples include: radio waves, microwaves, and visible and ultraviolet (UV) light. Due to its ubiquity, it is impossible to escape exposure to radiation. Fortunately, most types of radiation are not known to be harmful. Only the more energetic wavelengths that are collectively called ionizing radiation, such as UV, X-rays, gamma rays, and alpha and beta particles, display carcinogenic capabilities [1-3]. Ionizing radiation is known to be a universal carcinogen, i.e., it is capable of inducing cancers in all tissues and across all ages of development [1-5]. Unlike chemical carcinogens whose effects may be mitigated by cell membranes, transmembrane proteins or intracellular metabolism, ionizing radiation achieves its universal influence by delivering acute levels of energy in a random fashion. Thus, any cells exposed to radiation will experience the immediate effects of exposure [3]. The mutagenic and potentially carcinogenic effects of radiation exposure are achieved through energy transfer to biological molecules of the cell—namely DNA [3, 6]. Energy exposure may break chemical bonds, produce free radicals, crosslink macromolecules or cause direct damage to the DNA. Additionally, radiation can cause changes in gene expression, disruption of mitochondrial processes, cell cycle arrest and apoptotic cell death [7-20]. The carcinogenic potential of ionizing radiation was recognized very soon after its discovery when the first radiation-induced tumor was reported in 1902 [2, 3, 21]. Yet, radiation is a key clinical modality used for detecting and fighting human malignancies, which makes understanding of underlying mechanisms of radiation effects essential. Interestingly, the effects of radiation are also observed in distal cells and tissues that do not receive the initial energy quanta or even fractions of it. The radiation-induced response in cells that are descendents of directly irradiated cells is termed radiation-induced genomic instability (RIGI) [22-28]. Genomic instability is associated with an increased rate of acquisition of changes in the genome. This phenomenon manifests itself as elevated rates of aneuploidy, chromosomal aberrations, gene mutations and amplifications, microsatellite instability, altered levels of cell proliferation and cell death [24-27, 29, 30]. Importantly, it is now well established that RIGI occurs in irradiated cells after irradiation and in the progeny of irradiated cells for multiple generations after exposure [3, 31, 32]. It has long been suggested that genomic instability in general and RIGI specifically may play a significant role in carcinogenesis. Presently, genomic instability is well-accepted as a key characteristic of many cancers and as an important step in carcinogenesis [33-35].

BYSTANDER EFFECTS The effects of radiation can also be seen in distal naïve ‗bystander‘ cells and tissues which either were in contact with irradiated cells or received signals from irradiated cells (Figure 1). This phenomenon, known as the bystander effect, includes a number of radiationinduced endpoints. Similar to RIGI, the bystander effect manifests itself as an induction of chromosome aberrations, genome rearrangements, mutations, altered cell death and tumorigenesis [36-51].

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Figure 1. A well-established mouse exposure model for analysis of bystander effects in vivo. Animals can have the lead shielding covering half their bodies during exposure. Alternatively, the animal‘s head is irradiated and the animal‘s body – shielded.

In studies on single cells which were irradiated with a lethal dose of alpha radiation and naïve bystander cells were analyzed, mutation rates were reported to be 3-4 fold higher with up to 40% reduction in survival rates in non-irradiated bystander cells [52, 53]. Furthermore, it was possible to detect radiation-induced effects in non-irradiated cells if they were cocultured with previously irradiated cells or received medium from exposed cells [54, 55]. Currently, the existence of bystander effects is well-documented in cell culture, tissue, organ and whole organism models [25, 26, 56-64]. Bystander effects have been reported in three-dimensional (3D) tissue models including spheroids [65, 66] and in reconstructed human tissue models [57, 67]. Presently, bystander effects are accepted as a ubiquitous consequence of radiation exposure [68] that occur in the context of whole organisms. Notwithstanding, conclusive data on somatic bystander effects in vivo are still in short supply [34, 60, 61, 63, 64, 69-73]. Bystander effects in vivo were observed in the early 1940s and in radiation-treated cancer patients in the 1950s [53]. These early studies found a profound radiation-induced clastogenic activity observed in the plasma of patients undergoing radiotherapy and in individuals accidentally exposed to ionizing radiation [74-78]. These clastogenic factors are powerful inducers of chromosomal damage in cultured naïve bystander cells [74-81]. In whole animal models, bystander effects were noted in distal parts of a particular organ or tissue and even in other whole tissues [73, 82]. In an animal model, irradiation of the lung base resulted in significant molecular and cellular damage in the shielded lung apex [83, 84]. Furthermore, exposure of one lung, either right or left, led to a noticeable increase of micronuclei in the unexposed shielded lung [83, 84]. Similarly, within-the-organ bystander effects were seen following partial liver irradiation [82, 85]. Somatic bystander effects were also noted using the rodent skin and spleen models whereby one part of the animal‘s body was exposed to radiation, while the other part was protected by a medical grade shield [59-61, 63, 64, 72, 73] (Figure 1). Furthermore, the types and magnitudes of bystander effects appear to be tissue- [59] and sex-specific [60]. The far-reaching effects of radiation exposure suggest that cells are able to communicate exposure events through a signaling mechanism. Although the exact mechanism of bystander effect propagation has not been fully elucidated, it appears to be dependent upon gap junctions and cell secreted factors such as radical oxygen species, cytokines and/or nitric oxide [39]. It is not likely that a single common propagation mechanism gives rise to all

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bystander effects, it is rather probable that various bystander endpoints (for example, mutation versus cell killing) are mediated through various molecular species [54, 86]. In sum, even though a great deal of data has been accumulated on the existence and manifestation of genomic instability and bystander effects in cultured cells, 3D tissues, organs and organisms, the mechanisms of these phenomena remain to be established. Interestingly, high frequency of occurrence and persistence of bystander responses and a profound role of gene expression changes caused by bystander effects suggest a possible epigenetic background [25-27, 48, 87, 88].

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TRANSGENERATIONAL EFFECTS Persistent characteristics of radiation exposure have been heavily studied both in vitro and in vivo and strong evidence has been provided that the effects of exposure can be observed through many cell divisions and even in offspring. Many studies employed a spectrum of measures for assessing an increased frequency of carcinogenesis, mutation rates and decreased genome stability in subsequent generations [89]. These effects, termed transgenerational effects, may not be considered bona fide bystander effects as they are observed in progeny of directly exposed cells. Numerous studies have reported the existence of radiation-induced transgenerational mutations in human populations. Considerable work in this direction was conducted by Dubrova and colleagues who studied populations affected by technological accidents at the Chernobyl nuclear powerplant, the Mayak nuclear processing facility and the nuclear testing site at Semipalatinsk [90-96]. Dubrova‘s experimental strategy was based on analysis of minisatellites, the loci that were later renamed expanded simple tandem repeat (ESTR) loci (ESTR instability is discussed in detail in Chapter 9 by Yauk and Stead). It should be noted, however, that an increased minisatellite variability observed in the offspring of irradiated parents remains a controversial issue. An extensive study of children born to A-bomb survivors in Japan failed to detect any transmissible effects of parental irradiation [97]. While evidence to support the hypothesis of heritable effects from parental germline radiation exposure in humans has been the subject of much debate, data from animal models have clearly demonstrated that the effects of parental radiation exposure are transmitted through the germline to the progeny of irradiated parents [25-27]. In animal models, early studies of transgenerational effects employed various tests such as the specific locus and dominant lethal assays as well as the heritable translocation assays [98-100]. Pioneering work by Nomura showed an increase in tumors in the progeny of irradiated and chemically exposed mice [99, 101, 102]. Other studies focused on heritable alterations in cancer incidence and teratogenesis following parental preconception irradiation [103-109]. In addition to this classic evidence for transmitted effects, it is now well-accepted that the progeny of irradiated parents are also at risk of transgenerational genomic instability [26, 64, 89, 105-120]. While the exact molecular mechanisms of transgenerational radiation-induced genome instability have yet to be discovered, some evidence suggests a possible epigenetic nature of this phenomenon [26, 27, 121].

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EPIGENETICS The term epigenetic regulation refers to all regulations of the genome, specifically DNA methylation, histone modifications and small non-coding RNAs, but it does not include direct changes in the DNA sequence [122, 123]. DNA methylation is a covalent modification involving the addition of a methyl group to carbon 5 of cytosine residues. DNA methylation is important for normal development, cell proliferation and maintainance of genome stability in a given organism [122, 124-127]. In mammals, DNA methylation occurs mostly in the context of CG dinucleotides methylated to 60–80% [128] and is mediated by DNA methyltransferase enzymes (DNMT1, DNMT3a and DNMT3b) [124, 129-133]. DNMT1 is the major enzyme involved in maintaining DNA methylation patterns after DNA replication [134]. DNMT3a and DNMT3b are de novo methyltransferases targeting unmethylated and hemimethylated sites [128, 129, 135]. The association of DNA methylation with transcriptional repression is mediated by the MBD (methyl CpG-binding domain) proteins (MeCP2, MBD1, MBD2, and MBD3) [136]. These proteins selectively interact with methylated DNA [122, 136-139]. The regulatory potential of DNA methylation manifests itself in promoter regions that control the expression of adjacent genes. Hypermethylated promoters lead to an ―off‖ state of expression while less methylated ones are deemed as ―on‖[122]. Moreover, methylated cytosines themselves can physically prevent proper binding of transcription factors to promoter regions [128]. Cancer cells are well known to exhibit altered global DNA methylation patterns [128, 132, 140]. Specifically, DNA methylation profiles of cancer cells are frequently characterized by global genome hypomethylation as well as concomitant hypermethylation of various gene promoters [122, 125, 140]. Loss of global DNA methylation (hypomethylation) was the first epigenetic change identified in cancer cells. Global DNA hypomethylation has been linked to the activation of transposable elements, elevated chromosome breakage, aneuploidy, increased mutation rates and to the phenomenon of global genomic instability [128, 137, 138, 140]. Overall, changes in gene expression patterns, dysregulation of DNA methylation and chromatin changes are associated with alterations in genome stability, and hypomethylation leads to a more instable state [122]. Furthermore, DNA methylation is closely related to chromatin modifications that also give rise to an ―on/off‖ state of expression. In this instance, there are more than 50 posttranslational modifications that may occur on key amino acid residues within the histone core of the nucleosome. Histone modifications encompass acetylation, methylation, phosphorylation, ubiquitination and sumoylation, and they are important for transcriptional regulation and genome stability [140, 141]. For example, acetylation of several lysine residues within the N-terminal tail of H3 and H4 gives rise to an open or ―on‖ chromatin state typically associated with DNA hypomethylation and expressed genes, while deacetylation tends to coincide with hypermethylated DNA loci that are not expressed [123]. Histone methylation can lead to different transcriptional consequences based on the residue affected [141, 142]. Moreover, histone residues can be mono-, di- and tri-methylated, leading to enormous complexity of the yet uncharted histone code [140, 142-144].

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Lastly, epigenetic control is also exercised by small regulatory RNAs [145], specifically microRNAs. MicroRNAs (miRNAs) are abundant, small, single-stranded noncoding RNAs that are potent regulators of gene expression [146, 147]. To control the translation of their target mRNAs, miRNAs associate with the RNA-induced silencing complex (RISC) proteins and bind to the 3'UTR of their cognate mRNAs, thus serving as translational suppressors that regulate protein synthesis [148, 149]. Regulatory miRNAs impact a wide variety of cellular processes, such as cellular differentiation, proliferation, apoptosis, genome stability and even predisposition to cancer, to name a few [150, 151].

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BYSTANDER EPIGENETICS It is now well accepted that the epigenetic component of gene regulation is equally important in determining expression and ultimate characterization of a given cell. Therefore, it is not surprising that mounting evidence suggests an epigenetic component of bystander effects.[64, 152]. In the realm of bystander effects, dysregulation of DNA methylation has been observed in several cell types and in vivo systems; the implications of dysregulated DNA methylation include a less stable genome with increased DNA damage and increased activation of transposable elements [59, 73, 152]. The pioneering work of Kaup and colleagues has shown that DNA methylation is important for maintenance of radiation-induced bystander effects in cultured cells and is associated with increased and persistent levels of chromosome and chromatid aberrations, reproductive cell death, apoptosis and other signs of genome instability [152]. Sedelnikova and colleagues studied bystander effects in two human 3D tissue models: airway and full-thickness skin. Following microbeam irradiation of cells, numerous endpoints were analyzed in bystander cells. The study revealed a significant increase in the level of phosphorylated H2AX, apoptosis and micronucleus formation in bystander cells. These changes were paralleled by persistent growth arrest, an increasing number of senescent cells and most importantly loss of global DNA methylation [57]. Of special interest is the observed loss of DNA methylation in bystander cells as DNA methylation is an important epigenetic phenomenon involved in the regulation of gene expression and genome stability. In animal-based studies, irradiation was shown to induce DNA damage and modulate epigenetic effectors in distant bystander tissues. To analyze bystander effects in vivo, Koturbash and colleagues developed a mouse model whereby half of the animal‘s body was exposed to radiation, while the other half was protected by a clinical grade lead shield [73] (Figure 1). In this model, radiation exposure to one half of the body resulted in elevated levels of DNA strand breaks, altered levels of key proteins that modulate methylation patterns and silencing in the bystander half of the body at least 0.7 cm from the irradiated tissue. The existence and epigenetic nature of bystander effects was later confirmed in the spleen of mice subjected to cranial irradiation [61, 63, 72] (Figure 1). There, cranial exposure led to significant DNA hypomethylation and altered DNMT levels. Further studies using a rat model have revealed that localized cranial radiation exposure results in decreased levels of global DNA methylation, altered levels of DNMT3a, MeCP2 and methylation-associated reactivation of LINE1 retrotransposon in the bystander spleen located at least 16 cm from the irradiation site. Importantly, the studies provided the first evidence that down-regulation of

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DNA methylation-regulating proteins, DNMT3a and MeCP2, is triggered and maintained by the microRNA miR-194 which was up-regulated in the bystander rat spleen. The data have clearly demonstrated that the bystander effect occurs in distant tissues in vivo, persists over a long period of time and is epigenetically regulated [72]. Altered expression of miR-194 observed in the bystander rat spleen was very intriguing and promoted further studies of microRNAome changes in bystander tissues. Using a miRNA microarray platform, microRNAome profiles have been compiled from skin and spleen tissues of mice subjected to sham treatment, whole-body or head exposure. Radiation exposure led to very significant alterations in the miRNA expression profiles in bystander skin and spleen [59]. Pronounced microRNAome alterations can also be seen in bystander tissues using the 3D model (Kovalchuk and Sedelnikova, unpublished). These data suggest that miRNA expression changes really occur in bystander tissues. The exact function of miRNAs in the bystander effect still has to be delineated. Furthermore, due to their small size and high stability, miRNAs may be plausible bystander-signaling molecules. Unfortunately, while bystander effect-associated DNA methylation and miRNA changes have been analyzed, not much work has focused on specific histone modifications that occur within bystander cells, with the exception of phosphorylated histone2AX (γH2AX). However, it is important to note that γH2AX observations are usually used to indicate the presence of DNA breaks and thus are an indirect result of other primary bystander effects [57, 123, 153]. The demonstration of extreme persistence of epigenetic alterations in bystander tissue is of high importance as it has the potential to explain how radiation effects can be propagated to subsequent daughter cells and even offspring. In addition, epigenetic dysregulation provides a reasonable explanation for all phenotypes (carcinogenesis, mutations and genome stability) investigated in transgenerational studies. Indeed, such epigenetic persistence does exist. A human keratinocyte cell line demonstrated dysregulated methylation patterns for at least 20 generations, while in vivo, the effects were seen in mouse spleen tissue up to seven months post-irradiation [72, 152]. A much more convincing argument for the role of epigenetic dysregulation in transgenerational effects was provided by mice progeny conceived following cranial X-ray irradiation of the male parent [62].

EPIGENETICS OF TRANSGENERATIONAL EFFECTS Currently, the existence of transgenerational changes in somatic and germline tissues of progeny of exposed parents is well accepted [89, 107, 108, 120]. In spite of this, the precise molecular mechanisms that may cause radiation-induced transgenerational genome instability and carcinogenesis remain obscure. Recent evidence suggests that transgenerational effects may be attributed to epigenetic mechanisms [121]. Several independent studies have shown that the ionizing radiation-induced cellular reprogramming persists for multiple generations. Initially, Wiley and colleagues demonstrated the persistent heritable effects of paternal irradiation on embryonic cell proliferation [154, 155]. The persistent nature of these effects suggests that a non-Mendelian mode of inheritance is involved. Several studies demonstrated that offspring of F0 irradiated parents also exhibited marked biochemical alterations [156-158]. Furthermore, four generations of offspring from irradiated sires demonstrated changes in the level of protein

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kinase C, mitogen-activated protein kinase, Tpr53 and p21waf1. These expression changes were neither consistent in magnitude or direction within the offspring of a given generation, nor were they consistent between generations [159]. These observations led the authors to hypothesize that these expression changes were altered through an epigenetic mechanism. Moreover, high frequency of persistent heritable effects on embryonic and germ cell proliferation rates that spanned over 2-3 generations following paternal irradiation further reinforced the epigenetic nature of regulation of these transgenerational effects [154, 156]. As a result, the mechanism that may underlie the observed persistent transgenerational gene expression phenotype was named ‗epigenomic instability‘ [159]. Recent studies using the sperm of irradiated male mice 7-weeks post exposure also demonstrated unconventional heritable DNA damage effects including chromatin changes in progeny of exposed fathers. These key findings further support the proposed ‗epigenomic instability‘ theory [159, 160]. Dubrova and colleagues pioneered the analysis of transgenerational instability in ESTR loci within the genome [89-93, 114-119, 161-170]. High magnitude non-Mendelian inheritance and persistence of ESTR mutation rates clearly speak for epigenetic dysregulation as a possible causative factor of this phenomenon [89, 116]. Yet, relatively few studies have addressed the potential bona fide epigenetic alterations in offspring of irradiated parents. The first direct evidence of the involvement of epigenetic regulators in transgenerational responses stems from a recent study that used an in vivo mouse model [171]. In this study, male animals were irradiated and mated to unexposed females. The level of global DNA methylation was measured in spleen, thymus and liver of offspring. A significant loss of DNA methylation was observed in the thymus of offspring upon paternal and combined parental exposure. DNA methylation changes were correlated with alterations in the level of DNA methyltransferases and methyl-binding proteins. Since mammalian genomes heavily depend upon properly set DNA methylation patterns in their function, loss of global DNA methylation and altered levels of DNMT1, DNMT3a or 3b can lead to the activation of transposable elements, thereby contributing to genome instability [121, 172, 173]; it can be suggested that loss of global DNA methylation observed in progeny of irradiated parents may influence retrotransposons and satellite DNA, thus causing transgenerational genome instability. Importantly, this observation may explain, at least in part, satellite DNA instability in progeny of exposed parents [89]. The most recent data suggest that transgenerational DNA methylation alterations affect satellite DNA, tubulin loci and a variety of short interspersed nuclear elements (Kovalchuk, unpublished). Parental irradiation also results in a significant increase in levels of phosphorylated histone H2AX in thymic tissues. The importance of histone H2AX phosphorylation was recently confirmed by another study. Barber and colleagues analyzed transgenerational DNA damage and mutation rates in the germline and somatic tissues of the first generation offspring of irradiated mice from two different strains. In both strains, they noted significantly elevated transgenerational mutation rates. Elevated mutation rates may be attributed, at least in part, to persistently elevated levels of phosphorylated histone H2AX [116]. Most importantly, the study implies that the persistently elevated mutation rates in the tissues of offspring may be due to epigenetic inheritance of instability signals through sperm. Amongst those, DNA methylation is proposed to be a reasonable candidate for an epigenetic signal that leads to transgenerational mutagenesis. Hatch and colleagues have recently reinforced the conclusion that DNA methylation plays an important role in transgenerational changes of mutation and

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recombination rates in progeny of exposed animals [174]. Notwithstanding, the exact mechanisms of transgenerational DNA methylation and inherited phenotypic and genotypic changes need to be further analyzed. The majority of current research on transgenerational ionizing radiation effects focus on the role of whole-body exposure, yet such exposure is relatively rare. In contrast, localized body-part exposure occurs frequently during radiation diagnostics and therapy. Localized cranial exposure also leads to DNA damage and epigenetic changes in the germline and consequently to transgenerational epigenetic changes in progeny [62]. The possible function of small regulatory RNAs in transgenerational radiation effects is the least explored area of research that requires further investigation. Small RNAs and especially microRNAs may constitute another epigenetic signal that can be passed on through sperm cells, thus influencing chromatin packaging and gene expression in the fertilized egg. Another group of short RNAs are the 29-30 nucleotides long RNAs that form complexes with Piwi proteins (piRNAs) [175-184]. This novel class of small RNA molecules discovered in 2007 is expressed in mammalian germline. PiRNA-Piwi complexes have been linked to retrotransposon silencing and other germline genetic elements [183, 185-187]. Therefore, they may be important regulators of transgenerational effects. Their role in germline effects and therefore in transgenerational genome instability has yet to be defined.

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CONCLUSIONS AND OUTLOOK While the research data accumulated to date has provided extremely pertinent information in the area of transgenerational responses, there are still mysteries that remain unsolved. Specifically, the exact contribution of DNA methylation to generation and maintenance of transgenerational effects induced by whole-body and localized irradiation and chemotherapy agents has yet to be precisely delineated. Transgenerational short RNA changes need to be further elucidated. The interrelationship between transgenerational epigenetic changes and transgenerational carcinogenesis needs to be established. Additionally, the impact of DNA damage and changes in short RNA pools in the germline of exposed parents needs to be exactly defined. Furthermore, future studies including those that employ transgenic and knockout models of epigenetic regulator genes may shed more light on the role and interrelationship between genetic rearrangements and epigenetic effects.

REFERENCES [1]

[2] [3]

Little MP, Heidenreich WF, Moolgavkar SH, Schollnberger H, Thomas DC: Systems biological and mechanistic modelling of radiation-induced cancer. Radiat. Environ. Biophys. 2008, 47:39-47. Little JB: Genomic instability and radiation. J. Radiol. Prot. 2003, 23:173-181. Little JB: Radiation carcinogenesis. Carcinogenesis 2000, 21:397-404.

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140 [4] [5] [6] [7] [8] [9] [10] [11]

[12]

[13]

[14]

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[15] [16] [17] [18]

[19]

[20] [21] [22] [23] [24]

Jody Filkowski, Yaroslav Ilnytsky and Olga Kovalchuk Williams D: Cancer after nuclear fallout: lessons from the Chernobyl accident. Nat. Rev. Cancer 2002, 2:543-549. Trosko JE: Role of low-level ionizing radiation in multi-step carcinogenic process. Health Phys. 1996, 70:812-822. Kleinsmith LJ: Principles of Cancer Biology. San Fransico: Pearson Benjamin Cummings; 2006. Rodemann HP, Blaese MA: Responses of normal cells to ionizing radiation. Semin. Radiat. Oncol. 2007, 17:81-88. Jeggo PA, Lobrich M: Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability. DNA Repair (Amst) 2006, 5:1192-1198. Amundson SA, Bittner M, Fornace AJ, Jr.: Functional genomics as a window on radiation stress signaling. Oncogene 2003, 22:5828-5833. Amundson SA, Fornace AJ, Jr.: Monitoring human radiation exposure by gene expression profiling: possibilities and pitfalls. Health Phys. 2003, 85:36-42. Amundson SA, Lee RA, Koch-Paiz CA, Bittner ML, Meltzer P, Trent JM, Fornace AJ, Jr.: Differential responses of stress genes to low dose-rate gamma irradiation. Mol. Cancer Res. 2003, 1:445-452. Criswell T, Klokov D, Beman M, Lavik JP, Boothman DA: Repression of IR-inducible clusterin expression by the p53 tumor suppressor protein. Cancer Biol. Ther. 2003, 2:372-380. Criswell T, Leskov K, Miyamoto S, Luo G, Boothman DA: Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation. Oncogene 2003, 22:5813-5827. Valerie K, Yacoub A, Hagan MP, Curiel DT, Fisher PB, Grant S, Dent P: Radiationinduced cell signaling: inside-out and outside-in. Mol. Cancer Ther. 2007, 6:789-801. Fei P, Bernhard EJ, El-Deiry WS: Tissue-specific induction of p53 targets in vivo. Cancer Res 2002, 62:7316-7327. Fei P, El-Deiry WS: P53 and radiation responses. Oncogene 2003, 22:5774-5783. Iliakis G, Wang Y, Guan J, Wang H: DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 2003, 22:5834-5847. Powell SN, Kachnic LA: Roles of BRCA1 and BRCA2 in homologous recombination, DNA replication fidelity and the cellular response to ionizing radiation. Oncogene 2003, 22:5784-5791. Andreev SG, Eidelman YA, Salnikov IV, Khvostunov IK: Mechanistic modelling of genetic and epigenetic events in radiation carcinogenesis. Radiat. Prot. Dosimetry 2006, 122:335-339. Jeggo P, Lobrich M: Radiation-induced DNA damage responses. Radiat. Prot. Dosimetry 2006, 122:124-127. Chauveinc L, Giraud P, Dahnier S, Mounier N, Cosset JM: [Radiotherapy-induced solid tumors: review of the literature and risk assessment]. Cancer Radiother 1998, 2:12-18. Morgan WF, Sowa MB: Non-targeted bystander effects induced by ionizing radiation. Mutat Res. 2007, 616:159-164. Sowa M, Arthurs BJ, Estes BJ, Morgan WF: Effects of ionizing radiation on cellular structures, induced instability and carcinogenesis. EXS 2006:293-301. Morgan WF, Sowa MB: Effects of ionizing radiation in nonirradiated cells. Proc. Natl. Acad. Sci. U S A 2005, 102:14127-14128.

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141

[25] Morgan WF: Non-targeted and delayed effects of exposure to ionizing radiation: I. Radiation-induced genomic instability and bystander effects in vitro. Radiat. Res. 2003, 159:567-580. [26] Morgan WF: Non-targeted and delayed effects of exposure to ionizing radiation: II. Radiation-induced genomic instability and bystander effects in vivo, clastogenic factors and transgenerational effects. Radiat. Res. 2003, 159:581-596. [27] Morgan WF: Is there a common mechanism underlying genomic instability, bystander effects and other nontargeted effects of exposure to ionizing radiation? Oncogene 2003, 22:7094-7099. [28] Hendry JH: Genomic instability: potential contributions to tumour and normal tissue response, and second tumours, after radiotherapy. Radiother. Oncol. 2001, 59:117-126. [29] Suzuki K, Ojima M, Kodama S, Watanabe M: Radiation-induced DNA damage and delayed induced genomic instability. Oncogene 2003, 22:6988-6993. [30] Huang L, Snyder AR, Morgan WF: Radiation-induced genomic instability and its implications for radiation carcinogenesis. Oncogene 2003, 22:5848-5854. [31] Morgan WF, Day JP, Kaplan MI, McGhee EM, Limoli CL: Genomic instability induced by ionizing radiation. Radiat. Res. 1996, 146:247-258. [32] Wright EG: Radiation-induced genomic instability in haemopoietic cells. Int. J. Radiat. Biol. 1998, 74:681-687. [33] Coleman WB, Tsongalis GJ: The role of genomic instability in human carcinogenesis. Anticancer Res. 1999, 19:4645-4664. [34] Goldberg Z, Lehnert BE: Radiation-induced effects in unirradiated cells: a review and implications in cancer. Int. J. Oncol. 2002, 21:337-349. [35] Goldberg Z: Clinical implications of radiation-induced genomic instability. Oncogene 2003, 22:7011-7017. [36] Lorimore SA, McIlrath JM, Coates PJ, Wright EG: Chromosomal instability in unirradiated hemopoietic cells resulting from a delayed in vivo bystander effect of gamma radiation. Cancer Res. 2005, 65:5668-5673. [37] Hamada N, Ni M, Funayama T, Sakashita T, Kobayashi Y: Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions. Mutat. Res. 2008, 639:35-44. [38] Kanasugi Y, Hamada N, Wada S, Funayama T, Sakashita T, Kakizaki T, Kobayashi Y, Takakura K: Role of DNA-PKcs in the bystander effect after low- or high-LET irradiation. Int. J. Radiat. Biol. 2007, 83:73-80. [39] Hamada N, Matsumoto H, Hara T, Kobayashi Y: Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. J. Radiat. Res. (Tokyo) 2007, 48:87-95. [40] Matsumoto H, Hamada N, Takahashi A, Kobayashi Y, Ohnishi T: Vanguards of paradigm shift in radiation biology: radiation-induced adaptive and bystander responses. J. Radiat. Res. (Tokyo) 2007, 48:97-106. [41] Hei TK: Cyclooxygenase-2 as a signaling molecule in radiation-induced bystander effect. Mol. Carcinog 2006, 45:455-460. [42] Zhou H, Ivanov VN, Gillespie J, Geard CR, Amundson SA, Brenner DJ, Yu Z, Lieberman HB, Hei TK: Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway. Proc. Natl. Acad. Sci. U S A 2005, 102:1464114646.

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[43] Suzuki M, Zhou H, Hei TK, Tsuruoka C, Fujitaka K: Induction of a bystander chromosomal damage of He-ion microbeams in mammalian cells. Biol. Sci. Space 2003, 17:251-252. [44] Zhou H, Randers-Pehrson G, Geard CR, Brenner DJ, Hall EJ, Hei TK: Interaction between radiation-induced adaptive response and bystander mutagenesis in mammalian cells. Radiat. Res. 2003, 160:512-516. [45] Zhou H, Randers-Pehrson G, Suzuki M, Waldren CA, Hei TK: Genotoxic damage in non-irradiated cells: contribution from the bystander effect. Radiat. Prot. Dosimetry 2002, 99:227-232. [46] Klokov D, Criswell T, Leskov KS, Araki S, Mayo L, Boothman DA: IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects. Mutat. Res. 2004, 568:97-110. [47] Lorimore SA, Coates PJ, Wright EG: Radiation-induced genomic instability and bystander effects: inter-related nontargeted effects of exposure to ionizing radiation. Oncogene 2003, 22:7058-7069. [48] Lorimore SA, Wright EG: Radiation-induced genomic instability and bystander effects: related inflammatory-type responses to radiation-induced stress and injury? A review. Int. J. Radiat. Biol. 2003, 79:15-25. [49] Mothersill C, Lyng F, Seymour C, Maguire P, Lorimore S, Wright E: Genetic factors influencing bystander signaling in murine bladder epithelium after low-dose irradiation in vivo. Radiat. Res. 2005, 163:391-399. [50] Maguire P, Mothersill C, Seymour C, Lyng FM: Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes. Radiat. Res. 2005, 163:384-390. [51] Maguire P, Mothersill C, McClean B, Seymour C, Lyng FM: Modulation of radiation responses by pre-exposure to irradiated cell conditioned medium. Radiat. Res. 2007, 167:485-492. [52] Zhou H, Randers-Pehrson G, Waldren CA, Vannais D, Hall EJ, Hei TK: Induction of a bystander mutagenic effect of alpha particles in mammalian cells. Proc. Natl. Acad. Sci. U S A 2000, 97:2099-2104. [53] Sgouros G, Knox SJ, Joiner MC, Morgan WF, Kassis AI: MIRD continuing education: Bystander and low dose-rate effects: are these relevant to radionuclide therapy? J. Nucl. Med. 2007, 48:1683-1691. [54] Yang H, Asaad N, Held KD: Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts. Oncogene 2005, 24:2096-2103. [55] Liu Z, Mothersill CE, McNeill FE, Lyng FM, Byun SH, Seymour CB, Prestwich WV: A dose threshold for a medium transfer bystander effect for a human skin cell line. Radiat. Res. 2006, 166:19-23. [56] Sokolov MV, Dickey JS, Bonner WM, Sedelnikova OA: gamma-H2AX in bystander cells: not just a radiation-triggered event, a cellular response to stress mediated by intercellular communication. Cell Cycle 2007, 6:2210-2212. [57] Sedelnikova OA, Nakamura A, Kovalchuk O, Koturbash I, Mitchell SA, Marino SA, Brenner DJ, Bonner WM: DNA double-strand breaks form in bystander cells after

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Role of Epigenetic Changes in the Non-Targeted Radiation-Induced Effects

[58]

[59]

[60]

[61]

[62]

[63]

[64]

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

[65]

[66]

[67]

[68] [69] [70]

[71]

[72]

143

microbeam irradiation of three-dimensional human tissue models. Cancer Res. 2007, 67:4295-4302. Sokolov MV, Smilenov LB, Hall EJ, Panyutin IG, Bonner WM, Sedelnikova OA: Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts. Oncogene 2005, 24:7257-7265. Ilnytskyy Y, Koturbash I, Kovalchuk O: Radiation-induced bystander effects in vivo are epigenetically regulated in a tissue-specific manner. Environ Mol Mutagen 2009, 50:105-113. Koturbash I, Zemp FJ, Kutanzi K, Luzhna L, Loree J, Kolb B, Kovalchuk O: Sexspecific microRNAome deregulation in the shielded bystander spleen of cranially exposed mice. Cell Cycle 2008, 7:1658-1667. Koturbash I, Kutanzi K, Hendrickson K, Rodriguez-Juarez R, Kogosov D, Kovalchuk O: Radiation-induced bystander effects in vivo are sex specific. Mutat. Res. 2008, 642:28-36. Tamminga J, Koturbash I, Baker M, Kutanzi K, Kathiria P, Pogribny IP, Sutherland RJ, Kovalchuk O: Paternal cranial irradiation induces distant bystander DNA damage in the germline and leads to epigenetic alterations in the offspring. Cell Cycle 2008, 7:12381245. Koturbash I, Loree J, Kutanzi K, Koganow C, Pogribny I, Kovalchuk O: In vivo bystander effect: cranial X-irradiation leads to elevated DNA damage, altered cellular proliferation and apoptosis, and increased p53 levels in shielded spleen. Int. J. Radiat. Oncol. Biol. Phys. 2008, 70:554-562. Kovalchuk O, Baulch JE: Epigenetic changes and nontargeted radiation effects--is there a link? Environ. Mol. Mutagen. 2008, 49:16-25. Persaud R, Zhou H, Hei TK, Hall EJ: Demonstration of a radiation-induced bystander effect for low dose low LET beta-particles. Radiat. Environ. Biophys. 2007, 46:395400. Persaud R, Zhou H, Baker SE, Hei TK, Hall EJ: Assessment of low linear energy transfer radiation-induced bystander mutagenesis in a three-dimensional culture model. Cancer Res 2005, 65:9876-9882. Belyakov OV, Mitchell SA, Parikh D, Randers-Pehrson G, Marino SA, Amundson SA, Geard CR, Brenner DJ: Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away. Proc. Natl. Acad. Sci. U S A 2005, 102:1420314208. Mothersill C, Seymour CB: Radiation-induced bystander effects--implications for cancer. Nat. Rev. Cancer 2004, 4:158-164. Hall EJ: The bystander effect. Health Phys. 2003, 85:31-35. Mothersill C, Smith RW, Agnihotri N, Seymour CB: Characterization of a radiationinduced stress response communicated in vivo between zebrafish. Environ. Sci. Technol. 2007, 41:3382-3387. Mothersill C, Bucking C, Smith RW, Agnihotri N, Oneill A, Kilemade M, Seymour CB: Communication of radiation-induced stress or bystander signals between fish in vivo. Environ. Sci. Technol. 2006, 40:6859-6864. Koturbash I, Boyko A, Rodriguez-Juarez R, McDonald RJ, Tryndyak VP, Kovalchuk I, Pogribny IP, Kovalchuk O: Role of epigenetic effectors in maintenance of the longterm persistent bystander effect in spleen in vivo. Carcinogenesis 2007, 28:1831-1838.

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Jody Filkowski, Yaroslav Ilnytsky and Olga Kovalchuk

[73] Koturbash I, Rugo RE, Hendricks CA, Loree J, Thibault B, Kutanzi K, Pogribny I, Yanch JC, Engelward BP, Kovalchuk O: Irradiation induces DNA damage and modulates epigenetic effectors in distant bystander tissue in vivo. Oncogene 2006, 25:4267-4275. [74] Marozik P, Mothersill C, Seymour CB, Mosse I, Melnov S: Bystander effects induced by serum from survivors of the Chernobyl accident. Exp. Hematol. 2007, 35:55-63. [75] Hollowell JG, Jr., Littlefield LG: Chromosome damage induced by plasma of x-rayed patients: an indirect effect of x-ray. Proc. Soc. Exp. Biol. Med. 1968, 129:240-244. [76] Goh K, Sumner H: Breaks in normal human chromosomes: are they induced by a transferable substance in the plasma of persons exposed to total-body irradiation? Radiat. Res. 1968, 35:171-181. [77] Emerit I, Oganesian N, Sarkisian T, Arutyunyan R, Pogosian A, Asrian K, Levy A, Cernjavski L: Clastogenic factors in the plasma of Chernobyl accident recovery workers: anticlastogenic effect of Ginkgo biloba extract. Radiat. Res. 1995, 144:198205. [78] Emerit I, Quastel M, Goldsmith J, Merkin L, Levy A, Cernjavski L, Alaoui-Youssefi A, Pogossian A, Riklis E: Clastogenic factors in the plasma of children exposed at Chernobyl. Mutat. Res. 1997, 373:47-54. [79] Pant GS, Kamada N: Chromosome aberrations in normal leukocytes induced by the plasma of exposed individuals. Hiroshima J. Med. Sci. 1977, 26:149-154. [80] Pant GS, Kamada N, Tanaka R: Sister chromatid exchanges in peripheral lymphocytes of atomic bomb survivors and of normal individuals exposed to radiation and chemical agents. Hiroshima J. Med. Sci. 1976, 25:99-105. [81] Emerit I, Levy A, Cernjavski L, Arutyunyan R, Oganesyan N, Pogosian A, Mejlumian H, Sarkisian T, Gulkandanian M, Quastel M, et al.: Transferable clastogenic activity in plasma from persons exposed as salvage personnel of the Chernobyl reactor. J. Cancer Res. Clin. Oncol. 1994, 120:558-561. [82] Brooks AL: Evidence for 'bystander effects' in vivo. Hum. Exp. Toxicol. 2004, 23:6770. [83] Khan MA, Van Dyk J, Yeung IW, Hill RP: Partial volume rat lung irradiation; assessment of early DNA damage in different lung regions and effect of radical scavengers. Radiother. Oncol. 2003, 66:95-102. [84] Khan MA, Hill RP, Van Dyk J: Partial volume rat lung irradiation: an evaluation of early DNA damage. Int. J. Radiat. Oncol. Biol. Phys. 1998, 40:467-476. [85] Brooks AL, Retherford JC, McClellan RO: Effect of 239PuO2 particle number and size on the frequency and distribution of chromosome aberrations in the liver of the Chinese hamster. Radiat. Res. 1974, 59:693-709. [86] Fakir H, Hofmann W, Tan WY, Sachs RK: Triggering-response model for radiationinduced bystander effects. Radiat. Res. 2009, 171:320-331. [87] Wright EG, Coates PJ: Untargeted effects of ionizing radiation: implications for radiation pathology. Mutat. Res. 2006, 597:119-132. [88] Nagar S, Smith LE, Morgan WF: Characterization of a novel epigenetic effect of ionizing radiation: the death-inducing effect. Cancer Res. 2003, 63:324-328. [89] Barber RC, Dubrova YE: The offspring of irradiated parents, are they stable? Mutat. Res. 2006, 598:50-60.

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145

[90] Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Neumann R, Neil DL, Jeffreys AJ: Human minisatellite mutation rate after the Chernobyl accident. Nature 1996, 380:683-686. [91] Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Vergnaud G, Giraudeau F, Buard J, Jeffreys AJ: Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident. Mutat. Res. 1997, 381:267-278. [92] Dubrova YE: Monitoring of radiation-induced germline mutation in humans. Swiss Med. Wkly 2003, 133:474-478. [93] Dubrova YE, Grant G, Chumak AA, Stezhka VA, Karakasian AN: Elevated minisatellite mutation rate in the post-chernobyl families from ukraine. Am. J. Hum. Genet. 2002, 71:801-809. [94] Lindholm C, Murphy BP, Bigbee WL, Bersimbaev RI, Hulten MA, Dubrova YE, Salomaa S: Glycophorin A somatic cell mutations in a population living in the proximity of the Semipalatinsk nuclear test site. Radiat. Res. 2004, 162:164-170. [95] Salomaa S, Lindholm C, Tankimanova MK, Mamyrbaeva Z, Koivistoinen A, Hulten M, Mustonen R, Dubrova YE, Bersimbaev RI: Stable chromosome aberrations in the lymphocytes of a population living in the vicinity of the Semipalatinsk nuclear test site. Radiat. Res. 2002, 158:591-596. [96] Akleev AV, Dubrova Iu E, Ploshchanskaia OG, Kozionova OS: [The effects of chronic radiation exposure on the frequency of mutations at minisatellite DNA loci in residents of the Techa Riverside Villages]. Radiats Biol. Radioecol. 2007, 47:558-566. [97] Satoh C, Takahashi N, Asakawa J, Kodaira M, Kuick R, Hanash SM, Neel JV: Genetic analysis of children of atomic bomb survivors. Environ. Health Perspect 1996, 104 Suppl 3:511-519. [98] Generoso WM, Bishop JB, Gosslee DG, Newell GW, Sheu CJ, von Halle E: Heritable translocation test in mice. Mutat. Res. 1980, 76:191-215. [99] Russell WL, Kelly EM: Mutation frequencies in male mice and the estimation of genetic hazards of radiation in men. Proc. Natl. Acad. Sci. U S A 1982, 79:542-544. [100] Russell WL, Bangham JW, Russell LB: Differential response of mouse male germ-cell stages to radiation-induced specific-locus and dominant mutations. Genetics 1998, 148:1567-1578. [101] Nomura T, Okamoto E, Gotoh H, Namba T, Tanaka H, Sakamoto Y: Diminution of chemically induced lung neoplasia in mice by phenobarbital pre-treatment. Cancer Lett. 1982, 17:33-36. [102] Nomura T: Parental exposure to x rays and chemicals induces heritable tumours and anomalies in mice. Nature 1982, 296:575-577. [103] Nomura T: X-ray- and chemically induced germ-line mutation causing phenotypical anomalies in mice. Mutat. Res. 1988, 198:309-320. [104] Luning KG, Frolen H, Nilsson A: Genetic effects of 239Pu salt injections in male mice. Mutat. Res. 1976, 34:539-542. [105] Tomatis L: Transgeneration carcinogenesis: a review of the experimental and epidemiological evidence. Jpn J. Cancer Res. 1994, 85:443-454. [106] Mohr U, Dasenbrock C, Tillmann T, Kohler M, Kamino K, Hagemann G, Morawietz G, Campo E, Cazorla M, Fernandez P, et al: Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice. Carcinogenesis 1999, 20:325-332.

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Jody Filkowski, Yaroslav Ilnytsky and Olga Kovalchuk

[107] Nomura T, Nakajima H, Ryo H, Li LY, Fukudome Y, Adachi S, Gotoh H, Tanaka H: Transgenerational transmission of radiation- and chemically induced tumors and congenital anomalies in mice: studies of their possible relationship to induced chromosomal and molecular changes. Cytogenet. Genome Res. 2004, 104:252-260. [108] Nomura T: Transgenerational carcinogenesis: induction and transmission of genetic alterations and mechanisms of carcinogenesis. Mutat. Res. 2003, 544:425-432. [109] Dasenbrock C, Tillmann T, Ernst H, Behnke W, Kellner R, Hagemann G, Kaever V, Kohler M, Rittinghausen S, Mohr U, Tomatis L: Maternal effects and cancer risk in the progeny of mice exposed to X-rays before conception. Exp. Toxicol. Pathol. 2005, 56:351-360. [110] Balentova S, Slovinska L, Misurova E, Rybarova S, Adamkov M: Effect of paternal rat irradiation transmitted to the progeny during prenatal development. Folia Biol. (Praha) 2008, 54:151-156. [111] Slovinska L, Elbertova A, Misurova E: Transmission of genome damage from irradiated male rats to their progeny. Mutat. Res. 2004, 559:29-37. [112] Carls N, Schiestl RH: Effect of ionizing radiation on transgenerational appearance of p(un) reversions in mice. Carcinogenesis 1999, 20:2351-2354. [113] Barber R, Plumb M, Smith AG, Cesar CE, Boulton E, Jeffreys AJ, Dubrova YE: No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin. Mutat. Res. 2000, 457:79-91. [114] Barber R, Plumb MA, Boulton E, Roux I, Dubrova YE: Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice. Proc. Natl. Acad. Sci. U S A 2002, 99:6877-6882. [115] Barber RC, Hardwick RJ, Shanks ME, Glen CD, Mughal SK, Voutounou M, Dubrova YE: The effects of in utero irradiation on mutation induction and transgenerational instability in mice. Mutat. Res. 2009, 664:6-12. [116] Barber RC, Hickenbotham P, Hatch T, Kelly D, Topchiy N, Almeida GM, Jones GD, Johnson GE, Parry JM, Rothkamm K, Dubrova YE: Radiation-induced transgenerational alterations in genome stability and DNA damage. Oncogene 2006, 25:7336-7342. [117] Dubrova YE, Plumb MA: Ionising radiation and mutation induction at mouse minisatellite loci. The story of the two generations. Mutat. Res. 2002, 499:143-150. [118] Dubrova YE: Radiation-induced transgenerational instability. Oncogene 2003, 22:70877093. [119] Dubrova YE: Germline mutation induction at mouse and human tandem repeat DNA loci. Adv. Exp. Med. Biol. 2003, 518:115-129. [120] Niwa O: Induced genomic instability in irradiated germ cells and in the offspring; reconciling discrepancies among the human and animal studies. Oncogene 2003, 22:7078-7086. [121] Jirtle RL, Skinner MK: Environmental epigenomics and disease susceptibility. Nat. Rev. Genet 2007, 8:253-262. [122] Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003, 33 Suppl:245-254. [123] Munshi A, Shafi G, Aliya N, Jyothy A: Histone modifications dictate specific biological readouts. J. Genet. Genomics 2009, 36:75-88.

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147

[124] Rountree MR, Bachman KE, Herman JG, Baylin SB: DNA methylation, chromatin inheritance, and cancer. Oncogene 2001, 20:3156-3165. [125] Baylin SB, Ohm JE: Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat. Rev. Cancer 2006, 6:107-116. [126] Baylin SB: DNA methylation and gene silencing in cancer. Nat. Clin. Pract. Oncol. 2005, 2 Suppl 1:S4-11. [127] Baylin SB, Chen WY: Aberrant gene silencing in tumor progression: implications for control of cancer. Cold Spring Harb. Symp. Quant. Biol. 2005, 70:427-433. [128] Weber M, Schubeler D: Genomic patterns of DNA methylation: targets and function of an epigenetic mark. Curr. Opin. Cell Biol. 2007, 19:273-280. [129] Goll MG, Bestor TH: Eukaryotic cytosine methyltransferases. Annu. Rev. Biochem. 2005, 74:481-514. [130] Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D: Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 2007, 39:457-466. [131] Wilson IM, Davies JJ, Weber M, Brown CJ, Alvarez CE, MacAulay C, Schubeler D, Lam WL: Epigenomics: mapping the methylome. Cell Cycle 2006, 5:155-158. [132] Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D: Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 2005, 37:853-862. [133] Robertson KD: DNA methylation, methyltransferases, and cancer. Oncogene 2001, 20:3139-3155. [134] Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, Laird PW, Jones PA: Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol. Cell Biol. 2002, 22:480-491. [135] Okano M, Bell DW, Haber DA, Li E: DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999, 99:247257. [136] Klose RJ, Bird AP: Genomic DNA methylation: the mark and its mediators. Trends Biochem. Sci. 2006, 31:89-97. [137] Robertson KD, Wolffe AP: DNA methylation in health and disease. Nat. Rev. Genet 2000, 1:11-19. [138] Robertson KD: DNA methylation and chromatin - unraveling the tangled web. Oncogene 2002, 21:5361-5379. [139] Hendrich B, Tweedie S: The methyl-CpG binding domain and the evolving role of DNA methylation in animals. Trends Genet. 2003, 19:269-277. [140] Weidman JR, Dolinoy DC, Murphy SK, Jirtle RL: Cancer susceptibility: epigenetic manifestation of environmental exposures. Cancer J. 2007, 13:9-16. [141] Jenuwein T, Allis CD: Translating the histone code. Science 2001, 293:1074-1080. [142] Cheung P, Lau P: Epigenetic regulation by histone methylation and histone variants. Mol. Endocrinol. 2005, 19:563-573. [143] Saha A, Wittmeyer J, Cairns BR: Chromatin remodelling: the industrial revolution of DNA around histones. Nat. Rev. Mol. Cell Biol. 2006, 7:437-447. [144] He S, Dunn KL, Espino PS, Drobic B, Li L, Yu J, Sun JM, Chen HY, Pritchard S, Davie JR: Chromatin organization and nuclear microenvironments in cancer cells. J. Cell Biochem. 2008, 104:2004-2015.

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148

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[145] Bernstein E, Allis CD: RNA meets chromatin. Genes Dev. 2005, 19:1635-1655. [146] Hwang HW, Mendell JT: MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br. J. Cancer 2006, 94:776-780. [147] Sevignani C, Calin GA, Siracusa LD, Croce CM: Mammalian microRNAs: a small world for fine-tuning gene expression. Mamm. Genome 2006, 17:189-202. [148] Hutvagner G, Zamore PD: A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002, 297:2056-2060. [149] Hutvagner G, Zamore PD: RNAi: nature abhors a double-strand. Curr. Opin. Genet. Dev. 2002, 12:225-232. [150] Chang TC, Mendell JT: microRNAs in vertebrate physiology and human disease. Annu Rev. Genomics Hum. Genet. 2007, 8:215-239. [151] Fabbri M, Ivan M, Cimmino A, Negrini M, Calin GA: Regulatory mechanisms of microRNAs involvement in cancer. Expert Opin. Biol. Ther. 2007, 7:1009-1019. [152] Kaup S, Grandjean V, Mukherjee R, Kapoor A, Keyes E, Seymour CB, Mothersill CE, Schofield PN: Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes. Mutat. Res. 2006, 597:87-97. [153] Sedelnikova OA, Horikawa I, Redon C, Nakamura A, Zimonjic DB, Popescu NC, Bonner WM: Delayed kinetics of DNA double-strand break processing in normal and pathological aging. Aging Cell 2008, 7:89-100. [154] Wiley LM, Baulch JE, Raabe OG, Straume T: Impaired cell proliferation in mice that persists across at least two generations after paternal irradiation. Radiat. Res. 1997, 148:145-151. [155] Vance MM, Wiley LM: Gap junction intercellular communication mediates the competitive cell proliferation disadvantage of irradiated mouse preimplantation embryos in aggregation chimeras. Radiat. Res. 1999, 152:544-551. [156] Baulch JE, Raabe OG, Wiley LM, Overstreet JW: Germline drift in chimeric male mice possessing an F2 component with a paternal F0 radiation history. Mutagenesis 2002, 17:9-13. [157] Baulch JE, Raabe OG, Wiley LM: Heritable effects of paternal irradiation in mice on signaling protein kinase activities in F3 offspring. Mutagenesis 2001, 16:17-23. [158] Vance MM, Baulch JE, Raabe OG, Wiley LM, Overstreet JW: Cellular reprogramming in the F3 mouse with paternal F0 radiation history. Int. J. Radiat. Biol. 2002, 78:513526. [159] Baulch JE, Raabe OG: Gamma irradiation of Type B spermatogonia leads to heritable genomic instability in four generations of mice. Mutagenesis 2005, 20:337-343. [160] Baulch JE, Li MW, Raabe OG: Effect of ATM heterozygosity on heritable DNA damage in mice following paternal F0 germline irradiation. Mutat Res 2007, 616:34-45. [161] Dubrova YE: Long-term genetic effects of radiation exposure. Mutat Res 2003, 544:433-439. [162] Dubrova YE: Radiation-induced mutation at tandem repeat DNA Loci in the mouse germline: spectra and doubling doses. Radiat. Res. 2005, 163:200-207. [163] Dubrova YE, Bersimbaev RI, Djansugurova LB, Tankimanova MK, Mamyrbaeva Z, Mustonen R, Lindholm C, Hulten M, Salomaa S: Nuclear weapons tests and human germline mutation rate. Science 2002, 295:1037.

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[164] Dubrova YE, Hickenbotham P, Glen CD, Monger K, Wong HP, Barber RC: Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice. Environ. Mol. Mutagen. 2008, 49:308-311. [165] Dubrova YE, Jeffreys AJ, Malashenko AM: Mouse minisatellite mutations induced by ionizing radiation. Nat. Genet. 1993, 5:92-94. [166] Dubrova YE, Plumb M, Brown J, Boulton E, Goodhead D, Jeffreys AJ: Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to gammaradiation and fission neutrons. Mutat. Res. 2000, 453:17-24. [167] Dubrova YE, Plumb M, Brown J, Fennelly J, Bois P, Goodhead D, Jeffreys AJ: Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation. Proc. Natl. Acad. Sci. U S A 1998, 95:6251-6255. [168] Dubrova YE, Plumb M, Brown J, Jeffreys AJ: Radiation-induced germline instability at minisatellite loci. Int. J. Radiat. Biol. 1998, 74:689-696. [169] Dubrova YE, Plumb M, Gutierrez B, Boulton E, Jeffreys AJ: Transgenerational mutation by radiation. Nature 2000, 405:37. [170] Barber RC, Miccoli L, van Buul PP, Burr KL, van Duyn-Goedhart A, Angulo JF, Dubrova YE: Germline mutation rates at tandem repeat loci in DNA-repair deficient mice. Mutat. Res. 2004, 554:287-295. [171] Koturbash I, Baker M, Loree J, Kutanzi K, Hudson D, Pogribny I, Sedelnikova O, Bonner W, Kovalchuk O: Epigenetic dysregulation underlies radiation-induced transgenerational genome instability in vivo. Int. J. Radiat. Oncol. Biol. Phys. 2006, 66:327-330. [172] Yu F, Zingler N, Schumann G, Stratling WH: Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription. Nucleic Acids Res. 2001, 29:4493-4501. [173] Xu GL, Bestor TH, Bourc'his D, Hsieh CL, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ, Viegas-Pequignot E: Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 1999, 402:187-191. [174] Hatch T, Derijck AA, Black PD, van der Heijden GW, de Boer P, Dubrova YE: Maternal effects of the scid mutation on radiation-induced transgenerational instability in mice. Oncogene 2007, 26:4720-4724. [175] Assis R, Kondrashov AS: Rapid repetitive element-mediated expansion of piRNA clusters in mammalian evolution. Proc. Natl. Acad. Sci. U S A 2009, 106:7079-7082. [176] Wang J, Saxe JP, Tanaka T, Chuma S, Lin H: Mili interacts with tudor domaincontaining protein 1 in regulating spermatogenesis. Curr. Biol. 2009, 19:640-644. [177] Aravin AA, Hannon GJ: Small RNA silencing pathways in germ and stem cells. Cold Spring Harb. Symp. Quant. Biol. 2008, 73:283-290. [178] Reuter M, Chuma S, Tanaka T, Franz T, Stark A, Pillai RS: Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile. Nat. Struct. Mol. Biol. 2009. [179] Aravin AA, Hannon GJ, Brennecke J: The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 2007, 318:761-764. [180] Yin H, Lin H: An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster. Nature 2007, 450:304-308.

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[181] Chu CY, Rana TM: Small RNAs: regulators and guardians of the genome. J. Cell Physiol. 2007, 213:412-419. [182] Aravin AA, Sachidanandam R, Girard A, Fejes-Toth K, Hannon GJ: Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 2007, 316:744747. [183] O'Donnell KA, Boeke JD: Mighty Piwis defend the germline against genome intruders. Cell 2007, 129:37-44. [184] Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Hannon GJ: Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 2007, 128:1089-1103. [185] Aravin AA, Sachidanandam R, Bourc'his D, Schaefer C, Pezic D, Toth KF, Bestor T, Hannon GJ: A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 2008, 31:785-799. [186] Brennecke J, Malone CD, Aravin AA, Sachidanandam R, Stark A, Hannon GJ: An epigenetic role for maternally inherited piRNAs in transposon silencing. Science 2008, 322:1387-1392. [187] Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, Asada N, Kojima K, Yamaguchi Y, Ijiri TW, et al: DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes. Dev. 2008, 22:908-917.

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Chapter 9

A REVIEW OF TANDEM REPEAT SEQUENCES: MARKERS OF GENETIC INSTABILITY Carole Yauk and John D. H. Stead Environmental Health Science and Research Bureau, Health Canada, Tunney's Pasture, 50 Columbine Driveway, Ottawa, Ontario, Canada

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ABSTRACT The study of germline mutations is extremely difficult because rates of mutation are extremely low for gene sequence DNA. An alternative approach uses highly variable non-coding regions of the genome that possess rates of mutations that are orders of magnitude greater than protein-coding sequences. Mutations result from gains and losses of repeat units that arise during replication, recombination and repair. Germline mutation frequencies range from 1 – 20% and facilitate the measurement of induced mutations in small sample sizes. Mutations can be detected in pedigrees using Southern blotting, or by PCR analysis of sperm DNA. Expanded simple tandem repeat loci (ESTR) and minisatellites have been shown to undergo elevated rates of mutation following exposure to radiation and chemicals. As such, the study of repeat mutations in populations exposed to toxicants in the environment showed that induced germline mutations may result following exposure to ambient levels of contaminants. In this chapter, we review the current state of knowledge in this field of study and provide an overview of repeat types and structure, the mechanisms of repeat mutations, the methods used to study mutations and discuss some experiments applying repeat analysis to study germline mutations.

OVERVIEW: CHERNOBYL ACCIDENT On April 26th 1986, the nuclear reactor at the Chernobyl nuclear power plant in the Ukranian Soviet Socialist Republica (now Ukraine) exploded in what remains the world‘s worst nuclear disaster. Though the loss of life within the first month of the accident was limited to 30 power plant employees and firemen, the health effects for the broader population were profound. This resulted from the release of large amounts of radioactive isotopes into the environment, contaminating broad areas of Ukraine, Belarus and Russia and

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exposing the population to radiation. Furthermore, the clean-up operation following the disaster exposed about 600,000 civilian and military personnel (termed the ‗liquidators‘) to even higher levels of radiation from the local region. The impact on the local population and economy was profound, with hundreds of thousands of people being forced to leave their homes and jobs to escape the contamination. Despite the evacuation, long-term health effects for the exposed population have been described, with about 4000 cases of thyroid cancer identified in exposed individuals, many of which are likely to result directly from the exposure. In addition to the direct health effects on those exposed to radiation, DNA mutation research later discovered that even children of exposed parents born years after the disaster were potentially impacted through new mutations in their DNA that had been inherited from their parents. Thus exposure of parents to radiation led to increased DNA mutations in the parents, and these mutations were passed on to the next generation. While it should be stressed that no adverse health effects have been attributed to these induced mutations, these results suggest that environmental factors can impact the rate of heritable mutations. These induced mutations were first identified by Dubrova et al. (1996) [1]. By comparing DNA profiles of parents and offspring born after the accident, this initial study revealed that heritable mutation rates were twice as high for a population from Belarus (where an estimated ~60% of the fallout from Chernobyl landed) compared with a control population from the UK. Identification of an increase in DNA mutation rates is technically very challenging, as the mutation rate of most DNA sequences is very low (~1 mutation per billion nucleotides). Therefore, focusing on a single nucleotide, it would be necessary to screen 1 billion families in order to identify a single DNA mutation. The number of individuals required to detect a significant doubling of DNA mutation rate would thus be larger than the current global population. So, how was an increase in mutation detected? The study focused on regions of tandemly repetitive DNA, which normally displays an unusually high mutation rate. This dramatically reduces the number of families required for detecting mutation events, and therefore allows changes in mutation frequency to be investigated. Furthermore, a technique was employed that allows the simultaneous detection of many different tandem repeat regions, increasing the probability of detecting mutations – this technique is DNA fingerprinting. This chapter will first introduce various classes of tandemly repetitive DNA sequences that are used in de novo mutation detection studies and describe what is currently known about their mechanisms of mutation. We will then introduce common techniques used to detect tandem repeat mutations, including approaches designed to increase the ease with which new heritable mutations can be identified using a relatively small number of individuals. These techniques include DNA fingerprinting, which analyzes many different loci increasing the chances of detecting a mutation in a single parent-offspring transmission, plus PCR-based techniques that detect new mutations directly from sperm DNA, allowing for analyses of millions of potential offspring from a single father. Finally, we will review recent case studies that have used these techniques to examine the impact of the environment on DNA mutation rates.

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CLASSES OF TANDEMLY REPETITIVE DNA IN MUTATION DETECTION ANALYSIS Tandemly repetitive DNA is formed when a specific sequence is repeated end-on-end many times. Different classes of tandem repeats exist in eukaryote genomes, with classes having differences between the length of the individual repeat unit, the total number of tandem repeats in the array, the location of the array within the genome, and the mechanisms which underlie the generation and mutation of the arrays. Here, we will focus exclusively on the two classes of tandem repeats that have been studied most extensively in the context of induced DNA mutation: minisatellites and expanded simple tandem repeats (ESTRs).

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Minisatellites/VNTRs Minisatellites, also termed VNTRs (Variable Number of Tandem Repeats), are composed of 6-100 bp long repeat units, which form arrays up to 30 kb in total length. Small sequence differences typically exist between various repeat units in an array. Minisatellite alleles can therefore vary both in the total number of repeats (the allele length) and in the specific order of variant repeat types within the array (the allele structure), leading to potentially enormous allele diversity within a population. This diversity is driven by high minisatellite mutation rates, which can be up to 25% in sperm DNA (thus one in four offspring will carry a mutant allele compared to their father) [2], though mutation rates do vary dramatically between loci (to below 1 in 10,000), and to a lesser extent between individuals. The resulting allele diversity in populations underlies the individual-specific nature of DNA fingerprinting, which simultaneously detects many different minisatellite loci. As will be described, minisatellites also form the basis of research using tandem repeat loci to study induced mutations in human populations.

Expanded Simple Tandem Repeats ESTRs were first identified through DNA fingerprinting and as a result were initially misclassified as minisatellites. However, they are now understood as being a distinct class of tandem repeats composed of shorter repeat units (typically 1%) in 61 offspring [31] and found no evidence of an effect. Thus, it appears that exposure to ionizing radiation from A-bombs may not impact the germline. However, these data should be interpreted with caution because a large portion of time passed between exposure and measurements of mutation frequency. The average time between birth and A-bomb was approximately 15 years. Therefore, signals or effects resulting from radiation exposure may have no longer been expressed in those parents. Moreover, the probability of detecting a false negative was estimated to be relatively high because the number of individuals studied was quite low. Chronic Radiation Exposure: Chernobyl and Nuclear Bomb Test Sites In contrast to acute toxicity, which arises as a result of exposure to a high dose over a very short period of time, chronic toxicity results from prolonged or continuous exposure to lower doses. Thus, exposure to A-bomb radiation is considered acute, while inhabiting the contaminated sites near radioactive contamination is a chronic exposure. Families in the Chernobyl environment were initially exposed acutely to a high dose of short-lived isotopes, but if they continued to live in the area were exposed both internally and externally to more

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stable caesium-137. Three studies have demonstrated that chronic exposure to ionizing radiation from the Chernobyl explosion results in significant increases in germline mutation [1, 32, 33]. Using similar approaches and similar repeat loci to the A-bomb studies, Dubrova et al., found an increase in mutation frequency in families in the Chernobyl area compared to a British cohort, as well as a correlation between estimated dose and mutation frequency. The most convincing evidence demonstrated that mutation frequency in offspring conceived after the Chernobyl accident was significantly higher than those conceived before the accident (and therefore not exposed to radiation), and also higher than matched controls from the same area. A significant 1.6-fold increase in mutation frequency was found for children born after the accident resulting from paternal mutation. This result, combined with similar data from the authors‘ previous two studies, provide compelling evidence that chronic exposure to internal and external sources of radiation in the Chernobyl area results in heritable mutation. Further work examining chronic radiation exposure explored human populations living near a site of past nuclear bomb testing [34]. From 1949 to 1989 the Semipalatinsk nuclear test site was used for 470 surface and underground nuclear bomb tests. Humans living near the site were exposed to fresh fallout from four of the surface explosions conducted between 1949 and 1956. These explosions generated chronic contamination in the areas down-wind of the test site. Current levels of contamination at the site are now low as a result of radionuclide decay. Using a pedigree approach, Dubrova et al. investigated three-generation families inhabiting the rural areas down-wind of the test sites, alongside three-generation families from a non-contaminated, geographically similar rural area. Mutation frequency was elevated by 1.8-fold in the first generation, and 1.5-fold in the subsequent generation compared to the control group. The decay of radionuclides over time is thought to result in a lower mutation frequency in the second generation relative to the first. In support of this, the authors found a negative relationship between the parents‘ year of birth and mutation frequency. Thus, the authors postulate that germline mutation at repeat regions resulted from relatively uniform high-dose chronic exposures predominantly in the 1940s and 1950s. However, there were no dose estimates within the cohort, and thus specific exposure is unconfirmed. Unfortunately, the authors could not partition internal versus external exposure, and thus could not make a direct comparison to the Chernobyl or A-bomb studies.

Fractionated Radiation Exposure: Chernobyl Workers and Radiotherapy Patients Chernobyl liquidators were civilian and military personnel who were involved in the clean-up operation following the explosion. These individuals were repeatedly exposed to small daily external doses of radiation. A number of studies have measured mutations in the offspring of these individuals [35-38]. Although a few studies found a non-significant increase in mutation frequency at a few selected loci, none of these studies demonstrated convincingly that these fractionated exposures affect the germline. The lack of mutation induction following fractionated exposure is supported by research in males undergoing radiotherapy, another form of fractionated external radiation exposure. Studies on these males are ideal for determining potential germline effects because exposure is precisely known, and genetic confounders are eliminated by using a pre-radiation sample as a control for each male (e.g., sperm samples collected prior to and following radiotherapy). Three studies applied SP-PCR at various hypervariable minisatellites to determine the effects of radiotherapy (sometimes in combination with chemotherapy) on mutation frequency [3941]. None of these studies demonstrated marked increases in germline instability following

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treatment. For example, May et al. [39] examined three patients who received 15 hemipelvic radiotherapy treatments over a 3-week period, yielding a total testicular X-ray dose between 0.38 and 0.82 Gy. None of the men, sampled across various stages of spermatogenesis, showed any evidence for an increase in repeat instability. These findings corroborate data demonstrating no observable increases in birth defects in offspring of patients treated with chemo- and radiotherapy [42-45]. However, a very small number of men have been studied across heterogeneous treatment types and diverse stages of spermatogenesis. Furthermore, genetic differences between the men and the limited number of alleles studied should be considered. Thus, these results should be viewed as being tentative.

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Impact of Air Pollution A series of studies on both wild and laboratory animals has provided compelling evidence to demonstrate that exposure to ambient air pollution causes mutation in the germline. These studies focused on industrial environments that are in populated urban centers, within a short distance from heavily used highways and various industries. In 1996, Yauk et al. [46] used multi-locus DNA fingerprinting to study mutation in full families of herring gulls living near steel mills. Herring gull offspring sampled from colonies situated in cities with steel mills had increased mutation frequencies relative to gulls from rural locations. Moreover, mutation frequencies declined with increasing distance of the colonies from coking ovens of steel mills [47]. The results provided the first demonstration that ambient air pollutants may impact the germline. However, because wild birds were the test organism, there were other potentially confounding variables that could not be controlled. For example, the nutritional status, disease burden, age of birds and genetic composition may have influenced mutation rates. More importantly, it was not possible to determine whether the primary source of exposure was inhalation of contaminated air or ingestion of contaminated fish. Thus, the potential relevance to humans and other animals in these areas was unknown. Follow-up work examined outbred Swiss-Webster mice housed in cages located 1 km in a predominantly down-wind direction of two steel industries and near a heavily trafficked highway. This removed confounding variables (such as diet, age and disease) and examined the role of air pollution exclusively. Germline mutation frequencies in mice from the steel mill environment were compared to: a) mice from a reference location outside the city [48] b) mice caged in the industrial environment but breathing air that passed through a high-efficiency particulate air filter (HEPA: removes 99.97% of particles > 0.3 M in diameter) [49] Mice from the various groups were held for 10 weeks in the environment and then brought back to the laboratory for mating. After 6 weeks in the laboratory (to allow exposed spermatogonial stem cells to mature), mice were mated and ESTR mutations arising in offspring were quantified. A significant 1.5- to 2-fold increase in ESTR mutation rate was observed in offspring of male mice housed at the steel location compared to the rural location. Moreover, mutation frequencies returned to reference levels when particles were removed from the air using the HEPA filters. Thus, particles in the air in the steel mill environment

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cause germline mutation. However, the identity of mutagens associated with these particles is unknown. It was hypothesized that large polycyclic aromatic hydrocarbons were the likely culprits. The above work explored mutation in outbred strains of mice and used the pedigree approach. Therefore, further work was carried out to eliminate genetic confounders, to confirm that mutations arose in sperm and not in the developing embryo, and to investigate the types of lesions that may lead to induced mutation following exposure to particulate air pollutant. In this work, inbred male mice were housed in the environment alongside mice breathing HEPA-filtered air [50]. ESTR mutation was measured directly in sperm using SMPCR. Sperm mutation frequency was 1.6 fold higher in mice breathing particle-contaminated air than in those from HEPA-filtered cages. To investigate what chemicals may contribute to this effect, the presence of chemicals bound to DNA (DNA adducts such as those made by polycyclic aromatic hydrocarbons) were measured. Adduct levels were below detection threshold in germ cells, indicating that DNA reactive chemicals do not penetrate the germline and cause ESTR mutation. However, DNA strand breaks were elevated in mice exposed to unfiltered air. Thus, oxidative stress that results from exposure to particles, polycyclic aromatic hydrocarbons or metals may play a role in induced germline mutation in this environment. Lastly, the authors also found that sperm DNA was hypermethylated in mice breathing unfiltered air, suggesting an epigenetic mechanism may also influence mutation frequency. Therefore, animals inhabiting this environment experience increased rates of germline mutation, DNA damage and methylation. The population-level effects that result from changes to the germline are unknown, but they may correlate with: increased genetic disease if tandem repeat mutation is correlated with gene mutation, changes in chromatin structure, gene expression and genome stability.

CONCLUSIONS Tandem repeat loci such as minisatellites and ESTRs provide powerful analytical tools for studying spontaneous and induced mutation. The extremely high mutation frequencies at these regions have allowed the development of the most sensitive methods available today to study de novo mutation at the molecular level. Moreover, these methods are capable of identifying mutagenic agents that can cause an increase in germline mutation rates in small sample sizes and at low doses. In natural populations, exposure to ambient levels of environmental contaminants can result in induced germline mutation at repeat loci. Chronic exposure to radiation (such as that encountered by people living near the Chernobyl power plant) correlates with increased germline repeat mutation frequencies. Particulate air pollution in cities with steel mills also correlates with an increase in mutation frequency at repeat regions, both in laboratory mice and wild populations of gulls. Increased mutation rates may be related to epigenetic modifications causing changes in DNA structure and function. Much more work is needed to characterize the potential repercussions of increased tandem repeat mutation rates. It should be noted that although the loci described are non-coding, numerous tandem repeat regions are associated with genes and regulatory regions and may play a role in disease. For example, several neurodegenerative disorders are associated with trinucleotide

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repeats (reviewed in [51-54]). A number of minisatellites are also associated with disease genes (e.g., insulin, dopamine receptor D4) and have been implicated in disease susceptibility (e.g., diabetes, depression, epilepsy) [55-57]. Therefore, some repeat mutations may have consequences for human health, and as a result, non-coding repeat mutation should not be considered unimportant. The extent to which results from tandrem repeat studies can be generalized to the health effects of environmental mutagens in the context of risk assessment is unclear. The repeat regions examined represent a distinct class of DNA sequences that mutate by a potentially unusual mechanism. Further work needs to be carried out to precisely characterize the molecular mechanisms operating in spontaneous and induced mutation and to determine whether mutation frequencies at repeat loci may correlate with rates elsewhere in the genome. Although most tandem repeat regions were originally considered to be ‗junk‘ DNA, it is becoming clear that non-coding regions of the genome may have very important roles in the structure and function of DNA. Moreover, correlations between repeat instability and epigenetic modification highlight the potential importance of genome structure and its role in maintaining germline genome stability. Thus, repeat instability may also provide a surrogate marker for epigenetic changes in the genome, although much further work is needed to explore this hypothesis.

REFERENCES [1]

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[2] [3]

[4]

[5]

[6]

[7]

[8]

Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Neumann R, Neil DL, Jeffreys AJ: Human minisatellite mutation rate after the Chernobyl accident. Nature 1996, 380:683-686. Buard J, Bourdet A, Yardley J, Dubrova Y, Jeffreys AJ: Influences of array size and homogeneity on minisatellite mutation. Embo J. 1998, 17:3495-3502. Bois PR, Southgate L, Jeffreys AJ: Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements. Mamm. Genome 2001, 12:104-111. Kelly R, Gibbs M, Collick A, Jeffreys AJ: Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events. Proc. Biol. Sci. 1991, 245:235-245. Kelly R, Bulfield G, Collick A, Gibbs M, Jeffreys AJ: Characterization of a highly unstable mouse minisatellite locus: evidence for somatic mutation during early development. Genomics 1989, 5:844-856. Jeffreys AJ, Murray J, Neumann R: High-resolution mapping of crossovers in human sperm defines a minisatellite-associated recombination hotspot. Mol. Cell 1998, 2:267273. Buard J, Shone AC, Jeffreys AJ: Meiotic recombination and flanking marker exchange at the highly unstable human minisatellite CEB1 (D2S90). Am. J. Hum. Genet. 2000, 67:333-344. Tamaki K, May CA, Dubrova YE, Jeffreys AJ: Extremely complex repeat shuffling during germline mutation at human minisatellite B6.7. Hum. Mol. Genet. 1999, 8:879888.

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

A Review of Tandem Repeat Sequences: Markers of Genetic Instability [9] [10]

[11] [12]

[13]

[14] [15] [16]

[17]

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[18]

[19]

[20]

[21] [22]

[23] [24]

[25] [26]

165

Jeffreys AJ, Neumann R: Somatic mutation processes at a human minisatellite. Hum. Mol. Genet. 1997, 6:129-132; 134-126. Yauk CL, Dubrova YE, Grant GR, Jeffreys AJ: A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus. Mutat. Res. 2002, 500:147-156. Shanks M, Riou L, Fouchet P, Dubrova YE: Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline. Mutat Res. 2008, 641:58-60. Fukuda H, Katahira M, Tsuchiya N, Enokizono Y, Sugimura T, Nagao M, Nakagama H: Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1. Proc. Natl. Acad. Sci. U S A 2002, 99:12685-12690. Katahira M, Fukuda H, Kawasumi H, Sugimura T, Nakagama H, Nagao M: Intramolecular quadruplex formation of the G-rich strand of the mouse hypervariable minisatellite Pc-1. Biochem. Biophys. Res. Commun. 1999, 264:327-333. Glen CD, Smith AG, Dubrova YE: Single-molecule PCR analysis of germ line mutation induction by anticancer drugs in mice. Cancer Res. 2008, 68:3630-3636. Vilarino-Guell C, Smith AG, Dubrova YE: Germline mutation induction at mouse repeat DNA loci by chemical mutagens. Mutat. Res. 2003, 526:63-73. Dubrova YE, Plumb M, Brown J, Boulton E, Goodhead D, Jeffreys AJ: Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to gammaradiation and fission neutrons. Mutat. Res. 2000, 453:17-24. Dubrova YE, Plumb M, Brown J, Fennelly J, Bois P, Goodhead D, Jeffreys AJ: Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation. Proc. Natl. Acad. Sci. U S A 1998, 95:6251-6255. Barber RC, Miccoli L, van Buul PP, Burr KL, van Duyn-Goedhart A, Angulo JF, Dubrova YE: Germline mutation rates at tandem repeat loci in DNA-repair deficient mice. Mutat. Res. 2004, 554:287-295. Yauk CL, Polyzos A, Rowan-Carroll A, Kortubash I, Williams A, Kovalchuk O: Tandem repeat mutation, global DNA methylation, and regulation of DNA methyltransferases in cultured mouse embryonic fibroblast cells chronically exposed to chemicals with different modes of action. Environ. Mol. Mutagen. 2008, 49:26-35. Barber R, Plumb MA, Boulton E, Roux I, Dubrova YE: Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice. Proc. Natl. Acad. Sci. U S A 2002, 99:6877-6882. Dubrova YE, Plumb MA: Ionising radiation and mutation induction at mouse minisatellite loci. The story of the two generations. Mutat. Res. 2002, 499:143-150. Dubrova YE, Hickenbotham P, Glen CD, Monger K, Wong HP, Barber RC: Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice. Environ. Mol. Mutagen. 2008, 49:308-311. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 1985, 314:67-73. Jeffreys AJ, Royle NJ, Wilson V, Wong Z: Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature 1988, 332:278281. Southern E: Southern blotting. Nat. Protoc. 2006, 1:518-525. Jeffreys AJ, MacLeod A, Tamaki K, Neil DL, Monckton DG: Minisatellite repeat coding as a digital approach to DNA typing. Nature 1991, 354:204-209.

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[27] Jeffreys AJ, Tamaki K, MacLeod A, Monckton DG, Neil DL, Armour JA: Complex gene conversion events in germline mutation at human minisatellites. Nat. Genet. 1994, 6:136-145. [28] Satoh C, Takahashi N, Asakawa J, Kodaira M, Kuick R, Hanash SM, Neel JV: Genetic analysis of children of atomic bomb survivors. Environ. Health Perspect 1996, 104 Suppl 3:511-519. [29] Satoh C, Kodaira M: Effects of radiation on children. Nature 1996, 383:226. [30] Kodaira M, Satoh C, Hiyama K, Toyama K: Lack of effects of atomic bomb radiation on genetic instability of tandem-repetitive elements in human germ cells. Am. J. Hum. Genet. 1995, 57:1275-1283. [31] Kodaira M, Izumi S, Takahashi N, Nakamura N: No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors. Radiat. Res. 2004, 162:350-356. [32] Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Vergnaud G, Giraudeau F, Buard J, Jeffreys AJ: Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident. Mutat. Res. 1997, 381:267-278. [33] Dubrova YE, Grant G, Chumak AA, Stezhka VA, Karakasian AN: Elevated minisatellite mutation rate in the post-chernobyl families from ukraine. Am. J. Hum. Genet. 2002, 71:801-809. [34] Dubrova YE, Bersimbaev RI, Djansugurova LB, Tankimanova MK, Mamyrbaeva Z, Mustonen R, Lindholm C, Hulten M, Salomaa S: Nuclear weapons tests and human germline mutation rate. Science 2002, 295:1037. [35] Kiuru A, Auvinen A, Luokkamaki M, Makkonen K, Veidebaum T, Tekkel M, Rahu M, Hakulinen T, Servomaa K, Rytomaa T, Mustonen R: Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers. Radiat. Res. 2003, 159:651-655. [36] Livshits LA, Malyarchuk SG, Kravchenko SA, Matsuka GH, Lukyanova EM, Antipkin YG, Arabskaya LP, Petit E, Giraudeau F, Gourmelon P, et al: Children of chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles. Radiat. Res. 2001, 155:74-80. [37] Slebos RJ, Little RE, Umbach DM, Antipkin Y, Zadaorozhnaja TD, Mendel NA, Sommer CA, Conway K, Parrish E, Gulino S, Taylor JA: Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers. Mutat. Res. 2004, 559:143-151. [38] Furitsu K, Ryo H, Yeliseeva KG, Thuy le TT, Kawabata H, Krupnova EV, Trusova VD, Rzheutsky VA, Nakajima H, Kartel N, Nomura T: Microsatellite mutations show no increases in the children of the Chernobyl liquidators. Mutat. Res. 2005, 581:69-82. [39] May CA, Tamaki K, Neumann R, Wilson G, Zagars G, Pollack A, Dubrova YE, Jeffreys AJ, Meistrich ML: Minisatellite mutation frequency in human sperm following radiotherapy. Mutat. Res. 2000, 453:67-75. [40] Armour JA, Brinkworth MH, Kamischke A: Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies. Mutat. Res. 1999, 445:73-80. [41] Zheng N, Monckton DG, Wilson G, Hagemeister F, Chakraborty R, Connor TH, Siciliano MJ, Meistrich ML: Frequency of minisatellite repeat number changes at the

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

A Review of Tandem Repeat Sequences: Markers of Genetic Instability

[42] [43]

[44] [45]

[46]

[47] [48] [49] [50]

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[51] [52] [53] [54] [55]

[56]

[57]

167

MS205 locus in human sperm before and after cancer chemotherapy. Environ. Mol. Mutagen. 2000, 36:134-145. Senturia YD, Peckham CS: Children fathered by men treated with chemotherapy for testicular cancer. Eur. J. Cancer 1990, 26:429-432. Babosa M, Baki M, Bodrogi I, Gundy S: A study of children, fathered by men treated for testicular cancer, conceived before, during, and after chemotherapy. Med. Pediatr. Oncol. 1994, 22:33-38. Nicholson HS, Byrne J: Fertility and pregnancy after treatment for cancer during childhood or adolescence. Cancer 1993, 71:3392-3399. Green DM, Zevon MA, Lowrie G, Seigelstein N, Hall B: Congenital anomalies in children of patients who received chemotherapy for cancer in childhood and adolescence. N. Engl. J. Med. 1991, 325:141-146. Yauk CL, Quinn JS: Multilocus DNA fingerprinting reveals high rate of heritable genetic mutation in herring gulls nesting in an industrialized urban site. Proc. Natl. Acad. Sci. U S A 1996, 93:12137-12141. Yauk CL, Fox GA, McCarry BE, Quinn JS: Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills. Mutat Res 2000, 452:211-218. Somers CM, Yauk CL, White PA, Parfett CL, Quinn JS: Air pollution induces heritable DNA mutations. Proc. Natl. Acad. Sci. U S A 2002, 99:15904-15907. Somers CM, McCarry BE, Malek F, Quinn JS: Reduction of particulate air pollution lowers the risk of heritable mutations in mice. Science 2004, 304:1008-1010. Yauk C, Polyzos A, Rowan-Carroll A, Somers CM, Godschalk RW, Van Schooten FJ, Berndt ML, Pogribny IP, Koturbash I, Williams A, et al: Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location. Proc. Natl. Acad. Sci. U S A 2008, 105:605-610. Gomes-Pereira M, Monckton DG: Chemical modifiers of unstable expanded simple sequence repeats: what goes up, could come down. Mutat. Res., This Issue. Armour JAL: Tandemly repeated DNA: why should anyone care? Mutat. Res., This Issue. Pearson CE, Edamura KN, Cleary JD: Repeat instability: mechanisms of dynamic mutations. Nat. Rev. Genet. 2005, 6:729-742. Gatchel JR, Zoghbi HY: Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. 2005, 6:743-755. Lopez Leon S, Croes EA, Sayed-Tabatabaei FA, Claes S, Van Broeckhoven C, van Duijn CM: The dopamine D4 receptor gene 48-base-pair-repeat polymorphism and mood disorders: a meta-analysis. Biol. Psychiatry 2005, 57:999-1003. Bennett ST, Lucassen AM, Gough SC, Powell EE, Undlien DE, Pritchard LE, Merriman ME, Kawaguchi Y, Dronsfield MJ, Pociot F, et al.: Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat. Genet. 1995, 9:284-292. Virtaneva K, D'Amato E, Miao J, Koskiniemi M, Norio R, Avanzini G, Franceschetti S, Michelucci R, Tassinari CA, Omer S, et al: Unstable minisatellite expansion causing recessively inherited myoclonus epilepsy, EPM1. Nat. Genet. 1997, 15:393-396.

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In: Genome Instability and Transgenerational Effects ISBN: 978-1-60876-831-8 Editors: I. Kovalchuk, O. Kovalchuk, pp. 169-199 © 2010 Nova Science Publishers, Inc.

Chapter 10

TRANSGENERATIONAL GENOMIC INSTABILITY IN FISH Carmel Mothersill and Colin Seymour Medical Physics and Applied Radiation Sciences Department, McMaster University, Hamilton, Ontario, L8S 4K1, Canada

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ABSTRACT This chapter will review the data for somatic and germ line transgenerational effects in fish. The emphasis will be on radiation studies with reference, where appropriate, to chemical or mixed exposure studies. The terms will be defined as used in most of the literature with discussion of confusing elements. There are two main sources of data for fish transgenerational studies. These are the use of fish in the laboratory as models for human studies and the study of impacts of radiological accidents or planned exposures on fish as critical elements in the aquatic ecosystem. Both data sets will be reviewed. Comparative studies of mammalian and fish radiation responses are rare but important for both mechanistic understanding and environmental risk assessment. Common assumptions are that fish are ―radioresistent‖. The basis for this will be reviewed and discussed in detail. Work from the authors‘ laboratory concerning bystander signaling and associated induction of genomic instability will also be reviewed. Finally, reasons for these studies will be discussed in terms of relevance for evolutionary biology and environmental protection of seeking a mechanistic understanding of vertebrate responses to low doses of environmental stressors.

INTRODUCTION Transgenerational effects in the fish radiation literature generally refer to the effects in embryos or offspring derived from exposed parents. Often the eggs (unfertilized) are in a female when she is exposed. There are very few studies looking at the F2 (usually G2) or subsequent generations in any systematic way. Inferential data concerning long-term effects can be obtained from older studies of populations in Japan and in the Chernobyl area. The papers which could be described as ―transgenerational‖ by the above definitions are

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Carmel Mothersill and Colin Seymour

summarized in Table 1. Material from these papers is presented in further tables where appropriate. Definitions of F1, G1 and F2, G2 and various terms used in fish biology, etc. are summarized in Text Box 1. Text Box 1 Definitions of terms in transgenerational fish radiobiology F1: the progeny of irradiated fish where an identified male and an identified female are used to produce eggs and sperm. G1: the progeny of a population of fish exposed to a particular set of experimental conditions. F2/G2: the progeny of the F1/G1 generation. Treatment at the fertilised egg stage: treatment of eggs within 48 hrs of fertilization before cell division has started. Treatment at the eyed egg stage: treatment at the phase of embryonic development when the pigmented eye can be seen in the egg. This represents the first stage where tissue cells are present.

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Treatment at the yolk sac larval stage: treatment of free swimming fish that are not yet able to feed but whose food supply is contained in a yolk sac (just after egg hatching). Treatment at the juvenile stage (first feeder stage): treatment of young fish that have started to feed independently.

SCOPE OF THE REVIEW This chapter will therefore include ―transgenerational‖ studies even if eggs are in the female when exposed. Data for fish cells where somatic reproductive effects are measured in the progeny of irradiated cells will also be discussed with an emphasis on non-targeted effects such as bystander effects and genomic instability where exciting new data are available. For the purposes of this chapter ―fish‖ are defined as members of the Phylum Chordata (see Text Box 2 for taxonomy and statistics). Aquatic invertebrates are not included. There are more than 28,000 known extant species compared to one human species. They inhabit all aquatic environments from shallow mountain streams to the deepest ocean and have many bizarre evolutionary adaptations. This gives some idea of generalizations which will be made in any discussion of radiobiology of ―fish‖. In fact, fish radiobiology mostly concerns just a few species which live well in the laboratory (see Text box 3). About 10 fish cell lines are commercially available (also listed in Text box 3). There is quite a large old literature on these laboratory species reviewed in International Commission on Radiological Protection (ICRP) publication number 103 (1) and in a symposium held in Japan in 1979 [2].

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Table 1. Fish Studies Ref

Major Point

Endpoint Used

[105]

-Wild-type inbred strain HNI medaka fish were irradiated, mated with nonirradiated females, and the embryos were collected. The mutation frequency was found to be significantly lower in fish in the 0.03 cGy/min group than in the 95 cGy/min group. There was a dose-rate effect on the mutation frequency in medaka spermatogonial cells. The mouse and medaka fish have similar magnitudes of dose-rate effects on transgenerational mutation frequencies, showing similar dose-rate effects among vertebrates

- Frequency of specific-locus mutations at the 5 pigmentation loci in medaka spermatogonial stem cells

[106]

The sperm and spermatids of male medaka fish were exposed to -rays. These male fish (+/+) were then mated with female medaka fish (wl/wl). The frequency of wild-type and mosaic mutants was 5.7 x 10-3/Gy, and the frequency of whole body mutants was 1.3 x 10-3/Gy. The irradiated sperm can cause delayed mutations. Brook charr fish were exposed to ionizing radiation at the hatching stage. The mean body weights for those exposed to 7.8 Gy and 11.4 Gy after 2 months were lower than the control, and after 4 months those exposed to 6.2 Gy had lower body weights. After 15 months however, all fish ended with about the same body weight.

- mutations (whole body and mosaics) in embryos of irradiated fish

- Changes in growth and tissue analysis

[107]

Time after exposure when endpoint measured

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

- 0 cGy/min, 0.03 cGy/min and 95 cGy/min dose rates - Total doses: 0 Gy, 1.9 Gy, 3.2 Gy, 4.75 Gy

- F1 embryos collected on the 30th day from paternal exposure

-rays

N/A

The sperm and spermatids were exposed to either 0 Gy, 4.75 Gy, or 9.50 Gy of radiation

Lifestage of offspring measured (embryos): 1, 3, 7, 26, and 50 hours after fertilization

-rays

Growth study: time of exposure – 15months Tissue analysis: time of exposure - 9 months

4 groups: 0 Gy, 6.2 Gy, 7.8 Gy, 11.4 Gy

Exposed at hatching stage

60

Co source

Table 1. (Continued) Ref

[108]

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[109]

[110]

Major Point

Tissue analysis showed that after 3 months those exposed to 11.4 Gy had the lower DNA and RNA content and lower RNA/protein ratio. After 6 months, the RNA content and RNA/protein ratio of the irradiated fish reached the levels of the nonirradiated fish. When sperm and late spermatids were irradiated, the mutant frequency within nonirradiated maternally derived alleles was ~3 times higher then in the control group. Irradiation of these produces an indirect mutation in the F1 somatic cells. Genomic instability arises during the F1 embryoic stage. Determined the frequency of specific- locus mutations at the 5 pigmentation loci in medaka spermatogonial stem cells

Chinese rare minnow exposed to diethylstilbestrol from fertilized eggs for up to mature period under flow-through conditions. Development, reproduction fitness and transgenerational effects were evaluated

Endpoint Used

Time after exposure when endpoint measured

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

Embryos with 1 or more leucophore was scored as a mosaic mutant

Embryos collected 1-3 months after exposure of parents to dose— embryos viewed after day 6 of fertilization Embryos collected after the 30th day after parental exposure

T5 and HNI Males4.75 Gy from 80 TBq137 Cs source at 0.95 Gy/min in water

Adult exposed – embryo examined

γ-rays

HNI Males 80 TBq137 Cs source at 0.03 cGy/min or 95 cGy/min

Adult exposed – embryo examined

γ- rays

See Phases under the life stages, which determine time before examination

0.05, 0.5, 1 and 5 ug/L DES concentrations

Exposed as eggs, examinedembryological phase, Larval-juvenile phase, reproductive phase, F1 embryo phase, F1 larvaljuvenile phase

DES

Eggs contained in water with 1 ppm methylene blue in 96well plastic mircotiter plates—measurements used a view through the microscope and transmission of light VTG analysis, histological analysis, thyroid hormone assays, steroid hormone radioimmuno-assay

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Ref

Major Point

Endpoint Used

[111]

Assessed the effects of TPT on medaka; reproduction responses and transgenerational effects were studied. Results showed that TOT exposure suppressed the spawning frequency and induced teratongenesis such as hemorrhaging, eye defects, morphological malformation and conjoined twins, less hatchability and swim-up failure in the F1 generation.

[112]

Evaluated potential effects on the early life stage of Japanese medaka; Larvae and fertilized eggs were exposed. A dosedependent decrease in fecundity and fertility accompanied by histological changes suggested inhibition of oocyte growth and possible maturation. Also a dose- dependent increase in the proportion of genotypic F1 was found. Groups of 30 fish each were exposed to radiation varying from 100r to 10000r. Survival time decreased with increasing dose. There was an eruption of melanophores in response to irradiation. The outer surface of the intestines inflamed, the spleen increased in size, the gonads and kidney inflamed, all with increasing dose. When the fish were injected with Aeromonas salmonicida 7 days after irradiation, they died sooner. The effect of radiation on susceptibility to infection was found to be a function of radiation dose.

Gonad, liver and brain were examined. Half of gonad put in 10% neutral buffered formalin for histological analysis (stained and sectioned). Everything else was frozen in liquid nitrogen till RNA isolation (RTPCR Assay) Plasma VTG levels were measured using a competitive ELISA.

[35]

- Time of death and inflammation of organs

Time after exposure when endpoint measured Exposure for 5 weeks, in a flowthrough tank

Dose

Test duration 21 days

1,5,25,125,625, and 3125 ug/L

Examined until death

Radiation experiment: 100r, 500r, 1000r, 2500r, 3000r, 5000r, 10000r Radiation and organism experiment: 1000r and 2000r, 1.2x105 to 1.2x109 organisms (Aeromonas salmonicida)

1.6, 8, 40, 200, 1000 ng/L

Lifestage of parent exposed and lifestage of offspring measured Adults exposed

Type of Radiation

Embryos exposed

Lertrozole in water containing 1% methylene blue and 0.1% acetone

TPT-Cl

X-rays

Table 1. (Continued) Ref

Major Point

Endpoint Used

Time after exposure when endpoint measured

Dose

[113]

Evaluated both estrogenic effects in directly NPexposed sexually mature rainbow trout and possible transgenerational effects in the offspring of exposed fish. Findings indicate that NP, in an environmentally relevant concentration range, acts as a weak estrogen in directly exposed adult male rainbow trout, as it is indicated by elevated plasma vitellogenin levels. Reproduction was decreased. EE a drug that is used in birth control was tested and found in several water treatment plants. Low concentrations have the potential to disrupt the development of normal endocrine and reproductive function when exposed over critical periods of development. To test this, Japanese medaka were used. Findings showed no reproduction effect, but they did show that the relationship between biomarkers and estrogen exposure were altered by timing and frequency.

Blood sampling using ELISA

Adults exposed 4 months prior to spawning till the offspring juvenile stage

1 and 10 ug/L

For each pair of adults, the total number of eggs, # fertilized, avg. size, hatching date and # hatched were recorded.

Exposed for 2 weeks

1 ng/l to 200 ng/l

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[114]

Lifestage of parent exposed and lifestage of offspring measured 3 yr old adults exposed

Type of Radiation

2-day- old medaka exposed

EE

NP

Transgenerational Genomic Instability in Fish

175

Text Box 2. Classification of Fish taken from Wikipedia and the wider classification of fish within the vertebrates Fish are a paraphyletic group: that is, any clade containing all fish also contains the tetrapods, which are not fish. For this reason, groups such as the "Class Pisces" seen in older reference works are no longer used in formal classifications.

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Fish are classified into the following major groups: * Subclass Pteraspidomorphi (early jawless fish) * Class Thelodonti * Class Anaspida * (unranked) Cephalaspidomorphi (early jawless fish) o (unranked) Hyperoartia + Petromyzontidae (lampreys) o Class Galeaspida o Class Pituriaspida o Class Osteostraci * Infraphylum Gnathostomata (jawed vertebrates) o Class Placodermi (armoured fishes, extinct) o Class Chondrichthyes (cartilaginous fish) o Class Acanthodii (spiny sharks, extinct) o Superclass Osteichthyes (bony fish) + Class Actinopterygii (ray-finned fish) # Subclass Chondrostei * Order Acipenseriformes (sturgeons and paddlefishes) * Order Polypteriformes (reedfishes and bichirs). # Subclass Neopterygii * Infraclass Holostei (gars and bowfins) * Infraclass Teleostei (many orders of common fishes) + Class Sarcopterygii (lobe-finned fish) # Subclass Coelacanthimorpha (coelacanths) # Subclass Dipnoi (lungfish) Of the 28,000 species still living, approximately 27,000 species belong to the Teleostei which are an infraclass of bony fish to which the species used in radiobiology research belong. Vertebrate Animal Groups with Key Features Jawless Fish - no jaws - no paired fins - gave rise to placoderms, cartilaginous and bony fish Placoderms - no jaws - armored fish Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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Carmel Mothersill and Colin Seymour Text Box 2. (Continued)

Cartilaginous fish - cartilage skeletons - no swim bladder - no lungs - internal fertilization Bony fish - gills - lungs - swim bladder - some developed fleshy fins (gave rise to amphibians) Amphibians - first vertebrates to venture out onto land - remained quite tied to aquatic habitats - external fertilization - eggs had no amnion or shell - moist skin

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Reptiles - scales - hard-shelled eggs - stronger legs positioned directly beneath body Birds - feathers - hollow bones Mammals - fur - mammary glands - warmblooded

The field has been revitalized recently in two main areas: 1. The concern about specific radiation protection regulations to govern non-human biota [3-5] has led to heated discussion about how to do this, how to do moregood than harm, and how to deal with the complexities of protecting ecosystems rather than individuals [6-8]. Attempts to grapple with these issues have revealed large data gaps. The twin aims of filling these and developing methods to model or predict effects with limited data have led to significant new research in the field [9-12]. Several recent reports and international bodies are focusing on the problems of regulating exposure of biota [1, 13-16], and the most fundamental issue is lack of adequate scientific data concerning low dose exposure effects.

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Transgenerational Genomic Instability in Fish

177

2. Concerns about the use of mammals in research and drug testing coupled with soaring costs for animal care have led to the search for alternative models for these applications. Fish with their high fecundity and external reproduction offer an easy, cheap and more ethically acceptable screening method for looking at embryological impacts [17-19] and for mutagenesis screening. Since most cancer and stress-related metabolic pathways are highly conserved across vertebrate classes, there is growing enthusiasm for the use of fish models in these research areas [20-23]. Text Box 3. Fish cell lines and laboratory species commonly used in radiobiology Fish cell lines

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Commonly used fish species for in vivo experiments Salmonid species - salmon and trout - e.g. Salmo trutta, Oncorhynchus mykiss Name of Cell Line

Species

Depositor of the cell line

Origin of Cells

BF-2

Lepomis macrochirus (Rafinesque) bluegill

K Wolf

Caudal trunk

CHH-1

Oncorhynchus keta (Walbaum) Salmon, chum

JL Fryer

Heart

CHSE214

Oncorhynchus tshawytscha (Walbaum) Salmon, Chinook

JL Fryer

Embryo

RTH-149

Oncorhynchus mykiss (Walbaum) (Trout, rainbow)

JL Fryer

Liver

G1B

Clarias batrachus (L.) Walking catfish

EJ Noga

Gill

1G8

NW Miller

Peripheral blood

3B11

Ictalurus punctatus (Rafinesque) Channel catfish I. punctatus (Channel catfish)

NW Miller

Peripheral blood

CCO

I. punctatus (Channel catfish)

L Hanson

Ovary

EPC

Pimephales promelas (Rafinesque) Fathead minnow

J Winton

Skin (Epithelioma Papulosum Cyprini)

Historical Data There is a considerable amount of old data concerning the effects of acute doses of ionising radiation on a limited number of fish species and on cell lines developed mainly from salmonids, medaka, fathead minnow, zebrafish and carp [24-30] and reviewed in ICRP 2008 [1]. The studies were usually aimed at studying radiation effects on fertility, fecundity and mutation frequency in fish to prepare for the ecological consequences of a nuclear accident. The studies predated the discovery of subtle effects of low doses described later and usually

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concluded that ―fish‖ were radioresistant and needed 5 to 10 times the dose which would cause a similar effect in humans. There are several fascinating aspects of this history of fish radiation radioresistance. A major anomaly is the history of the famous UNSCEAR chart [31] reproduced here (Figure 1) that represents a ―ranking‖ of different animal and plant groups in terms of radiosensitivity. This shows ―fish‖ (all species irrespective of life stage or other important factors) as relatively radioresistant compared with Man and other mammals. The chart shows Man as the most sensitive organism; it gives justification to ICRP‘s contention in ICRP publication number 60 [32] that if Man is protected from harmful effects of ionizing radiation then all other species are safe too. The chart is attributed to Wicker and Schultz‘s review [33] who refer to Sparrow [34], but nowhere are there any data to support the ranking. The authors have been informed (personal communication H. Mueller, Heimholtz Institute Muenchen 2009) that the chart is being dropped from the new UNSCEAR report because the ranking was based on theoretical calculations of DNA content of cells from which radiosensitivity was ranked using radiation target theory (see Text box 4) and not on any biological considerations or actual data!

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Text Box 4. Elements of Target Theory in Radiobiology Target theory developed in the 1930‘s and 40‘s. It implies there is a sensitive ―target‖ within the cell which leads to death of the cell when it is ―hit‖ or inactivated. It considers the number of ionizing tracks produced by exposure to ionizing radiation in relation to the number of sensitive targets in the exposed object. Because the target was small in relation to the size of the cell, low doses had a probability of not hitting any target. This was the explanation for shouldered survival curves showing the reduced cell kill at low doses. Initially no consideration was given to modifying factors, such as repair occurring post exposure, except by introducing mathematical expressions to modify the survival curve function. The theory holds that DNA is the main target of radiation interaction resulting in biological damage and that DNA double strand breaks are critical lesions resulting in death of the cell. Dose is proportional to double strand breaks and thus proportional to cell death. Early realization that low doses produce proportionally less death than high doses has led to consideration of multi-hit and single –hit multi-target variations of pure target theory, but it was only in the late 1970‘s that alternative models to describe the relationship between radiation dose and effect were seriously considered. These included various repair models, ―pool‖ models and more recently models incorporating concepts of delayed and epigenetic or non-targeted damage. Key References: [139-142]

The data in Table 2 presents a summary of selected papers in the literature with reasonable datasets for radiation effects in fish species. These are not limited to transgenerational studies but serve to show how variable the data are and also how limited is the number of species about which we know anything. In fact, when the data on radiosensitivity in the table are reviewed, it can be seen that far from being resistant, fish are actually very variable in their response as might be expected from their wide diversity of habitat form and longevity. Age/life stage and species are critical parameters, as is the time after exposure when the effects are monitored.

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Table 2. Radiobiological Papers Using Fish Ref

Endpoints Used

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Articles From Journal Literature [115] Cell death during embryogenesis

Time after exposure when endpoints are measured

Dose

Life stage of parents exposed and offspring measured

Type of Radiation

Continuously

50 cGy

Embryo (1 cell-stage)

γ-rays (from 137 Cs source)

Plants (within 30 km of the accident): received 0.7-3.9 GBq/m2 (beta) Soil invertebrates (3-7 km of the accident): approx. 30 Gy total Terrestrial animals: 12-110 Gy (gamma) and 580-4500 Gy (beta) Aquatic organisms: Non-predatory fish (carp, goldfish, bleak) – peak of 3 mGy/d, Bottom-dwelling fish(goldfish, silver bream, bream, carp) – approx. 10 Gy N/A Experiment 1: Embryos injected with buffer only – 0, 50, 100-1000 mGy. Embryos injected with Ku80 MO – 0100mGy in 10mGy increments Experiment 2: 0, 3, 8, 20 and 50 mGy (both buffer only and Ku80 MO)

N/A

β- and γ-rays

N/A Embryos received radiation 6 h after fertilization

N/A 137 Cs γ-rays

[116]

Visible and genetic changes among plant and animal species

3 Time Periods: 1. The period of intense exposure – 30 days following the Chernobyl accident 2. Short-lived radionuclides decayed, longer-lived radionuclides dispersed – one year following the accident 3. The period of chronic exposure (dose rates less than 1% of the initial dose) – continuing today

[117] [118]

N/A A trend between a dose given to zebrafish embryos and the number of TUNELpositive cells

N/A After irradiation, the cells were incubated for 18 h

Table 2. (Continued) Ref

Endpoints Used

Time after exposure when endpoints are measured

Dose

[119]

An increase in 8hydroxyguanine (8OHG) base modifications and DBS N/A N/A TUNEL analysis was carried out to determine apoptosis in the embryos

24 h-72 h

0-15 Gy γ-rays, 0-50 μM CdCl2

N/A N/A 24 hpf

N/A N/A ~1 Gy/min

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[120] [121] [122]

[123] N/A N/A Articles from symposium on Effects of Radiation on Aquatic Organisms [124] - Tritium specific N/A activity - Tritiated organic fraction [125] - Body weight Continuously up to 120 days - Gonadal weight -Spermatogonia cell number - Fertility [126]

- Mortality - Gonad weight - Hormone secretion - Body size and shape

3-3.5 months

Life stage of parents exposed and offspring measured N/A

Type of Radiation

N/A N/A Exposed at the gastrula stage of embryo

N/A N/A 137 Cs

γ-rays

N/A

Endpoint- embryo N/A

N/A

N/A

N/A

N/A

-X-rays (acute): 100-1000 rad -Tritiated water concentration: 5x10-3, 10-2, 5x10-2, 10-1 Ci/L - β-irradiation: 5x10-2 Ci/L -γ-irradiation (chronic): 1.3, 2.9, 6.8, 15.6, 24.2, and 84.3 rad/day (for 120 days) -Total exposure (for groups I-A respectively, excluding H): 0, 3083,6608, 6608, 10469, 10469,12540, 43063 R

Adult males

X-ray and γray (from 137 Cs source) -β-rays

Developmental stage: 0-1 day

γ-irradiation from 60Co source

Ref

Endpoints Used

Time after exposure when endpoints are measured

Dose

[127]

Changes in germ cell population from x-irradiation at different stages of development - Hatching rate of embryos - Malformation appearance rate - Survival rate - Average total length - Average body weight

Continuously

1000 R

Continuously

Three groups of 0, 600 and 800 R

-Volume of thymus in young adult fish -Development of thymus in newly hatched fish -Inhibition of mitosis - Suppression of fin elongation

Adults fixed with Bouin‘s solution 2, 5 and 11 days after irradiation. Embryos fixed at 0, 5/6 and 10 days after hatching Immediately and continuously

Irradiation of young adult fish: 200 and 500 R Irradiation of embryos: 0, 1, 2, 4 kR 0, 2, 3, 4 kR

Examined macroscopically. Autoradiography with NTB – measurements done on kidneys and intestines Determined the survival rate of cells in accordance to the control cells. Eggs dissected under microscope, and embryonic cells fixed and stained Tumors were examined

2 years

1000 rad

[128]

[129]

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[130]

[131]

[132]

[133]

[134]

10-14 days

250, 500, 1000, 2000, and 3000 R

N/A

0, 10, 100, 1000 μCi/L

N/A

HTO 0, 0.1, 1, 10 Ci/L Treated in MAM 0.1-10 ppm 250 R/min X-ray

Life stage of parents exposed and offspring measured Embryos and fry (0, 2, 3, 10 and 15 days after hatching) Embryos irradiated 29 days post insemination and 10 days after the beginning of eyemaking in the late eye period Young adult fish and embryos 0, 4 and 8 days after fertilization

Type of Radiation

Adults

γ-ray (from 137 Cs and 60 Co source) x-ray γ- ray

Exposed at 3 months Killed at the age of 2 years and 3 months Irradiated 24h after incubation. 10-14 days incubation later on. Exposure immediately after fertilization for 22 h 150days-1hr in MAM

X-ray

γ-ray from a 60 Co source

γ-ray

X-ray

90

Sr-90Y and HTO X-ray

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Table 2. (Continued) Ref

Endpoints Used

[135]

Measured size of tumor, also LI and MI

[136] [137] Changes in the Lipid [138]

N/A N/A Tumors were measured

Time after exposure when endpoints are measured 2 hours

N/A N/A N/A

Dose 100 ppm 1μCi N/A N/A 1000 R

Life stage of parents exposed and offspring measured 6-8 weeks of DENA 1 pulse of H-thymidine 2 hours before killing N/A N/A 2 day exposure

Type of Radiation DENA H-thymidine N/A N/A X-Ray

Paper, Author/Date Summary Articles From Websites [115] Zebrafish embryos were irradiated with low doses of ionizing radiation, and their development was monitored. The Ku70 protein is a product of the XRCC6 gene, which is part of the nonhomologous end-joining pathway of DNA repair. A morpholino-mediated knockdown of Ku70 expression was performed, and this did not have any effect on zebrafish embryogenesis. However, when the embryos lacking Ku70 expression were exposed to radiation, cell death in the central nervous system was observed. [116] This article examined the effects of radiation released by the Chernobyl accident on plants and animals, through three different periods after the accident. During the first 20 days following the accident, short-lived, highly radioactive nuclides were deposited on the earth surface. In the second phase, shortlived radionuclides decayed, and long-lived radionuclides were transferred throughout the environment. The Chernobyl accident caused death and shortterm sterility in plants, and it also caused soil invertebrates to decrease by a factor of 30. Small rodents were decreased by a factor of 2-10. Barn swallows had increased germline mutations. However, human abandonment of the city of Pripyat caused nature to flourish in this area and led to an increase in game mammals. [117] Zebrafish are comparable to many vertebrates, including humans, at genomic and molecular levels. Thousands of mutant phenotypes have been identified in zebrafish, of which many are comparable to human genetic disorders. Another benefit of zebrafish is that they are transparent. Tissuespecific fluorescent fish can be made and studied using fluorescent confocal microscopy. Gene knockout can also be achieved in zebrafish using either morpholino antisense oligos or targeting induced local lesions in genomes. [118] An antisense oglionucleotide that targets Ku80 (a protein needed for DNA DSB repair) was injected into zebrafish embryos. Wild-type embryos and those injected with the Ku80 MO were irradiated. The TUNEL-assay performed on the embryos detects DNA fragmentation, which is a sign that cells are undergoing death by apoptosis. It was found that cells with attenuated Ku80 have more TUNEL-positive cells. Those cells injected with just buffersolution had a linear trend between a dose and the number of TUNEL-positive cells. Cells irradiated and injected with Ku80 showed a linear trend and a quadratic trend. There was a 30-fold difference between the slopes of linear sections of the buffer- and Ku80 MO- injected embryos. [119] Medaka fish were exposed to both ionizing radiation and cadmium (Cd). It was found that Cd increased the effects of radiation on the fish, causing more cell death. Cd affects DNA repair systems, therefore not allowing the damage caused by ionizing radiation to be fixed.

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Paper, Author/Date [120]

Summary

This is a review paper that shows how the zebrafish have helped in discovering new things about vascular system. It has helped in understanding gene expression, mutagenesis, imaging and use of transgenic lines. The studies that have been done have helped to link drugs effective in fish with cure of specific human diseases. [121] This is a paper that recommends procedures for evaluating agents, selecting and validating model systems for prophylaxis/protection, mitigation and treatment of radiation injuries. The use of the whole animal model has been suggested since the effects vary from tissue to tissue and are also dependent upon exposure. There were 5 systems that were said to be the most important: hematopoietic, gastro-intestinal, central nervous system, urinary and respiratory tracts. [122] This study shows the effect of radiation-induced DNA damage on the zebrafish embryo. The results of this study showed that nonhomologous end joining components repair DNA of irradiated embryonic cells. [123] This is a review article that summarizes the zebrafish model for cancer. Zebrafish have come to play a big role in this research, as it is possible to develop various types of human tumors in them. The techniques that can be used are chemical treatment, transplantation of mammalian cells, forward genetic screening, reverse genetic knockouts, and expression of transgenes. The function can be studied using mutants obtained via forward or reverse genetic approaches as well as using transgenic organisms. Articles From Effects on Aquatic Organisms [124] Due to the increase in the number of nuclear power plants, more tritium has been contaminating the aquatic ecosystems. Samples of water, sediments, plants and animals were collected, and it was found that tritium in dry matter of fish had a higher specific activity than in the water medium. There are two types of tritium present in food, the tritiated water form and the organically-bound tritium form. The tritium organic fraction was found to be in the range from 25 to 90 percent. [125] Oryzias male adult fish were irradiated with x-rays (acute), and their testes were found to be reduced in weight within two weeks. The fish were also continuously exposed to tritiated water, and those exposed to a concentration of 5x10-2Ci/L had weight loss of the testes and a decrease in the number of spermatogonia Ib cells. The adult males that were continuously exposed to γ-rays had no body weight loss but had reduced gonad weight (for those exposed to ≥ 2.9 rad/day). For those exposed to 2.9 and 6.8 rad/day, the gonad weight was recovered after 30 days. The spermatogonia Ib cells also lessened, as the exposure rate increased, but recovered at the 2.9 and 6.8 rad/day after 30 days. Regeneration of the cells was seen if the fish were removed from radiation after 60 days and not exposed for another 2 months. [126] O. latipes fish were exposed to γ-rays at different distances from the 60Co source and with different exposure rates, and the results were examined. There were no effects of exposure on the body size, shape and weight. However, the gonad tissues were not able to be seen through autopsy for groups B, C and D (12540 R, 10469 R, 10469 R), except for 1 male and female in group D. In groups E and F (6608R, 6608R), some male fish had small testes and all female fish had small ovaries. Not including the gonads, there was no difference between the organs of the irradiated and control fish. Irradiation did not alter hormone secretion. [127] Medaka fish were irradiated with 1000 R x-rays as embryos and as fry. The effect of radiation on germ cells varied according to their developmental stage. Embryos at stage 30 were irradiated, and proliferation of germ cells was inhibited promptly. Germ cells with enlarged nuclei were noticed. The hatched fry were irradiated, and the number of germ cells decreased within a few days of exposure, and this carried on for 20 days. In females, the radiosensitivity changed according to the current stage of meiosis.

Table 2. (Continued) Paper, Author/Date [128]

[129]

[130]

[131]

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[132]

[133] [134] [135] [136] [137] [138]

Summary Rainbow trout embryos were irradiated, and eight days after irradiation 50% of embryos hatched. There were no effects from the irradiation at the time of hatching. The only difference between the irradiated and nonirradiated groups was that survival rates of the irradiated groups were lower than those of the nonirradiated group. Young adult fish were irradiated with 200 R and 500 R γ-rays, and the volume of the thymus was found to decrease after irradiation but then increase again after a few days. Embryos were also irradiated with γ-rays, and when hatched, it was found that there was a dose dependent negative relationship between the decrease of the thymus volume and irradiation. Fish irradiated with 1 kR had less mitotic activity than the nonirradiated. The fish irradiated with 2 kR had low mitotic indices for 3 days, but then they increased. Those irradiated with 3 kR had delayed mitosis for 6 or more days. If fish were kept at 10°C, there was no effect of radiation. Those irradiated with 2 kR had their fins examined after irradiation, and it was found that the inhibitory effect was lessened with lower exposure rates (this was not observed at 10°C). The fins were amputated, and a restraint of fin elongation was seen 5 days later in fish receiving over 2 kR, especially 4 kR. The purpose of this study is to determine the long term effects on fish that have received doses which caused ―irreparable‖ effects. A change has been discovered in the haematopic tissue after the 9th day of exposure showing short term effects, and red blood cell counts fell too. After 2 years of exposure, 90% of the fish were still alive, and the irradiated fish could be distinguished from the non-irradiated through dark pigment patches. The results showed effects on the intestinal tract and the haematopoictic system, however some results were contradictory. The effects of whole body irradiation on cell kinetics in vivo were studied on fish. The effects of radiation on cultured cells in vivo were studied. A cell line was established from the fin of the goldfish. The results showed an almost linear relationship in the negative direction of the survival rate vs. the amount of radiation. High concentrations of radionuclides are needed to produce significant results to show somatic effects. The results showed that increased exposure increased chromatic cell aberrations. The paper studies the effects of x-irradiation on hepatic tumor induced by MAM acetate in medaka. The results showed that the fish treated with MAM after X-ray irradiation showed a decrease in tumors, if MAM was given after a few days. The paper studies the effects of DENA in the water that causes liver tumors in medaka. The results of the study showed that exposure to DENA and high temperature increased the appearance of tumors. This is a review paper focusing on the problem of pollutants in water effecting aquatic organisms. It has been shown that organisms that live in polluted areas are more likely to develop tumors. However, studies have not yet been able to pinpoint what may cause these tumors. The paper deals wth X-ray-induced mutations in the teleostean fish. This study was inspired by a pervious study showing that coho salmon could survive x-rays of 100 R, if they were kept in fresh water and not transferred into sea water. The results showed that if treated with Actinomycin D, the Eel will be less likely to develop tumors when kept in fresh water.

Transgenerational Genomic Instability in Fish

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Other features which clearly influence outcomes are whether fish are marine or freshwater species, tropical or cold water dwellers, teleosts (bony), elasmobranchs (cartilaginous) or jawless. To most biologists, it is self-evident that all these factors and a host of lifestyle and epigenetic factors would influence outcomes. It is a cautionary tale when reviewing old radiobiological literature. Key limitations of the data sets are the variability of doses and time points at which observations are recorded. One example serves to illustrate the importance of dose and time points [35]; it concerns experiments using goldfish (Carassius auratus L.). The experimenters irradiated the fish with a range of doses from 1-10 Gy and observed them to death (Figure 2). None of the fish given even 100 rad (1 Gy) survived for a year, while none of the controls died in a year – Goldfish can live up to 30 years in ideal conditions. Clearly, an effect marked as this cannot be described as ―life shortening‖, it is likely due to compromise of the immune system – a fact confirmed by the authors who found a marked reduction in bacterial and parasitic disease resistance in the fish receiving even the lowest doses. Old studies as comprehensive as this are rare, but it is clear that when LD50 or LD90 data are presented, they must be seen in context of the time post irradiation when measurments are made. Modern texts use, for example, LD50 – 30, to denote a dose which causes death of 50% of animals after 30 days.

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Modern Findings Relevant to Transgenerational Effects in Fish Among advances which have helped revitalize the field are advances in tissue culture and molecular tools available for study of subtle effects in fish tissues [36-42]. The application of mammalian concepts in low dose radiobiology, such as genomic instability, radiation-induced bystander effects and transgenerational expression of delayed damage in somatic progeny of irradiated cells and animals has also contributed to the new interest in this field of research [43-45]. In the authors‘ laboratory, considerable advances in this area have been made using different fish species and cell lines from a range of species. These are reviewed in the next section.

Review of Technical Advances Relevant to Fish Transgenerational Research The early work by our laboratory using fish cells is reviewed in [46, 47]. This charts initial attempts to develop culture techniques for fish tissues and later to use these ―ex-vivo‖ models to look at radiation effects. Figures 3-6 show some of the cultures which can be obtained, and in particular the figures highlight the histological characteristics which suggest radiation damage in fish cells in vitro. Of course, the major limitation when trying to do fish radiobiology is the reliance of radiobiologists on the clonogenic assay of Puck and Marcus [48] to determine survival, even at low doses. Only a few of easily available cell lines actually form colonies at low densities which is a requirement for doing survival curve analysis using the endpoint of reproductive death. The other lines do not form colonies, and explanted tissues cannot be used for conventional colony assays. However, our laboratory has developed a method where medium from irradiated non-clonogenic cells or tissues can be transferred to a clonogenic reporter cell line [49]. This at least allows bystander and other

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non-targeted endpoints to be measured in fish cells and tissues [50, 51]. The lack of molecular probes developed for fish also limits research and casts doubt on the validity of data generated using mammalian probes. Bols and colleagues [52, 53] have made major advances in this area as have Goldspink and colleagues [54, 55] and Lane and colleagues [56, 57]. Mouse and human antibodies and probes abound, but there are so many fish species that very few molecular tools specific for fish species are available. Most mammalian antibodies do cross-react, but establishing and validating that fact is quite a struggle [58-60].

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Genomic Instability in Fish, Adaptive and Bystander Effects In spite of technical hurdles, many advances have been made in fish radiobiology. Advances from our laboratory show that using clonogenic cell lines, in vitro explants and live fish, evidence can be found for highly radiosensitive responses of fish to very low acute and chronic doses of ionizing radiation [50, 51, 61-70]. Delayed transgenerational somatic effects can be detected at doses of 4 mGy 60Co delivered over 48 hrs to live salmonids . In vitro, there is evidence for bystander effects, delayed death (delayed lethal mutations) and adaptive responses at doses of 0.5 Gy 60Co in cell lines showing resistant responses to higher doses [50, 51, 67-70]. Most excitingly, there is evidence that irradiated fish can signal to unirradiated fish of the same species swimming with them that they have been exposed. Unirradiated fish induce specific proteins in response to signals, which protect them. These results were published over the last few years in a series of papers [71-74]. Figure 7 shows the protocol for these experiments, and Figure 8 summarises the proteomics data showing the identity of bystander proteins uniquely induced by the signals. Long-term experiments using salmonids are underway in the laboratory which show that even irradiation at the egg stage confers a lasting ―memory‖ of irradiation or signal reception on adults grown from both irradiated and bystander eggs; so 2 years later, adult fish are showing persistent signal production, altered responses to further irradiation and altered uptake of copper and sodium from the water they swim in [86] (authors‘ laboratory (unpublished data). The adult fish from the experiments in authors‘ laboratory will be breeding next year, and this will allow germ line transgenerational studies to be done. Apart from the intrinsic interest in fish biology, these studies also provide a unique approach for studying bystander effects. Text Box 5 summarises the points in favour of the fish model and its value as an experimental approach. Two examples of how in vitro results were validated in vivo using the fish model are presented below. Example 1 concerns the role of DNA repair in bystander effects. It is known [75-77] that if DNA repair capacity is compromised by any of several faults, bystander signals tend to induce apoptosis in recipient cells. This was validated in mix and match protocols [74] using repair proficient and deficient Medaka fish (Oryzias latipes, Temminck and Schlegel) swimming with syngeneic or transgenic medaka. Figure 9 shows the original in vitro data from our laboratory with some of the new in vivo data. Another example concerns the role of serotonin (5HT). Validation of the role of serotonin in fish radiation response was confirmed using zebrafish (Danio rerio, Hamilton-Buchanan) which were injected with neurochemically relevant levels of reserpine – an inhibitor of 5HT binding to cell receptors. The data [78, 79] for in vitro and in vivo studies are shown in Figure 10 and confirm that both in vivo and in vitro, serotonin binding to cells is involved in the mechanism leading to the release of bystander signals from irradiated

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cells/tissues/organisms and serve again to validate in vitro observations in mammalian cells using the in vivo fish model. This makes the fish model very useful for studying mechanisms and relationships between genetic, epigenetic and non-targeted effects. Text Box 5. Value of the fish communication model for the study of epigenetic and nontargeted mechanisms in radiobiology 1. 2. 3.

4.

5.

6. 7.

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8. 9.

This is a model system for looking at bystander effects isolated from direct effects in vivo It is a unique system where the production of a signal in vivo in an irradiated animal can be studied separately from the response in vivo to that signal. Bystander response as distinct from signal production can be studied in vivo in a whole totally unexposed animal without the complication of while body scatter, circulating blood or stress hormones associated with whole body experiments. In terms of evolution, it shows conservation of the mechanism and bystander pathway across species and suggests a very primitive origin of vertebrates since teleost fish split from the main vertebrate line early in vertebrate evolution. Because the signal is secreted into water in this model and is stable in the water for at least six hours, it is possible to analyze the water and get information about the chemistry and stability of signals without the complicating factors of medium and serum components. Basically a whole live organism instead of a cell culture is being used as a ―reporter‖ for the bystander signal. In vitro findings concerning the role of serotonin and the importance of DNA repair capacity are both confirmed in the fish model which validates it as a useful and meaningful system. Health effects and transgenerational effects in short-lived zebrafish or Medaka can be studied much more conveniently than in studies on mice or humans. A huge range of knockout zebrafish and medaka are available, making mechanistic studies feasible.

Transgenics and the Use of Fish Embryos as Models for Finding Teratogenic Effects Much of the current interest in fish as model systems concerns the use of zebrafish and to a lesser extent medaka (mainly in Japan) as convenient organisms for testing chemical exposures. Techniques for their use are reviewed in detail by Hill et al. and Hobbie et al. [80, 81]. Pictures of the fish are shown in Figure 11. They are both small, they breed well in captivity and are highly fecund, meaning that great statistics can be got in mutagenesis studies. Their relatively fast breeding cycle means that transgenerational studies are easier and less expensive than mouse studies. A further major advantage of their fecundity is the ability to breed transgenic fish. Several transgenic zebrafish are available, including the famous ―glow-in-the-dark‖ fish which has GFP coupled to a p53 reporter [82]. When p53 is activated, the fish glows green.

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Context and Relevance of Fish Transgenerational Studies for Ecology, Systems Biology and Modeling Ecosystem Drift in Evolutionary Biology There are probably two main areas where the studies discussed above have wider relevance in biology; one is the ecological relevance and ability of stressed individuals or populations to adapt to changed environmental conditions. The second concerns the evolutionary relevance because the effect is so widespread in living organisms. This could suggest it evolved as a mechanism which optimizes multicellularity in higher organisms. In terms of ecological relevance – clearly, fish studies showing inter individual communication which alerts members of a fish population to the presence of a stressor mean that the mechanism is a natural protective response operating to benefit the population if an individual is stressed. The important point is that ―bystander fish‖ induce protective responses in response to receipt of signals from fish which were exposed to radiation. It has only been possible to examine the phenomenon in our in vivo model where different fish produce and receive signals and where the causative agent (electromagnetic ionizing radiation) leaves no residue in the organism or the medium (water) in which the fish swims. In the field of Chemical Ecology, although mainly studied in plants, allelopathic mechanisms such as alarm or other signaling between individuals are well known as protective strategies alerting members of the species to the presence of predators [83]. These signals can also enable social controls, territorial defense or habitat identification at the population level [84, 85]. However, the fish model suggests that these mechanisms do not only involve neural and behavioural pathways, as has been documented in the chemical ecology field, but that the unaffected fish appear to be able to induce proteins in specific organs on receipt of signals from exposure which prepare it for future exposure. The fact that the memory of this effect is so long [86, 87] suggests that it must involve immune memory or related pathways. The findings suggest conservation of genomic instability and bystander mechanisms across species because apart from the literature on fish, a similar finding of interanimal communication of radiation damage signals was reported in mice by Surinov and colleagues in a series of papers mostly in Russian [88-93]. This suggests a very primitive origin in vertebrates, since teleost fish split from the main vertebrate line early in vertebrate evolution. Across species, bystander effects can be seen in yeasts, plants, and in the animal kingdom in athropods, molluscs, and all groups of vertebrates examined so far [94]. The interesting question is why such a mechanism should be so widespread when it can cause induction of protective responses which may never be needed (the directly exposed fish does not induce the protective proteome). This raises other evolutionary questions. In terms of evolutionary mechanisms, the findings in fish radiobiology relating to communication of stress signals and adaptive responses raise some good points. For example, it is rather Lamarckian ideas to have an environmental change or exposure causing persistent changes in protein expression in unaffected individuals in the population. The idea of ―survival of the fittest by natural selection‖ first proposed by Darwin emphasizes the importance of reproductive fitness and the selection of fit individuals which live to breed, while those without advantageous adaptations die before they have a chance to pass on their genes. While these mechanisms have not yet been proven to be truly transgenerational, they are persistent in the absence of further exposure, suggesting that we are not dealing with a simple protein up-regulation in response to a specific signal. The transgenerational persistence of genomic instability has been demonstrated in mammals [96-101], however the

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persistent expression of effects due to induction of genomic instability in the stem cell compartment in bone marrow has also been confirmed [102-104] This makes it very likely that the immune style memory referred to above may also be in the germ line. Transmission of stress-induced instability through the germ line of individuals not directly exposed to a stressor would mean that characteristics induced by a particular environmental stressor would be permanently incorporated in the genome of the offspring, regardless of whether their parents were actually exposed or not, and whether the changes were advantageous or not, i.e., selection per se in the sense of favourable adaptive selection would play no role in perpetuating the transmission of a phenotypic change. In terms of evolution, this would be easier to understand in situations where chronic exposures or repeated acute exposures occur. This would lead to a well-known phenomenon of population drift, although suggesting an epigenetic component to the process, but acute one-off exposure leading to permanent changes is less intuitive and could be disadvantageous in that it induces redundant changes permanently in situations where such response might not be useful. Of course, it is a mistake to think that all evolutionary changes must be beneficial! The authors‘ current conclusion is that genomic instability and bystander effects however induced are part of a universal stress response probably operating at the level of the cell, even when it is detected at higher levels. It appears to facilitate rapid adaptation to changed environmental conditions. Problems may arise if the environmental change is temporary – unless of course there are yet unrecognized triggers or switch mechanisms that allow for rapid reverse adaptation after the stress has passed. Evidence for reverse adaptation is lacking for acute radiation stress in the data sets available to the authors at this time. Clearly mechanisms which evolve for one reason may emerge in subsequent evolution either to serve another purpose or may be an unfortunate consequence of a different environmental circumstance. It is likely that chemical signaling underlies the ability of life forms to associate in a controlled way and function as multicellular organs or as communities and ecosystems. The sophisticated use of these signaling mechanisms can be seen in the nervous system or immune system, for example. In higher hierarchical levels of organization, allomones, kiaromones and pheromones are examples of useful functions of chemical signals (see Text Box 6 for a description of these terms). Perhaps, it is not unexpected that exposure to acute and transitory stressors, which only really started to impact biological systems in a major way with the advent of human agricultural and industrial activities, could compromise a surveillance system which evolved over billions of years. Text Box 6. Terms Used In Chemical Ecology To Describe Inter- And Intra Signalling Mechanisms Pheromones: benefit the reproduction of the species - e.g., sex attractants Allomones: benefit the producer but harm the recipient - e.g., insectivorous plant nectars. Synomones: benefit both parties, e.g., plant scent which attracts a pollinating insect. Kairomones: benefit the recipient, e.g., chemicals produced by prey which identify them to predators Apneumones: Attractants produced by non-living matter, e.g., meat or dead wood which attracts prey to host habitat. Terms defined in: [143] Reviewed in Alberts (1992) [83]

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In summary, this chapter looks at old and new data relevant to transgenerational genomic instability in fish and concludes that genomic instability is probably part of an ancient and highly conserved evolutionary mechanism which permits rapid adaptation to changed environmental conditions. Problems with this idea are that acute and transient stressors induce genomic instability as a ―permanent‖ response to exposure. The argument is put forward that this is not a failure of evolution but rather an unfortunate consequence of human activity, although it is possible that reverse switches which could turn off these processes are awaiting discovery.

ACKNOWLEDGMENTS We acknowledge the help of Laura Banevicius and Britni Rowe, who are co-op students in our laboratory and did sterling work getting the references together and summarized for this paper. They are responsible for the information in Table 1 and 2.

REFERENCES

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[1] [2]

ICRP Publication 103: New Recommendations of the ICRP. Annals of the ICRP. 2008. Egami N: Radiation effects on aquatic organisms. Japan Scientific Societies Press. 1980. [3] Hinton TG, Whicker FW: A screening model approach to determine probable impacts to fish from historic releases of radionuclides. Freshwater and Estuarine Radioecology Studies in Environmental Science. 1997, 441-448. [4] Brownless GP: Does protecting humans protect the environment? A crude examination for UK nuclear power plants and the marine environment using information in the public domain. J. Radiol. Prot. 2008, 28(4):525-38. [5] Delistraty D: Radioprotection of nonhuman biota. J. Environ. Radioact. 2008, 99(12):1863-9. [6] Garnier-Laplace J, Copplestone D, Gilbin R, Alonzo F, Ciffroy P, Gilek M, Agüero A, Björk M, Oughton DH, Jaworska A, Larsson CM, Hingston JL: Issues and practices in the use of effects data from FREDERICA in the ERICA Integrated Approach. J. Environ Radioact 2008, 99(9):1474-83. [7] Howard BJ, Larsson CM: The ERICA Integrated Approach and its contribution to protection of the environment from ionising radiation. J. Environ. Radioac. 2008, 99(9):1361-3. [8] Ulanovsky A, Pröhl G, Gómez-Ros JM: Methods for calculating dose conversion coefficients for terrestrial and aquatic biota. J. Environ. Radioact. 2008, 99(9):1440-8. [9] Alonzo F, Hertel-Aas T, Gilek M, Gilbin R, Oughton DH, Garnier-Laplace J: Modelling the propagation of effects of chronic exposure to ionising radiation from individuals to populations. J. Environ. Radioact. 2008, 99(9):1464-73. [10] Alonzo F, Gilbin R, Zeman FA, Garnier-Laplace J: Increased effects of internal alpha irradiation in Daphnia magna after chronic exposure over three successive generations. Aquat Toxicol .2008, 87(3):146-56.

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Transgenerational Genomic Instability in Fish

191

[11] Eckerman KF, Bolch WE, Zankl M, Petoussi-Henss N: Response functions for computing absorbed dose to skeletal tissues from photon irradiation. Radiat Prot Dosimetry 2007, 127(1-4):187-91. [12] Franck D: Potential of modern technologies for improving internal exposure monitoring. Radiat Prot Dosimetry 2007, 127(1-4):233-9. [13] Amiro BD: Radiological dose conversion factors for generic non-human biota used for screening potential ecological impacts. J. Environ. Radioact. 1997, 35:37-51. [14] Hinton T, Brechignac F: The scientific basis for radiological protection of the environment. IRSN Series. 2004. [15] Børretzen P, Brown J, Strand P, Johansson E, Ramstedt M, Avila R, Pröhl G, Ulanovsky A, Copplestone D: The ERICA assessment tool. Proceeding of the 2nd International Conference on Radioactivity in the Environment 2005, 43–46. [16] ICRP Publication 91: A framework for assessing the impact of ionising radiation on non-human species. Annals of the ICRP 2005, 33(3):201-270. [17] Grunwald DJ, Eisen JS: Headwaters of the zebrafish -- emergence of a new model vertebrate. Nat. Rev. Genet 2002, 3(9):717-24. [18] Amsterdam A, Becker TS: Transgenes as screening tools to probe and manipulate the zebrafish genome. Dev. Dyn. 2005, 234(2):255-68. [19] Berghmans S, Jette C, Langenau D, Hsu K, Stewart R, Look T, Kanki JP: Making waves in cancer research: new models in ebrafish. Biotechniques 2005, 39(2):227-37. [20] Chapin R, Augustine-Rauch K, Beyer B, Daston G, Finnell R, Flynn T, Hunter S, Mirkes P, O'Shea KS, Piersma A, Sandler D, Vanparys P, Van Maele-Fabry G: State of the art in developmental toxicity screening methods and a way forward: a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish. Birth Defects Res B Dev. Reprod. Toxicol. 2008, 83(4):446-56. [21] Peterson RT, Nass R, Boyd WA, Freedman JH, Dong K, Narahashi T: Use of nonmammalian alternative models for neurotoxicological study. Neurotoxicology 2008, 29(3):546-55. [22] Wu M, Chaudhary A, Khan IA, Dasmahapatra AK: Ethanol teratogenesis in Japanese medaka: effects at the cellular level. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2008, 149(1):191-201. [23] Martins J, Oliva Teles L, Vasconcelos V: Assays with Daphnia magna and Danio rerio as alert systems in aquatic toxicology. Environ Int 2007, 33(3):414-25. [24] Niiyama H: Effects of X-irradiation upon Rainbow Trouts (Salmo-irideus) II. Stimulative effects of X-rays on spermatogenesis in fry. Journal of the Faculty of Science 1957, 13(1-4):281-288. [25] Bonham K, Welander AD: Increase in radioresistance of fish to lethal doses with advancing embryonic development. Proceeding of the First National Symposium on Radioecology 1961, 353. [26] Brown VM, Templeton WL: Resistance of fish embryos to chronic irradiation. Nature 1964, 203:1257-1259. [27] Engel DW, Angelovic JW, Davis EM: Effects of acute gamma irradiation on blood constituents infish, (Lagondon rhomboides L.) chesapeake. Science 1966, 7(2):90-94.

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192

Carmel Mothersill and Colin Seymour

[28] McGregor JF, Newcombe HB: Decreased risk of embryo mortality following low doses of radiation to trout sperm. Radiat. Res. 1972, 52:536-544. [29] Strand JA, Fujihara MP, Burdett RD, Poston TM: Suppression of the primary immune response in rainbow trout, Salmo gairdneri, sublethally exposed to tritiated water during embryogenesis. J. Fish Res. Board Can. 1977, 34(9):1293-1304. [30] Strand JA, Fujihara MP, Poston TM, Abernethy CS: Permanence of suppression of the primary immune response in rainbow trout, Salmo gairdneri, sublethally exposed to tritiated water during embryogenesis. Radiat Res. 1982, 91:533- 541. [31] UNSCEAR 1996 Report: Sources and Effects of Ionizing Radiation. 1996, 1-86. [32] ICRP Publication 60: Recommendations of the International Commission on Radiological Protection. 60 Annals of the ICRP 1990, 21:1-3. [33] Whicker FW, Schultz V: Radioecology: Nuclear Energy and the Environment. CRC Press. 1982. [34] Sparrow AH, Miksche JP: Correlation of nuclear volume and DNA [deoxyribonucleic acid] content with higher plant tolerance to chronic radiation. Science 1961, 134:282. [35] Shechmeister I, Watson LJ, Cole VW, Jackson LL: The Effect of X irradiation on Goldfish. Radiat Res 1962, 16:89-97. [36] Leal MC, de Waal PP, García-López A, Chen SX, Bogerd J, Schulz RW: Zebrafish primary testis tissue culture: an approach to study testis function ex vivo. Gen. Comp Endocrinol 2009, 162(2):134-8. [37] Skov J, Kania PW, Dalsgaard A, Jørgensen TR, Buchmann K: Life cycle stages of heterophyid trematodes in Vietnamese freshwater fishes traced by molecular and morphometric methods. Vet. Parasitol. 2009, 160(1-2):66-75. [38] Nolan EM, Lippard SJ: Small-molecule fluorescent sensors for investigating zinc metalloneurochemistry. Acc. Chem. Res. 2009, 42(1):193-203. [39] Kruse U, Bantscheff M, Drewes G, Hopf C: Chemical and pathway proteomics: powerful tools for oncology drug discovery and personalized health care. Mol. Cell Proteomics 2008, 7(10):1887-901. [40] Placzek MR, Chung IM, Macedo HM, Ismail S, Mortera Blanco T, Lim M, Cha JM, Fauzi I, Kang Y, Yeo DC, Yip Joan Ma C, Polak JM, Panoskaltsis N, Mantalaris A: Stem cell bioprocessing: fundamentals and principles. J. R. Soc. Interface 2009, 6(32):209-32. [41] Khoo SH, Al-Rubeai M: Metabolomics as a complementary tool in cell culture. Biotechnol Appl Biochem 2007, 47(2):71-84. [42] Deocaris CC, Widodo N, Wadhwa R, Kaul SC: Merger of ayurveda and tissue culturebased functional genomics: inspirations from systems biology. J Transl Med 2008, 6:14. [43] Kovalchuk O, Baulch JE: Epigenetic changes and nontargeted radiation effects--is there a link? Environ. Mol. Mutagen 2008, 49(1):16-25. [44] Wright EG: Microenvironmental and genetic factors in haemopoietic radiation responses. Int. J. Radiat. Biol. 2007, 83(11-12):813-8. [45] Schwartz JL: Variability: the common factor linking low dose-induced genomic instability, adaptation and bystander effects. Mutat. Res. 2007, 616(1-2):196-200.

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Transgenerational Genomic Instability in Fish

193

[46] Mothersill C, Austin B: In Vitro Methods in Aquatic Toxicology. Springer, Praxis (Berlin, New York, Chichester, UK) 2003. [47] Mothersill C, Lyng F, Mulford A, Seymour C, Cottell D, Lyons M, Austin B: Effect of low doses of ionizing radiation on cells cultured from the hematopoietic tissue of the Dublin Bay prawn, Nephrops norvegicus. Radiat. Res. 2001, 156(3):241-50. [48] Puck TT, Marcus PI: Action of x-rays on mammalian cells. J. Exp. Med. 1956, 103(5):653-66. [49] Mothersill C, Rea D, Wright EG, Lorimore SA, Murphy D, Seymour CB, O'Malley K: Individual variation in the production of a 'bystander signal' following irradiation of primary cultures of normal human urothelium. Carcinogenesis 2001, 22(9):1465-71. [50] O'Neill-Mehlenbacher A, Kilemade M, Elliott A, Mothersill C, Seymour C: Comparison of direct and bystander effects induced by ionizing radiation in eight fish cell lines. Int. J. Radiat. Biol. 2007, 83(9):593-602. [51] Ryan LA, Seymour CB, O'Neill-Mehlenbacher A, Mothersill CE: Radiation-induced adaptive response in fish cell lines. J. Environ. Radioact 2008, 99(4):739-47. [52] Steinmoeller JD, Fujiki K, Arya A, Müller KM, Bols NC, Dixon B, Duncker BP: Characterization of rainbow trout CHK2 and its potential as a genotoxicity biomarker. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2009, 149:491-499. [53] Tafalla C, Sanchez E, Lorenzen N, DeWitte-Orr SJ, Bols NC: Effects of viral hemorrhagic septicemia virus (VHSV) on the rainbow trout (Oncorhynchus mykiss) monocyte cell line RTS-11. Mol. Immunol. 2008, 45(5):1439-48. [54] Rock J, Magnay JL, Beech S, El Haj AJ, Goldspink G, Lunt DH, Whiteley NM: Linking functional molecular variation with environmental gradients: Myosin gene diversity in a crustacean broadly distributed across variable thermal environments. Gene 2009, 437:60-70. [55] Wilkes D, Xie SQ, Stickland NC, Alami-Durante H, Kentouri M, Sterioti A, Koumoundouros G, Fauconneau B, Goldspink G: Temperature and myogenic factor transcript levels during early development determines muscle growth potential in rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax). J. Exp. Biol. 2001, 204(Pt 16):2763-71. [56] Chen J, Ruan H, Ng SM, Gao C, Soo HM, Wu W, Zhang Z, Wen Z, Lane DP, Peng J: Loss of function of def selectively up-regulates Delta113p53 expression to arrest expansion. Genes Dev. 2005, 19(23):2900-11. [57] Chen J, Ng SM, Chang C, Zhang Z, Bourdon JC, Lane DP, Peng J: p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish. Genes Dev. 2009, 23(3):278-90. [58] Lyons-Alcantara M, Lambkin HA, Nordmo R, Lyng F, Mothersill C: Cross-reactivity of some antibodies to human epitopes with shrimp Pandalus borealis proteins: a possible aid in validation and characterization of crustacean cells in vitro. Cell Biochem. Funct. 2002, 20(3):247-56. [59] Lyons-Alcantara M, Lambkin HA, Mothersill C: Antigenic characterization Nephrops norvegicus (L.) hepatopancreas cells. Cell Biochem. Funct 1999, 17(3):157-64. [60] Kilemade M, Lyons-Alcantara M, Rose T, Fitzgerald R, Mothersill C: Trout primary epidermal cell proliferation as an indicator of aquatic toxicity: an in vitro/in vivo exposure comparison. Aquat. Toxicol. 2002, 60(1-2):43-59.

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[61] Lyng EM, Lyons-Alcantara M, Olwell P, Shuilleabháin SN, Seymour C, Cottell DC, Mothersill C: Ionizing radiation induces a stress response in primary cultures of rainbow trout skin. Radiat. Res. 2004, 162(2):226-32. [62] Kilemade M, Lyons-Alcantara M, Rose T, Fitzgerald R, Mothersill C: Rainbow trout primary epidermal cell proliferation as an indicator of aquatic toxicity: an in vitro/in vivo exposure comparison. Aquat Toxicol 2002, 60(1-2):43-59. [63] Olwell PM, Cottell DC, Ní Shúilleabháin S, Maderna P, Seymour C, Mothersill C, Lyng FM: Cytoskeletal reorganization and altered phagocytotic ability in primary cultures of rainbow trout hemopoietic tissue exposed to low-level ionizing radiation. Radiat Res 2005, 164(1):45-52. [64] O'Dowd C, Mothersill CE, Cairns MT, Austin B, McClean B, Lyng FM, Murphy JE: The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation. Radiat. Res. 2006, 166(4):611-7. [65] Mothersill C, Salbu B, Heier LS, Teien HC, Denbeigh J, Oughton D, Rosseland BO, Seymour CB: Multiple stressor effects of radiation and metals in salmon (Salmo salar). J. Environ. Radioact 2007, 96(1-3):20-31. [66] Salbu B, Denbeigh J, Smith RW, Heier LS, Teien HC, Rosseland BO, Oughton D, Seymour CB, Mothersill C: Environmentally relevant mixed exposures to radiation and heavy metals induce measurable stress responses in Atlantic salmon. Environ. Sci. Technol. 2008, 42(9):3441-6. [67] Mothersill C, Crean M, Lyons M, McSweeney J, Mooney R, O'Reilly J, Seymour CB. Expression of delayed toxicity and lethal mutations in the progeny of human cells surviving exposure to radiation and other environmental mutagens. Int. J. Radiat. Biol 1998, 74:673-680. [68] Lyons-Alcantara M, Mooney R, Lyng F, Cottell D, Mothersill C: The effects of cadmium exposure on the cytology and function of primary cultures from rainbow trout. Cell Biochem Funct 1998, 16(1):1-13. [69] Dowling K, Seymour C, Mothersill C: Delayed cell death and bystander effects in the progeny of Chinook salmon embryo cells exposed to radiation and a range of aquatic pollutants. Int. J. Radiat. Biol. 2005, 81:89-96. [70] Dowling K, Mothersill C: Use of rainbow trout primary epidermal cell cultures as an alternative to immortalized cell lines in toxicity assessment: a study with nonoxynol. Environ Tox. Chem. 1999, 18:2846–2850. [71] Mothersill C, Bucking C, Smith RW, Agnihotri N, O‘Neill A, Kilemade M, Seymour CB: Communication of radiation-induced stress or bystander signals between fish in vivo. Environ Sci. Technol. 2006, 40:6859-6864. [72] Mothersill C, Smith RW, Agnihotri N, Seymour CB: Characterization of a radiationinduced stress response communicated in vivo between zebrafish. Environ. Sci. Technol. 2007, 41(9):3382-3387. [73] Smith RW, Wang J, Bucking CP, Mothersill CE, Seymour CB: Evidence for a protective response by the gill proteome of rainbow trout exposed to X-ray induced bystander signals. Proteomics 2007, 7:4171-4180. [74] Mothersill C, Smith RW, Hinton TG, Aizawa K, Seymour CB: Communication of radiation-induced signals in vivo between DNA repair deficient and proficient medaka (Oryzias latipes). Environ. Sci. Technol. 2009, 43(9):3335–3342.

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Transgenerational Genomic Instability in Fish

195

[75] Mothersill C, Seymour RJ, Seymour CB: Bystander effects in repair-deficient cell lines. Radiat. Res. 2004, 161(3):256-63. [76] Mothersill C, Seymour RJ, Seymour CB: Increased radiosensitivity in cells of two human cell lines treated with bystander medium from irradiated repair-deficient cells. Radiat. Res. 2006, 165(1):26-34. [77] Burdak-Rothkamm S, Rothkamm K, Prise KM: ATM acts downstream of ATR in the DNA damage response signaling of bystander cells. Cancer Res. 2008, 68(17):7059-65. [78] Poon RC, Agnihotri N, Seymour C, Mothersill C: Bystander effects of ionizing radiation can be modulated by signaling amines. Environ. Res. 2007, 105(2):200-11. [79] Saroya R, Smith RW, Seymour CB, Mothersill C: A role for serotonin in bystander signaling between zebrafish in vivo. Radiation Research Society, Abstract. 2009. [80] Hill AJ, Teraoka H, Heideman W, Peterson RE: Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol Sci. 2005, 86(1):6-19. [81] Hobbie KR, Deangelo AB, King LC, Winn RN, Law JM: Toward a molecular equivalent dose: use of the medaka model in comparative risk assessment. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 2009, 149(2):141-51. [82] Lee KC, Goh WL, Xu M, Kua N, Lunny D, Wong JS, Coomber D, Vojtesek B, Lane EB, Lane DP: Detection of the p53 response in zebrafish embryos using new monoclonal antibodies. Oncogene 2008, 27(5):629-40. [83] Alberts AC: Constraints on the Design of Chemical Communication Systems in Terrestrial Vertebrates. Am. Nat. 1992, 139:S1- S62. [84] Herout V, Levinson HZ: Editorial, Low Molecular Weight substances as regulators in biological systems. Russ. Chem. Rev. 1974, 43:96-198. [85] Daev EV: Stress, Chemocommunication and the Physiological Hypothesis of Mutation. Russ. J. Genet. 2007, 43:1082-1092. [86] Smith RW, Mothersill C, Moccia RD, Seymour CB: Legacy effects of direct exposure to x-rays and to the x-ray induced bystander effect in rainbow trout (Oncorhynchus mykiss, W.). American Fisheries Society; 8th International Congress on the Biology of Fish. 2008. [87] Mothersill C, Smith RS, Moccia R, Seymour CB: Rainbow trout (Oncorhynchus mykiss, W.) directly irradiated or receiving bystander signals up to 2 months of age retain a memory of past exposure at least 2 years. Env. Sci. Tech. 2007, 41(9):3382-7. [88] Surinov BP, Isaeva VG: Immunomodulation effects volatile secretion of animals at postradiat immunodeficiency conditions. Radiats Biol. Radioecol. 2008, 48(6):665-70. [89] Surinov BP, Zhovtun LP: The alteration of attractiveness of intact males mice to females chemosignals, subjected of ionizing radiation in sublethal dose. Radiats Biol. Radioecol. 2008, 48(4):409-15. [90] Sharetskiĭ AN, Surinov BP, Abramova MR, Karpova NA: The influence of postradiation chemical signals of mice on humoral immune response in recipients different time relative antigenic stimulus. Radiats Biol. Radioecol. 2008, 48(3):342-5. [91] Daev EV, Surinov BP, Dukel'skaia AV, Marysheva TM: Chromosomal abnormalities and spleenocyte production in laboratory mouse males after exposure to stress chemosignals. Tsitologiya 2007, 49(8):696-701. [92] Surinov BP: Mice with radiation or toxic damage or malignant tumors produce aversive chemosignals repelling intact animals. Dokl Biol. Sci. 2007, 414:199-201.

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196

Carmel Mothersill and Colin Seymour

[93] Surinov BP, Shpagin DV: Influence of the irradiating on olfactory ability of male mice to distinguish chemo-signals of intact individuals. Radiats Biol. Radioecol 2007, 47(1):17-21. [94] Mothersill C, Seymour C: Radiation-induced bystander effects: past history and future directions. Rad. Res. 2001, 155:759-767. [95] Darwin, C: On the Origin of Species By Means of Natural Selection. London: John Murray, Albemarle Street, First Edition 1859. [96] Foster RF, Donaldson LR: The effect of embryos and young of rainbow trout from exposing the parent fish to X-rays. Growth 1949, 13:119-142. [97] Dubrova YE: Radiation-induced transgenerational instability. Oncogene 2003, 22:70877093. [98] Niwa O: Radiation induced dynamic mutations and transgenerational effects. J. Rad. Res. 2006, 47 Suppl:B:B25-30. 2006. [99] Nomura T: Transgenerational carcinogenesis: induction and transmission of genetic alterations and mechanisms of carcinogenesis. Mutat. Res. 2003, 544(2-3):425-432. [100] Tamminga J, Koturbash I, Baker M, Kutanzi K, Kathiria P, Pogribny IP, Sutherland RJ, Kovalchuk O: Paternal cranial irradiation induces distant bystander DNA damage in the germline and leads to epigenetic alterations in the offspring. Cell Cycle 2008, 7(9):1238-1245. [101] Vorobtsova IE: Transgenerational transmission of radiation-induced genomic instability and predisposition to carcinogenesis. Vopr. Onkol 2008, 54:490-493. [102] Lorimore SA, McIlrath JM, Coates PJ, Wright EG: Chromosomal instability in unirradiated hemopoietic cells resulting from a delayed in vivo bystander effect of gamma radiation. Cancer Res. 2005, 65:5668-5673. [103] Lorimore SA, Chrystal JA, Robinson JI, Coates PJ, Wright EG: Chromosomal instability in unirradiated hemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation. Cancer Res. 2008, 68(19):8122-8126. [104] Devi PU, Satyamitra M: Tracing radiation induced genomic instability in vivo in the haemopoietic cells from fetus to adult mouse. Br. J. Radiol. 2005, 78:928-933. [105] Shimada A, Eguchi H, Yoshinaga S, Shima A: Dose-rate effect on transgenerational mutation frequencies in spermatogonial stem cells of the medaka fish. Radiat Res. 2005, 163:112-114. [106] Shimada A, Shima A: High incidence of mosaic mutations induced by irradiating paternal germ cells of the medaka fish, Oryzias latipes. Mutat. Res. 2001, 495:33-42. [107] Le François NR, Blier PU, Adambounou LT, Lacroix M: Exposures to low-level ionizing radiation: Effects on biochemical and whole-body of growth in juvenile brook charr (Salvelinus fontinalis). J. Exp. Zoolog A Comp. Exp. Biol. 1999, 283:315–325. [108] Shimada A, Shima A: Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish. Mutat. Res. 2004, 552:119-124. [109] Shimada A, Shima A, Nojima K, Seino Y, Setlow RB: Germ cell mutagenesis in medaka fish after exposures to high-energy cosmic ray nuclei: A human model. Proc. Natl. Acad. Sci. USA 2005, 102:6063-6067. [110] Zhong X, Xu Y, Liang Y, Liao T, Wang J: The Chinese rare minnow (Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full lifecycle test with diethylstilbestrol. Aquat. Toxicol. 2005, 71:85-95.

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[111] Zhang Z, Hu J, Zhen H, Wu X, Huang C: Reproductive inhibition and transgenerational toxicity of triphenyltin on medaka (Oiyzias latipes) at environmentally relevant levels. Environ Sci. Technol. 2008, 42:8133-8139. [112] Sun L, Zha J, Spear PA, Wang Z: Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults. Comp Biochem Physiol. C Toxicol. Pharmacol. 2007, 145:533-541. [113] Schwaiger J, Mallow U, Ferling H, Knoerr S, Braunbeck T, Kalbfus W, Negele RD: How estrogenic is nonylphenol? A transgenerational study using rainbow trout (Oncorhynchus mykiss) as a test organism. Aquat Toxicol 2002, 59:177-189. [114] Foran CM, Peterson BN, Benson WH: Transgenerational and developmental exposure of Japanese medaka (Oryzias latipes) to ethinylestradiol results in endocrine and reproductive differences in the response to ethinylestradiol as adults. Toxicol. Sci. 2002, 68:389-402. [115] Bladen CL, Navarre S, Dynan WS, Kozlowski DJ: Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis. Neurosci. Lett. 2007, 422(2):97-102. [116] Hinton TG, Alexakhin R, Balonov M, Gentner N, Hendry J, Prister B, Strand P, Woodhead D: Radiation-induced effects on plants and animals: Findings of the United Nations Chernobyl Forum. Health Phys. 2007, 93(5):427-40. [117] Veldman MB, Lin S: Zebrafish as a Developmental Model Organism for Pediatric Research. Pediatr. Res. 2008, 64(5):470-6. [118] Bladen CL, Flowers MA, Miyake K, Podolsky RH, Barrett JT, Kozlowski DJ, Dynan WS: Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo. Radiat Res 2007, 168(2):149-57. [119] Grygoryev D, Moskalenko O, Zimbrick JD: Non-linear effects in the formation of DNA damage in Medaka fish fibroblast cells caused by combined action of cadmium and ionizing radiation. Dose Response 2008, 6(3):283-98. [120] Baldessari D, Mione M: How to create the vascular tree? (Latest) help from the zebrafish. Pharmacol Ther 2008, 118:206-230. [121] Stone HB, Moulder JE, Coleman CN, Ang KK, Anscher MS, Barcellos-Hoff MH, Dynan WS, Fike JR, Grdina DJ, Greenberger JS, Hauer-Jensen M, Hill RP, Kolesnick RN, Macvittie TJ, Marks C, McBride WH, Metting N, Pellmar T, Purucker M, Robbins ME, Schiestl RH, Seed TM, Tomaszewski JE, Travis EL, Wallner PE, Wolpert M, Zaharevitz D: Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI workshop, December 3-4, 2003. Radiat. Res. 2004, 162:711-728. [122] Bladen CL, Lam WK, Dynan WS, Kozlowski DJ: DNA damage response and Ku80 function in the vertebrate embryo. Nucleic Acids Res. 2005, 33(9):3002-3010. [123] Feitsma H, Cuppen E: Zebrafish as Cancer Model. Mol. Cancer Res. 2008, 6(5): 686. [124] Kirchmann R, Dupont JC: Incorporation of Tritium from various origins in freshwater fish and the radiological implication for man. In Radiation Effects on Aquatic Organisms. 1980, 39-43. [125] Hyodo-Taguchi Y: Effects of chronic γ-irradiation on spermatogenesis in the fish, oryzias latipes, with special reference to regeneration of testicular stem cells. In Radiation Effects on Aquatic Organisms.1980, 91-104.

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[126] Egami N, Hama-Furukawa A: Late effects of continuous γ-irradiation of the developmental stage on the gonads in oryzias latipes. In Radiation Effects on Aquatic Organisms. 1980, 105-117. [127] Hamaguchi S: Differential radiosensitivity of germ cells according to their developmental stages in the teleost, oryzias latipes. In Radiation Effects on Aquatic Organisms. 1980, 119-128. [128] Konno K: Effects of γ-irradiation on the gonads fo the rainbow trout, salmo gairdnerii irideus, during embryonic stages. In Radiation Effects on Aquatic Organisms. 1980, 129-133. [129] Ghoneum MMH, Egami N: Effect of γ-irradiation of adult and embryo of oryzias latipes on thymus size. In Radiation Effects on Aquatic Organisms. 1980, 135-137. [130] Hama-Fukukawa A, Egami N: Effect of irradiation on the fin regerneartion of the fish, oryzias latipes, with special reference to the dose rate at different temperatures. In Radiation Effects on Aquatic Organisms. 1980, 139-146. [131] Woodhead AD, Setlow RB: Response to and Recovery from acute sublethal Gammaradiation in the Amazon Molly, Poecilia Formosa. In Radiation Effects on Aquatic Organisms. 1980, 171-182. [132] Etoh H, Suyama I: Effects of Radiation on cultured fish cells. In Radiation Effects on Aquatic Organisms. 1980, 195-203. [133] Suyama I, Etoh H, Ichikawa R: Effects of ionizing Radiation on the development of Limanda Eggs. In Radiation Effects on Aquatic Organisms. 1980, 205-207. [134] Aoki K, Matsudaira H: Induction of Hepatic Tumors after treatment with Mam Acetate in Oryzias Latipes and its inhibition by previous irradiation with X-rays. In Radiation Effects on Aquatic Organisms. 1980, 209-211. [135] Kyono-Hamaguchi Y: Temperature effects on liver tumorigenesis in the Medaka, Oryzias Latipes. In Radiation Effects on Aquatic Organisms. 1980, 213-216. [136] Kligerman AD: The Use of Aquatic Organisms to detect mutagens that cause cytogenetic damage. In Radiation Effects on Aquatic Organisms. 1980, 241-252. [137] Schroder JH: Radiation- Induced Mutation in Fish. In Radiation Effects on Aquatic Organisms. 1980, 217-222. [138] Hansen HJM: Changes in the Lipid Fraction of Eel (Anguilla Anguilla) Gills after Ionizing Irradiation and a Shift from Fresh to Sea Water. In Radiation Effects on Aquatic Organisms. 1980, 147-155. [139] Timofeeff-Ressovky, N. W., Zimmer K. G., and Delbrück M: Über die Natur der Genmutation und der Genstruktur. Nachrichten von der Gesellschaft der Wissenschaften zu Göttingen Mathematische-Physikalische Klasse, Fachgruppe VI, Biologie 1935, 1(13):189-245. [140] Osborne James C, Miller Jr, Jay H., and Kempner ES: Molecular Mass and Volume in Radiation Target Theory. Biophys. J. 2000, 78:1698 –1702. [141] Elkind MM: Radiobiology of Cultured Mammalian Cells. Gordon and Breach Science Publishers. 1967. [142] Alper, T: Cellular Radiobiology. Cambridge University Press (Cambridge:UK). 1979. [143] Nordland DA, Lewis WJ: Terminology of chemical releasing stimuli in intraspecific and interspecific interactions. J. Chem. Ecol. 1976, 2(2):211-220.

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Chapter 11

GERM CELL-MEDIATED MUTAGENESIS: INSIGHTS FROM THE  TRANSGENIC MEDAKA Richard N. Winn1 Aquatic Biotechnology and Environmental Laboratory, Warnell School of Forestry and Natural Resources, University of Georgia, Athens, Georgia

ABSTRACT

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Transgenic animal models that carry mutation target genes have proven invaluable in addressing the need for improved approaches to study mutations induced in somatic cells. Recently, a small laboratory fish model, the  transgenic medaka, was introduced as a new model for germ cell-mediated mutagenesis. Attributes of medaka as an animal model and the cII transgene as a mutation target gene combine to provide numerous practical and scientific benefits for such investigations. In this chapter, features of using the  transgenic medaka and the cII mutation assay, and results from this recent investigation using the germ cell mutagen ethylnitrosourea (ENU) are reviewed with a focus on comparisons with investigations using mouse models. The ability to characterize both the frequencies and specific types of cII mutations carried by individual mutant offspring proved invaluable in distinguishing non-mutant, whole body and mosaic mutant offspring. The frequencies of mutant offspring derived from ENU-treated spermatogonial stem cells of fish were remarkably similar to those reported for transgenes and endogenous genes of mice, thereby supporting the use of medaka as a comparative animal model for germline mutagenesis. The prevalence of mosaic mutant offspring and the distinctive spectra of mutations they carried revealed that, in addition to the role germ cells play as direct transmitters of mutations, germ cells carrying persistent DNA damage act as mediators of indirect mutagenesis in cells of early stage embryos. Emerging evidence suggests that error-prone DNA repair processes and likely, epigenetic processes acting independently or together contribute to a phase of hypermutagenesis manifested as genomic instability in offspring of mutagen-exposed germ cells. Whereas the processes of mutagenesis mediated by germ cells remain incompletely characterized, a model for the mechanism(s) will incorporate the interplay between persistent DNA damage, delayed and untargeted mutations, and constraints of rapidly dividing cells of early stage embryos in responding to damage contributed by germ cells. 1

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INTRODUCTION Currently, extrapolation from studies using animal models remains the method of choice for establishing genetic health risks in humans. Studies of mutations transmitted to progeny of mutagen-treated parents have been almost exclusively based on using the mouse as an animal model over the past several decades. Traditional assays, such as the dominant lethal assay (DL) [1], the morphological specific locus test (STL) [2], and the heritable translocation assay (HT) [3], have provided the foundation for much of our understanding of germline mutagenesis. However, limitations in these models, such as high frequencies of spontaneous lethal mutations and demands for extensive resources have called attention to the need for new sensitive, efficient and cost-effective alternative models to study germ cell mutagenesis [4]. Transgenic mutation models were introduced originally to address the need for improved approaches to detect somatic mutations in vivo. Developed first in rodents [5-9] and subsequently in fish [10-13], transgenic mutation assays share a similar general approach in which genomically integrated prokaryotic vectors containing specific reporter genes are recovered from tissues of interest and analyzed for mutations. To detect mutations, the animal is treated with a mutagen, and after sufficient time is allowed for mutations to manifest, genomic DNA is isolated from tissues. Vectors containing mutation target genes are then separated from the genomic DNA and shuttled into specialized indicator bacteria where mutant and non-mutant target genes are readily distinguished and quantified. The transgenic mutation assays overcome many of the challenges that have impeded the identification and quantification of spontaneous and induced somatic mutations. Mutations are quantified directly in single genes - the ultimate endpoint of DNA damage and/or repair. Mutations are detected in the transgenic locus independently of gene expression in the animal, conferring neither an advantage nor disadvantage to the survival or proliferation of the cell. Consequently, mutations persist and accumulate over time without mutant cells being subjected to selection in the animal. Large numbers of target locus copies can be screened rapidly, providing statistically reliable information on the frequency of mutations, while also providing significant practical advantages by reducing the numbers of animals used in the study. Every cell in the animal carries a transgene, thereby facilitating analyses of mutations recovered from virtually any tissue. Mutant target genes can be recovered, and specific mutations can be characterized at the molecular level, thereby providing insights into the mechanisms of mutagen action. Many of the features of transgenic mutation assays that have proven invaluable in studies of somatic mutations offer similar practical and scientific benefits in studies of germline mutagenesis. However, applications of transgenic mutation assays to investigate mutations transmitted to progeny from mutagen-exposed parents have been limited. Notably, Barnett and colleagues [14] compared the response of the lacI gene transgene in the Big Blue transgenic mouse with those of specific locus genes and thereby established a fundamental basis supporting the use of transgenic mutation systems as adjuncts or alternatives to current approaches. This study provided the first clear evidence that mutations induced in a mutation target transgene within spermatogonial stem cells of mutagen-exposed males are transmitted and readily detected in progeny, and that the mutant frequency in transgenes in germ cells is similar to that of endogenous genes.

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Most recently, a laboratory fish model, the  transgenic medaka, previously used to study chemically-induced mutations in the cII transgene in somatic cells, was introduced as a new model for the study of germ cell mutagenesis [15]. In this chapter, features of mutation analyses using the  transgenic medaka and results from this recent study are reviewed in the context of how this transgenic fish models provide possible new insights into fundamental processes of mutagenesis mediated by germ cells of mutagen-exposed male parents.

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 TRANSGENIC MEDAKA Medaka (Oryzias latipes), a fish species native to Japan and southeast Asia, has numerous attributes including small size (~2- 4 cm), ease of laboratory culture, low husbandry costs, highly prolific reproduction (10-30 eggs/female/day), and short generation time (6-8 weeks) that contribute to medaka being one of the leading fish models in biomedical and environmental research. Medaka is the most widely used fish species in environmental toxicology and carcinogenesis bioassays [16], and it has been identified as an ideal complement to zebrafish for genetics, experimental embryology, and molecular biology [17]. Recent completion of sequencing of the medaka genome [18] combined with the ability to develop specialized lineages by targeted disruption of the function of specific genes [19] further contribute to the placement of medaka among the leading model organisms for genomics and developmental genetics research [20]. In several studies using a specific locus test, medaka has been shown to be an appropriate model for germline mutagenesis with the equivalent response to mutagen exposure as rodent models and with the comparable prevalence of offspring that exhibit transgenerational genomic instability [21-23]. The  transgenic medaka was originally introduced to address the need for improved methods to analyze somatic mutations and to provide an adjunct or alternative to other costly and less-sensitive approaches using mammalian models [13]. The  transgenic medaka carries the identical LIZ bacteriophage vector that contains the lacI and cII mutation target genes found in the most widely used transgenic rodent mutation assays (Big Blue®, Stratagene) [8]. The development of transgenic medaka with the same mutation target gene as in transgenic rodent models facilitates unprecedented comparative studies of complex processes of mutagenesis that is simply not possible otherwise. Using a variety of chemical and physical mutagens and exposure regimens, the  transgenic medaka model has been applied in studies ranging from chemoprevention [24], comparative chemical carcinogenesis and mutagenesis [12, 25, 26], and ultraviolet radiation [27] to health risk assessment of drinking water disinfection by-products [28, 29] and the assessment of mutagenicity of contaminants in aquatic environments [30, 31]. These studies have shown that, despite a significant phylogenetic distance between fish and mammals, the transgenic rodent and fish mutation models share remarkable similarities in many aspects of somatic cell mutagenesis, including comparable frequencies of spontaneous mutations and chemical concentration-dependent and tissue-specific induced mutation responses reflecting known modes of mutagen action [32]. These studies contribute to growing evidence of common processes related to DNA damage and repair shared among distantly related species, ranging from fish to mammals and, possibly, to humans [32].

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FEATURES OF THE APPROACH USING  TRANSGENIC MEDAKA

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The general approach for using  transgenic medaka to determine the frequency of mutations that arise in progeny from mutagen-exposed male germ cells is similar to that typically employed in progeny assays based on rodents [14, 33] (Figure 1). Transgenic male fish (homozygous for the LIZ vector) are treated with a mutagen for a prescribed period and bred with multiple non-transgenic, untreated females (8-10 females) over consecutive days. The offspring (hemizygous for the LIZ vector) are collected and analyzed for mutations in the cII gene that arise only from germ cells transmitted by the mutagen-exposed male parent. Various attributes of medaka as an animal model and the cII gene as a mutation target combine to provide numerous benefits not available with traditional mutation assays. In the following section, features of medaka biology and the cII mutation assay are reviewed with an emphasis on how these features facilitate efficient and sensitive assessment of induced mutations in progeny of mutagen-exposed parents.

Figure 1. Illustration of a general approach using the  transgenic medaka and cII mutation assay. Offspring are collected following exposure of transgenic males to a mutagen and breeding with nontransgenic untreated females. DNA is isolated from individual offspring and analyzed for mutations using pools of siblings or individuals.

Male Germ Cells and Study Design Male germ cells exposed to mutagens at different stages of spermatogenesis have been shown to have different susceptibilities to mutation induction, and consequently, knowledge of stage specificity is clearly essential to the evaluation of genetic health hazards [34]. Despite differences in duration and morphology of different male germ cell types, many aspects of spermatogenesis in the human, rodent, and fish are quite similar (Figure 2). In a mature male, there is an unlimited supply of germ cells and differentiating cells at all stages. Since male germ cells are constantly replenished throughout a male‘s reproductive life, the mutagenic effect of exposure at a specific stage of gametogenesis can be evaluated either by sampling germ cells or by mating animals at an appropriate interval subsequent to, or during,

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.

the exposure period. By collecting offspring from a mutagen-exposed male at prescribed times, specific patterns of mutagenesis among different germ cells exposed to mutagens can be identified.

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Figure 2. Comparison of germ cell stages in medaka, mouse and humans. The mutagenic effects at each stage can be analyzed by breeding males at selected times after mutagen exposure.

The strategy for analyzing different susceptibilities of germ cells exposed at different stages of spermatogenesis in medaka entails collection of fertilized eggs 0-6 days after mutagen treatment of the male parent to obtain offspring derived from mutagenized postmeiotic germ cells (spermatozoa/late spermatids). The individuals collected more than ~30 days post-mutagen exposure of the male parent represent offspring derived from mutagenized pre-meiotic germ cells (spermatogonial stem cells, Figure 2) [21].

Alternative Approaches: Isolated Spermatozoa and Female Germ Cells Recently, direct analysis of mutations in isolated spermatozoa of ENU-treated  transgenic medaka was shown to be a practical alternative to the characterization of mutations in progeny [35]. Non-lethal collection of spermatozoa from an individual male provides a large and homogenous cell population and thereby provides an estimation of potential genetic health risk without the need to produce and screen large numbers of offspring. Consequently, this approach offers significant practical benefits, particularly for high-throughput screening of suspected mutagens. However, because in this study gametes exposed to ENU as spermatozoa/late spermatids did not exhibit significant mutation induction, it is apparent that mature spermatozoa must be sampled at the time corresponding to mutagen exposure of early spermatogonial stem cells. Further, as it is apparent that progeny analyses account for the mutagenic processes that take place post-fertilization in the embryo whereas analyses of isolated spermatozoa do not, assessment of mutations in isolated spermatozoa, even those derived from mutagen-exposed spermatogonial stem cells, will provide only an approximate

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estimate of actual genetic health risks revealed by progeny analyses. Further discussion of the importance of post-fertilization processes is provided in the following sections. The feasibility, practicality and sensitivity of examining mutations in offspring derived from mutagen-exposed post-meiotic female germ cells of  transgenic medaka has also been shown [36]. These preliminary studies indicate that this fish model offers significant promise for expanding investigations of germ cell mutagenesis to include analyses of female germ cells.

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Numbers of Offspring A central challenge in detecting mutant offspring is essentially a sampling problem – to detect a significant increase in the induced frequency of mutant offspring requires exceptionally large sample sizes. Consequently, the production, collection and maintenance of large numbers of offspring can be daunting tasks, particularly using mammalian models. The prolific reproductive capacity of medaka, the ability to breed individual males with multiple untreated females prior to and after mutagen treatment of males, combined with the ability to analyze mutations using individuals or pools of offspring provides significant practical benefits in screening mutant offspring. At least 100 offspring from an individual male parent can be readily collected and analyzed. Additional efficiencies in screening can be achieved by using DNA from 10-20 siblings combined in a single sample. A caveat of using DNA pooled from multiple siblings is that mutant offspring with marginally elevated cII mutant frequencies (e.g., mosaic mutant offspring) may not be readily detected in the pooled analyses, and therefore may underestimate the frequency of mutant offspring. Analysis of mutations using DNA from individual offspring provides the most precise assessment of the frequency of mutant offspring as well as specific information on mutation load and mutational spectra.

DNA Isolation from Individual Offspring To analyze chemically-induced in vivo mutations in somatic cells of transgenic mutation models, DNA is isolated from tissues of interest, such as liver or other target organs. Similarly, assessment of the frequency of mutant transgenes in offspring from mutagenexposed in transgenic rodent mutation models has relied on analyses of somatic mutations recovered from selected tissues [14]. An alternative approach based on isolating DNA from an entire individual offspring, typically juveniles and/or embryos, has been successful with the  transgenic medaka to improve efficiencies in analyses while providing an assessment of mutation load in the whole animal. Tissue-specific patterns of mutagenesis are also feasible using DNA isolated from selected tissues of the  transgenic medaka adults [37].

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Detection of Mutations in the cII Mutation Target Gene The bacteriophage LIZ vector carried by the  transgenic medaka contains two mutation target genes, lacI and cII, flanked at each end by cos sites that allow excision and packaging of the  phage to recover the vector from the animal‘s genomic DNA. The most extensive information on mutations in fish using various test compounds and conditions is based on using the cII as the mutation target gene [12, 13, 28, 37]. The cII mutation assay was originally introduced in rodents as a logistically simple and cost-effective alternative to the mutation assay based on the lacI target gene [38]. The cII mutation assay has been the primary focus of mutation studies in the  transgenic medaka because it similarly has been shown to increase the efficiency of mutation analyses while providing equivalent responses as the lacI locus. The cII mutation assay is a positive-selection assay based on exploiting the role of the cII protein in the commitment of bacteriophage  to the lysogenic cycle in Escherichia coli host cells [38]. Bacteriophages that carry a mutant cIl - are selected by using a strain of E. coli (hfl-) that extends the longevity of the cII product. To detect mutations, genomic DNA is mixed with in vitro packaging extracts that excise the intact  bacteriophage vector from the genomic DNA and simultaneously package the vector into viable bacteriophages. Individually packaged phages are then allowed to infect and lyse the E. coli host. Bacteriophages with wild-type cII produce lysogens such that they are indistinguishable in the lawn of E. coli incubated at 37C overnight. Bacteriophages that carry a mutation in the cII gene lyse the bacteria forming plaques on the lawn when incubated at 24C for 40 hours. Mutant frequencies (MF) are calculated simply by dividing the total number of cII mutant plaque forming units (PFU) counted on the selective mutant screening plates by the estimated total + and cII phages on the titer plates. (Example: MF = 18 mutant PFU on screening plates/650,000 total PFU = 2.8 X 10-5). Despite the small size of medaka, the recovery and analysis of large numbers of LIZ vectors from medaka tissues have proven to be highly efficient. Routinely, greater than 50,000 PFU/ug genomic DNA are recovered from medaka tissues [37]. It is apparent that the relatively high copy number of the integrated LIZ vector (~75 copies/haploid genome) and the compact genome of medaka (~1/3 the size of the mouse genome) [39] contribute to the high efficiency of vector recovery and reduced variability in mutant frequencies observed among samples.

Mutant Offspring: cII Mutant Frequencies and Mutational Spectra Two features of the cII mutation assay, the ability to quantify the frequencies of mutant cII genes carried by individual offspring (mutation load), and the ability to describe specific mutations at the molecular level provide significant benefits in characterizing mutant offspring. In analyses of somatic mutations using transgenic rodent and fish mutation assays, a twofold induction of target gene mutant frequencies is commonly accepted as a rule of thumb as an indication of significant mutation induction [13, 40]. Based on previous studies that showed the  transgenic medaka carry ~75 copies of the cII target gene/haploid genome

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[13], theoretically a mutant offspring that carried a single mutation in every cell (whole body mutant offspring) would be expected to exhibit an extremely elevated cII MF (cII MF= 1.3 X 10-2), ~1000-fold higher than the spontaneous cII MF of a non-mutant individual (1-3 X 10-5). In contrast, an individual offspring that carried a mutant cII gene in a fraction of somatic cells (mosaic mutant offspring) would be expected to exhibit a cII MF less than a theoretical maximum (e.g., cII MF in the range of high 10-5 to 10-3). Offspring that exhibit cII MFs greater than a minimum 3-fold (9 X 10-5) than the historical spontaneous cII MF in adult whole fish (3 X 10-5) [13] are designated as mutant offspring, or in the case of pooled samples, as offspring containing at least one mutant offspring/pool. To date, the frequency of spontaneous mutant offspring has not been determined empirically, as no  transgenic medaka with highly elevated spontaneous cII MFs has been detected. Pending a more precise determination, the frequency of spontaneous mutant offspring may be estimated to be in the range of less than 1 X 10-4. A significant benefit of using the cII gene as a recoverable mutation target is that it provides a simple means of analyzing mutations at the molecular level. The small size of the cII gene (~300 base pairs, bps) facilitates efficient characterization of mutation spectra and determination of frequencies of specific mutations by direct sequencing of the entire gene. In the  transgenic medaka, sequencing disclosed that the mutant offspring exhibited highly distinctive mutational spectra comprised of high frequencies of multiple non-independent mutations consistent with clonal expansion of mutations in early-stage embryos. Sequencing provided confirmatory information supporting the presumed mutant status of the mutant individual as either a whole body or a mosaic mutant. Individuals with extremely elevated cII MFs exhibit the characteristic mutational spectra comprised solely of multiple identical mutations consistent with their status as whole body mutants. Mutant offspring with cII mutant frequencies elevated in the intermediate range typically exhibit mutational spectra comprised of diversity of mutations, but with numerous multiple identical mutations being also indicative of clonally expanded mutations. Sequencing may also disclose a shift in mutational spectra characteristic of the action of a specific mutagen [13, 24, 29, 32].

PERSPECTIVE ON MUTAGENESIS FROM TRANSGENIC MUTATION MODELS Transgenic mutation assays based on rodents and fish models have provided unprecedented opportunities to investigate fundamental aspects of in vivo mutagenesis. By controlling one fundamental variable of interest, a gene that serves as a target for mutations, while allowing other factors such as cells, tissues, organs, species and so on to vary, Tthe transgenic models facilitate comparative studies of complex processes of mutagenesis that are not possible using other approaches. While comparative genomic studies have demonstrated significant similarities between fish and human genomes [18, 41-44], results from studies of somatic mutations in transgenic target genes in rodent and fish models [32] have strengthened the evidence for common processes of DNA damage and repair shared among these distantly related species.

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Various attributes of the  transgenic medaka such as the prolific reproductive capacity, the ability to breed mutagen-treated males with multiple untreated females over consecutive days both prior to and after mutagen exposure, and the ability to analyze mutations using individuals or pools of offspring afford significant benefits in using this fish as a practical and sensitive model for germ cell mutagenesis. Most importantly, as similarly shown in the transgenic mice [14], chemically-induced mutations in a transgene mutation target carried by  transgenic medaka male parents are transmitted to progeny and provide evidence that processes of mutagenesis in the fish closely mimic those in mammals (Figures 3 and 4) [15]. Despite differences in various aspects of investigations, including the target genes used (cII in fish vs. lacI or specific locus mutations in mice), the observed frequency of mutant offspring from ENU-treated spermatogonial stem cells of male fish (0.9%) (Figure 3) were remarkably similar to that reported in mice (1.4%) [14]. As similarly shown in transgenic mice, mutant offspring derived from ENU-treated male medaka parents exhibited highly elevated transgene mutant frequencies in the range of 10-2, consistent with the status of whole body mutant offspring [15]. Characterization of mutations from the presumptive whole body mutant offspring revealed that mutational spectra were comprised predominantly of single clonally expanded identical mutations consistent with that expected of whole body mutant offspring. Therefore, the presence of whole body mutant offspring derived from mutagen-exposed spermatogonial cells of both transgenic fish and mouse models appears consistent with current understanding of a direct mechanism of mutagenesis in which mutagen-exposed germ cells transmit mutations to every cell of mutant offspring.

Figure 3. Comparison of cII MFs in offspring derived from ENU-exposed pre-meiotic germ cells (spermatogonial stem cells) and control offspring. Individuals with cII MFs greater than 3-fold (9 X 105 ) over the historical spontaneous MF (- - - - ) were designated mutant offspring. (Adapted from Winn et al., 2008 [15]).

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Figure 4. Comparison of cII MFs in offspring derived from ENU-exposed post-meiotic germ cells (spermatozoa/late spermatids) and control offspring. Individuals with cII MFs greater than 3-fold (9 X 10-5) over the historical spontaneous MF (- - - -) were designated mutant offspring. (Adapted from Winn et al., 2008)

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Departure from Mouse Assay Results: Prevalence of Mosaic Mutant Offspring Despite significant concordance between transgenic fish and mouse mutation assays, one notable discrepancy, the lack of mosaic mutant offspring detected using the mouse mutation assay, merits attention. Whereas the frequencies of whole body mutant offspring derived from ENU-treated spermatogonial stem cells of mice and fish were comparable, the majority of mutant fish were mosaic mutant offspring (73%, Figure 5) [15]. Mosaic mutant offspring also comprised the majority of mutant offspring (80%) derived from postmeiotic germ cells of fish. Therefore, the fish studies revealed the unexpected finding that spermatogonial stem cells appear to be capable of generating mosaic mutant offspring similar to that of ENUtreated post-meiotic germ cells. (Analyses of mutant offspring derived from post-meiotic germ cells in the mouse study were not conducted precluding comparisons with results obtained from the transgenic fish mutation assay). Mosaicism is widely recognized as an important and common mechanism responsible for the origin of genetic disorders [45]. However, because the presence of mosaic mutant offspring significantly confounds mutant screening and confirmation depending upon the assay, mosaic mutants are commonly excluded from such analyses [46]. Based on reports of the detection of mosaic mutant offspring using other mouse mutation assays [47, 48], the apparent discrepancy in frequencies of mosaic mutant frequencies detected in mice and fish are most likely due to differences in the procedures used to screen mutant offspring rather than differences in germ cell responses to mutagen exposure in animal models. In particular, the procedures used in the transgenic mouse assay to screen mutant offspring relied on comparatively low recoveries of the LIZ vector from the tissues (< 52,000 PFUs/animal) which permitted the detection of only mutant offspring that exhibited extremely elevated lacI

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mutant frequencies (~ 4.15 X10-2). Consequently, the mouse mutation assay was biased in selecting whole body mutants, thereby precluding mosaic mutant offspring from the analyses. By comparison, the procedures used to screen mutant fish, including the relatively high efficiencies in recoveries of the  vector from fish tissues (typically > 100,000 PFU/sample), facilitated distinguishing non-mutant, mosaic and whole body mutant offspring based on the wide range of cII MFs these individuals exhibited (from the 10-2 to 10-5 range).

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Figure 5. Composition of whole body and mosaic mutant offspring derived from ENU-exposed postmeiotic or pre-meiotic male germ cells.

The differences in patterns of mutagenesis reported among germ cell stages have been largely attributed to the differences in the proficiency of germ cells for DNA repair, to the mutagenic action of specific mutagens, to spermatozoa as transmitters of genetic damage, and to the possible roles of post-fertilization processes in DNA repair or attempted repair of premutational lesions [49]. The mechanism of mutagenesis that gives rise to mosaic mutant offspring from mutagenexposed post-meiotic germ cells has been based on the understanding that these cells can retain lesions upon fertilization because they are DNA repair deficient and fixation of a mutation requires DNA synthesis or error-prone DNA repair. These lesions are then either repaired or fixed as mutations in early stage embryo [22]. By contrast, because pre-meiotic germ cells are DNA repair competent, the mechanism of mutagenesis entails fixation of premutational lesions in the gametes or at the one-cell stage of embryo to generate a whole body mutant individual. Therefore, as mosaic mutant offspring can be generated only from premutational lesions fixed at or after the second cell cleavage, evidence of mosaic mutant offspring generated from mutagen-exposed premeiotic male  transgenic medaka germ cells indicates that DNA damage introduced in spermatogonial stem cells can persist through various stages of spermatogenesis (> 60 days after male parent exposure), and ultimately, the damage can be fixed as a mutation in the developing embryo. Consequently, these observations support the need to revise our current understanding of the role germ cells play in mutagenesis, in

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particular related to the role of post-fertilization processes in DNA repair or attempted repair of premutational lesions carried by pre-meiotic germ cells.

FEATURES OF MOSAIC MUTANT OFFSPRING Mosaic mutant offspring can be distinguished from non-mutant individuals by several key characteristics (Table 1). The evidence from the  transgenic medaka that shows mosaic mutant offspring derived from both mutagen-treated post-meiotic and pre-meiotic male germ cells indicates that mosaicism is a prevalent feature of mutagenesis mediated by mutagenexposed male germ cells [15]. Mosaic mutant offspring are important not only because the mechanism that contributes to generating mosaic mutant offspring is not completely understood and its common exclusion from analyses underestimates the actual frequencies of mutant offspring, but also because mosaic mutant offspring may offer new insights into the processes by which mutations arise from mutagen-exposed germ cells. In the following section, distinctive features of mosaic mutant offspring are reviewed in the context of the interplay between germ cells, persistence of DNA damage, delayed mutations, and indirect processes of mutagenesis in early stage embryos. Table 1. Summary of key features of mosaic mutant offspring

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 

Commonly excluded from analyses of germline mutagenesis Mutation loads among offspring vary by up to three orders of magnitude (cII MF range = high 10-5 to 10-3)  Comprise the majority (> 70%) of mutant offspring  Derived from both mutagen-exposed post- and pre-meiotic germ cells  Distinctive mutations high frequencies of clonal mutaions evidence of persistent DNA damage and delayed fixation multiple mutations at the same sites suggestive of delayed mutations  A prevalent feature of mutagenesis mediated by mutagen-exposed germ cells

Mutation Load and Timing of Mutation Fixation Studies of induced mutations in somatic cells of both transgenic mutation fish and rodent models typically show that the mutagen-treated individuals exhibit classical dose responses in frequencies of mutant transgenes, i.e. mutant frequencies increase with increasing exposure levels of mutagenic agents [32, 50]. By sharp contrast, offspring derived from mutagenexposed  transgenic medaka male parents exhibited a wide range of magnitudes of induced cII mutant frequencies (cII MFs from the 10-2 to high 10-5 range) disproportionate to a single exposure level of mutagens [15]. The range of magnitude of the mutation load exhibited by mutant offspring can be most readily attributed to the timing of mutation fixation relative to cell cleavage in the developing embryo. The magnitudes of the elevated cII MFs exhibited by individual offspring served as the basis to characterize mutants as either presumptive whole body or mosaic mutant

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offspring. Based on the current understanding that whole body mutant offspring are generated from DNA damage fixed as a mutation either in gametes or in zygotes at the one-cell stage, and mosaic mutant offspring are generated from a mutation fixed after the second cell cleavage, it follows that the magnitude of the induced cII MFs in mutant offspring may generally reflect the timing of mutation fixation relative to cell cleavages (Figure 6) [15]. In the  transgenic medaka, it may be assumed that DNA damage that was fixed as a mutation at early stages of spermatogenesis would be transmitted from the parental germ cell to contribute to whole body mutant offspring exhibiting an extremely elevated cII MF (approximately 1.3 X 10-2) equivalent to 1 mutant cII/75 cII copies/cell. For mosaic mutants there may be less certainty regarding the precise linkage between the mutation load and timing of mutation fixation relative to specific cell cleavage stages. However, it is understood that mutant offspring generated from DNA damage fixed in subsequent cell cleavages will carry the mutation in only portion of cells. Therefore, these mosaic mutant offspring would be expected to exhibit cII mutant frequencies at markedly lower magnitudes, presumably, at progressively lower magnitudes, if mutations were fixed in later cell divisions (e.g., less than or equivalent to approximately 1 mutant cell/4 cells, cII MF= 6.7 X 10-3). The observation of prevalence of mosaic mutant offspring derived from mutagen-treated premeiotic germ cells of the  transgenic medaka indicated that DNA damage introduced in the spermatogonial stem cells persisted throughout spermatogenesis and was ultimately fixed as mutations after at least the second cell cleavage in the embryo (Figure 6) [15]. Persistence of DNA damage and delayed fixation of mutation provide important evidence of postfertilization processes in the early stage embryo because delayed mutations are thought to arise solely in the zygote shortly after fertilization rather than in sperm at these early time points [49]. Consequently, delayed fixation of mutation appears to be a common feature of mutagenesis in early stage embryos derived from mutagen exposed germ cells.

Figure 6. Illustration depicting two types of mutant offspring derived from mutagen-exposed spermatozoa or spermatogonial stem cells. DNA damage fixed as a mutation in the gamete or prior to fertilization generates mutant offspring with mutations in every cell (whole body mutant). DNA damage that persists after fertilization and is fixed after the second cell cleavage generates mutant offspring with mutations in a portion of cells (mosaic mutant).

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Mutational Spectra and Delayed Multiple Mutations The characterization of the mutations carried by the individual mutant offspring of the ENU-exposed  transgenic medaka males proved invaluable in confirming the mutant status of the individual, but more importantly, it offers clues as to the processes of mutagenesis that give rise to the mutations in these individual offspring. Mutational spectra were typically reflective of the magnitudes of cII MFs exhibited by the individual and generally consistent with the mutant status of the individual offspring as either mosaic or whole-body mutant offspring (Table 2) [15]. Offspring designated as whole body mutants based on their extremely elevated cII MFs (~ 10-2 range) typically exhibited mutational spectra comprised solely of one mutation. The mutational spectra of these whole-body mutants were therefore consistent with a mutation fixed in either the gamete or prior to the first cleavage in the zygote and clonally expanded in all cells in subsequent cell divisions. It is interesting to note that none of the transgenic medaka whole-body mutant siblings shared identical mutations indicative of being derived from the same mutant germ cell of the parent. The mutation spectra carried among mosaic mutant offspring were highly heterogeneous and represented marked departures from spontaneous mutational spectra (Table 2) [15]. In particular, mosaic mutant offspring exhibited characteristic mutational spectra comprised of relatively lower frequencies of independent mutations and associated with higher frequencies of clonal or identical mutations. In general, with increasing mutational loads (e.g.,10-4 to 10-3), the mutational spectra showed a progressive decrease in the diversity of independent mutations, and in extreme cases, they were comprised of a single predominant mutation at one site. By comparison, the spectra of spontaneous cII gene mutations in transgenic medaka and rodents are comprised typically of a majority of independent base substitution mutations (~50-90%) and low frequencies of clonal mutations, with the exception of the prevalence of +1/-1 frameshift mutations at nucleotides 179-184, known mutation hotspots [13, 51]. Sequencing analyses of mutations recovered from several mosaic mutant offspring also revealed highly distinctive mutations reflective of a delayed mechanism of mutagenesis (Table 2) [15]. The mutant offspring carried unusual mutations comprised of two predominant mutations, each at high frequencies but at identical sites. These multiple mutations are exceptional because they are rarely observed in spontaneous or chemically-induced mutational spectra, except at the cII mutational hot spot [13]. The prevalence of these two different mutations at the same site supports the conclusion that these mutations did not arise as independent mutational events. The probability of two mutations being introduced independently at the same position is extremely low, equivalent to the product of the frequency of a mutation at one site X the frequency of a second mutation at the same site. Further, the two mutations were observed at unequal frequencies, suggesting that DNA damage was not fixed as a mutation simultaneously with the original mutation. Similar evidence of delayed mutations, in which the fixation of a second mutation was delayed in subsequent cell divisions following fertilization, has been reported in other systems and has been defined as genomic instability [49].

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Table 2. Comparison of cII mutational spectra from representative non-mutant, mosaic and whole body mutant offspring Non-mutant offspring (spontaneous) Offspring cII MF a. 2.2 X 10-5

b.

4.3 X 10-5

Whole body mutant offspring Offspring cII MF a. 1200 X 10-5

b.

1200 X 10-5

c.

1000 X 10-5

Mosaic mutant offspring Offspring cII MF a. 150 X 10-5

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b.

*c.

130 X 10-5

60 X 10-5

Mutation independent clonal (+1/-1 G @ base 179) independent mutations clonal (+1 G @ base 179)

% of total mutations 50% 50%

Mutation independent clonal (A to T @ base 61) independent clonal (C to T @ base 113) independent mutations clonal (T to C @ base 221)

% of total mutations

Mutation independent clonal (C to T @ base 212) independent clonal (G to A @ base 207) clonal (+1 G @ base 179) independent clonal (G to T @ base 175) clonal (G to T @ base 175)

% of total mutations 10% 90%

86% 14%

100% 0% 100% 0% 100%

15% 11% 74% 6% 76% 18%

*An example of dissimilar multiple mutations at the identical site characteristic of delayed mutagenesis. Clonal mutations at base 179 are characteristic of a spontaneous cII mutational hot spot. (From Winn et al., 2008 [15]).

Offspring of Mutagen-Exposed Parents Exhibit Mutations Consistent with Genomic Instability The term genomic instability has been ascribed to a variety of genetic changes including chromosomal rearrangements, micronuclei, transformation, gene amplifications, gene mutations, and lethal mutations or delayed reproductive cell death derived and clonally expanded from a mutagen-exposed cell [52]. The mutagenesis associated with induced genomic instability has two important features: untargeted mutations and delayed mutations.

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An untargeted (also referred to as non-targeted or indirect) mutation is induced in a region of DNA that has no direct insult by the mutagen when the fidelity of the genomic maintenance mechanism is reduced. A delayed mutation is induced in a delayed manner in descendents of irradiated or mutagen-exposed cells. Delayed and untargeted mutations are two features not shared by the classic mutagenic pathway that entails the introduction of a mutation in a targeted manner within a certain amount of time following the introduction of damage to DNA. The delayed mutations observed in the offspring of mutagen-exposed male germ cells of  transgenic medaka supports the growing evidence that genomic instability is a prevalent feature of mutagenesis mediated by germ cells [15]. Genomic instability has been observed in cells of irradiated mice, where such mutations were thought to arise in the zygote shortly after fertilization rather than in spermatozoa at these early time points [49]. High levels of mutational mosaicism have been observed in the germline and somatic tissues of F1 generation mice, supporting the model for destabilization of the genome at the early stages of development [53, 54]. Shimada and Shima [23] provided evidence that the second hallmark of genomic instability, untargeted mutagenesis, is also observed in offspring by showing that irradiation of spermatozoa/late spermatids induced mutations in non-irradiated maternally derived alleles in F1 medaka somatic cells. A most recently completed study has confirmed that mutagen-exposed male germ cells of medaka contribute to the induction of mutations in offspring independently of direct exposure to mutagens [55]. ENU-treated germ cells of non-transgenic medaka males mediated the induction of untargeted mutations in cII genes transmitted only from the  transgenic female parent not exposed to the mutagen. These exclusively mosaic mutant offspring derived from both mutagen-exposed post-meiotic and pre-meiotic male germ cells exhibited the significantly elevated cII mutant frequencies (2-170-fold above the spontaneous cII MF) and highly distinctive mutational spectra consistent with the delayed induction of mutations by the damaged spermatozoa in the maternal genome. These results provide unequivocal evidence that germ cells exposed at different stages of spermatogenesis carry persistent genomic damage that contributes to the induction of mutations encompassing the maternal genome that is not directly damaged by the mutagen. In addition, these results demonstrate that the fixation of a mutation in the spermatozoa is not required, nor is the interaction of the damaged allele with the corresponding allele necessary to induce mutations in the embryo.

INSIGHTS INTO THE MECHANISMS OF MUTAGENESIS The emerging picture appears to indicate that damage transmitted by mutagen-exposed germ cells is fixed as a mutation in offspring by processes that are more complex than previously understood. In particular, the analyses of the mosaic mutant offspring of medaka, their prevalence and mutations they carry challenge our current understanding of mutagenesis related to the roles of germ cells and post-fertilization processes in mutagenesis. Central to building a framework for improved understanding of a possible mechanism contributing to mutations in offspring is the distinction to be made between mutagenic processes that occur during spermatogenesis and those that occur after fertilization. Specifically, it is important to examine the roles that spermatozoa play as direct transmitters of mutations and/or as indirect

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mediators of mutagenic processes in early stage embryo. Consequently, in addition to direct mechanisms that give rise to mutations in offspring, it is apparent that indirect processes mediated by mutagen-exposed germ cells should be considered.

Models for Mutagenesis in Early Stage Embryos

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The prevalence and the distinctive mutational spectra of mosaic mutant offspring provide evidence supporting error-prone DNA repair processes as those playing a fundamental role in destabilization of the genome in early stage embryos (Figure 7) [49]. The early development of many animals is characterized by rapid and synchronous cleavages that subdivide the zygote into a large population of blastomeres in a relatively short time. These rapidly dividing cells have numerous constraints on DNA damage and/or attempted repair processes that differentiate them from all other cells. Among the constraints, the majority of cellular events that take place during the early cell cycles of development occur in the absence of transcription and are reliant solely upon mRNAs and proteins stored in the egg before fertilization [56, 57]. In addition, cells of early stage embryos lack the stringent cell cycle checkpoints and apoptotic response. Only upon reaching the mid-blastula transition stage, does the embryo begin to have significant gene expression and utilize gene products from its own genes.

Figure 7. Hypothesized period in which damage transmitted by spermatozoa is subjected to an errorprone DNA repair mechanism in cells of early stage embryos. The majority of mutations are likely fixed prior mid-blastula transition and activation of embryonic genes.

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Figure 8. Illustration of a model for the mechanism of indirect mutagenesis mediated by damaged spermatozoa. The damaged paternal genome elicits a mechanism of error-prone DNA repair encompasses both paternal and maternal genomes of the early stage embryo.

As a consequence, the cells of a developing embryo may have a special requirement for damage bypass and tolerance of endogenous damage, such as oxidative lesions [58]. Based on constraints of early stage embryos, a model for the mechanism of mutagenesis mediated by damaged spermatozoa may be proposed that incorporates an error-prone DNA repair mechanism that accounts for both delayed and untargeted mutations in early stage embryos. The model is characterized by a distinctive phase of hypermutagenesis whereby damage introduced directly in the paternal genome by mutagen exposure and carried by the spermatozoa invokes a global stress response that also encompasses regions of the paternal and maternal genome not directly exposed to the mutagen. Following fertilization of the cell by damaged spermatozoa, persistent premutational lesions in the paternal genome are either repaired and fixed as a mutation or stall the process of DNA replication (Figure 8). As a consequence of the failure of high fidelity DNA repair of lesions, a global error-prone DNA repair process is activated that encompasses attempted repair of premutational lesions in the maternal genome. Mutagenesis of undamaged DNA templates in bacterial cells has been shown to be enhanced by the induction of an error prone DNA repair (SOS) pathway [59, 60]. Pretreatment of mammalian cells with UV induced a cellular SOS-like response that reduced replication fidelity in the undamaged templates to introduce possible untargeted mutations. The existence of an untargeted mutagenesis pathway was suggested from observations of significant induction of mutation frequencies in monkey cells treated with mitomycin C before transfection with the UV-irradiated shuttle vector [61]. It therefore follows that transient hypermutability may be a key feature of mutagenesis in developing embryos, as an

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unavoidable inefficiency in such a process is directed more to increasing survival rather than reducing mutagenesis. Epigenetic processes have been hypothesized as alternative or additional mechanisms involved in indirect mutagenesis mediated by damaged germ cells [62-64]. Significant increases in mutations have been observed at non-coding tandem repetitive DNA sequences (e.g., minisatellites or expanded simple tandem repeats, ESTRs) in animals exposed to low doses of radiation [48, 65-75], chemical mutagens [76], ambient levels of environmental mutagens [77] and mainstream tobacco smoke [78]. ESTR mutations have been assumed to arise through an indirect mechanism resulting in destabilization at the repeat locus. Alterations in DNA methylation or in DNA methylation transferase expression have been proposed as associated with this indirect mechanism of mutation [63]. Chemicals that modify chromatin conformation through changes in methylation may compromise the ability of DNA repair enzymes to access and repair secondary structures that may form across ESTR loci resulting in mutation.

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PERSPECTIVES FOR GENETIC HEALTH RISK ASSESSMENT Genomic instability implies persistence of instability, which suggests that mutant offspring will exhibit a permanent loss of genetic integrity or will be at greater risk to accumulate additional mutations over time. Whole body mutant offspring are expected to transmit mutations to their offspring, whereas the extensive mosaicism exhibited among the mutant offspring of the  transgenic medaka suggests that, unless the mutation is present in germinal tissues, mosaic mutant individuals will not transmit mutations to subsequent generations [15]. Consequently, such highly mosaic mutant individuals do not contribute to germline mutations or contribute to transgenerational genomic instability in the strict sense. Currently, pending analyses of mutations in the F2 generation offspring of the  transgenic medaka, it remains unknown to what extent a mutant offspring may be mosaic and still be capable of transmitting a mutation [15]. Regardless of whether a mutation is transmitted to subsequent generations or not, considering the prevalence of mosaic mutants and distinctive mutations they carry, it is apparent that the potential for adverse health effects of increased mutation load on an individual deserves further investigation [4]. Evidence emerging from a variety of systems implicates exposure of male parents to a wide range of DNA-damaging agents with an increased rate of delayed and untargeted mutations rates in somatic cells of progeny. As a consequence, environmental exposure of parents to such agents may pose greater potential genetic risks to progeny than previously understood [15, 54, 62, 64]. Whereas induced genetic disease in human offspring from parental exposures has not been unequivocally demonstrated, there is a growing consensus that the disparity between human and animal studies is due to technological limitations in detection of mutations rather than biological differences between animal and human susceptibility, and that transmissible genetic damage from exposure to these agents is possible [79]. Evidence is accumulating related to increased incidence of such conditions as cancer, schizophrenia or dominant mutations in children of human males with DNA damage in their spermatozoa because of age, lifestyle or occupation [80]. These male-mediated maladies have been attributed to aberrant repair of DNA damage in the early embryo, rather than the direct

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transfer of mutations from the male germ line to the offspring [81, 82]. The susceptibility of germ cells to mutagen exposure highlights the need to better characterize the prevalence and mechanism by which germ cells mediate genetic damage. The unexpected complexity of mutations that arise in the developing embryo will likely contribute greater complexity to such analyses. Delayed and untargeted mutations provide evidence of an indirect mechanism of mutagenesis, and therefore it follows that we may predict that specific mutations induced in mutant offspring may not be tightly linked to the mode of action of a specific mutagen, but rather may be a consequence of failed DNA repair. Most recently, mutagenic and transgenerational effects from parental exposure to ENU and ionizing radiation were shown to be highly similar, despite differences in the mode of action, leading to the conclusion that such effects can be attributed to a stress response triggered by generalized DNA damage rather than a specific sub-set of DNA lesion, such as double-strand breaks [62]. A consequence of this indirect mechanism of mutagenesis is that a mutagenic response may be disproportionate to a mutagen dose, and therefore genetic risk models will need to be revised.

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CONCLUSION Various attributes of medaka as an animal model and the cII transgene as a mutation target combine to provide significant practical and scientific benefits for conducting investigations of mutations transmitted by mutagen-exposed germ cells. The previous studies using the  transgenic medaka to detect mutations induced in somatic cells by numerous agents established the foundation for using the  transgenic medaka as a model for germline mutagenesis. The most recent demonstrations that chemically-induced mutations in the transgene carried by  transgenic medaka male parents are transmitted to progeny as similarly shown in the transgenic mouse [15] strengthen the growing evidence of common processes of DNA damage, repair and attempted repair shared among fish and mammals. The ability to characterize both frequencies and specific types of cII mutations carried by individual mutant offspring has proven invaluable in distinguishing non-mutant, whole body and mosaic mutant offspring. In particular, the prevalence of mosaic mutant offspring and distinctive spectra of mutations they carry revealed that the focus on the role of germ cells as direct transmitters of mutations should be expanded to include the role of germ cells as mediators of indirect mutagenesis in cells of early stage embryos. Currently, the characterization of these postfertilization processes remains incomplete. Growing evidence suggests that error-prone DNA repair processes and, likely, epigenetic processes acting independently or together contribute to a phase of hypermutagenesis manifested as genomic instability in offspring of mutagenexposed germ cells. Ultimately, a model for the mechanism(s) of mutagenesis will need to incorporate the interplay between persistent DNA damage, delayed and untargeted mutations, and constraints of rapidly dividing cells of early stage embryos in responding to damage contributed by germ cells.

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ACKNOWLEDGMENTS Support for the development of the  transgenic medaka was provided by the National Center for Research Resources, U.S. National Institutes of Health (R24RR11733).

REFERENCES

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[1]

Green S, Auletta A, Fabricant R, Kapp R, Manandhar M, Sheu CJ, Springer J, Whitfield B: Current status of bioassays in genetic toxicology--the dominant lethal assay. A report of the U.S. E.P.A. Gene-Tox Program. Mutat. Res. 1985, 154:49-67. [2] Russell WL, Kelly EM, Hunsicker PR, Bangham JW, Maddux SC, Phipps EL: Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc. Natl. Acad. Sci. USA 1979, 76:5818-5819. [3] Generoso WM, Bishop JB, Gosslee DG, Newell GW, Sheu CJ, Halle Ev: Heritable translocation test in mice. Mutat. Res. 1980, 76:191-215. [4] Singer TM, Lambert IB, Williams A, Douglas GR, Yauk CL: Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays. Mutation Research 2006, 598:164-193. [5] Boerrigter M, Dolle M, Martus H, Gossen JA, Vijg J: Plasmid based transgenic mouse model for studying in vivo mutations. Nature 1995, 377:657-659. [6] Burkhart JG, Winn RN, Van Beneden RJ, Malling HV: Spontaneous and induced mutagenesis in transgenic animals containing FX174. Environ. Mol. Mutant. 1993, 21 (Suppl. 12):9. [7] Gossen JA, De Leeuw WJF, Tan CHT, Zwarthoff EC, Berends F, Lohman PHM, Knook DL, Vijg J: Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc. Natl. Acad. Sci. USA 1989, 86:7971-7975. [8] Kohler SW, Provost GS, Kretz PL, Fieck A, Bullock WO, Sorge JA, Putman DL, Short JM: Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP®/lacl shuttle vector. Environ. Mol. Mutagen. 1991, 18:316-321. [9] Manjanatha MG, Chen JB, Shaddock JGJ, Harris AJ, Shelton SD, Casciano DA: Molecular analysis of lacl mutations in Rat2TM cells exposed to 7, 12dimethylbenz[a]anthracene: evidence for DNA sequence and DNA strand biases for mutation. Mutat. Res. 1996, 372:53-64. [10] Winn RN, Van Beneden RJ, Burkhart JG: Transfer, methylation and spontaneous mutation frequency of fX174am3cs70 sequences in Medaka (Oryzias latipes) and Mummichog (Fundulus heteroclitus): implications for gene transfer and environmental mutagenesis in aquatic species. Mar. Environ. Res. 1995, 40:247-265. [11] Amanuma K, Takeda H, Amanuma H, Aoki Y: Transgenic zebrafish for detecting mutations caused by compounds in aquatic environments. Nat. Biotechnol. 2000, 18:6265. [12] Winn RN, Norris M, Muller S, Torres C, Brayer K: Bacteriophage λ and plasmid pUR288 transgenic fish models for detecting in vivo mutations. Maine Biotechnology 2001, 3:S185-S195.

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Richard N. Winn

[13] Winn RN, Norris MB, Brayer KJ, Torres C, Muller SL: Detection of mutations in transgenic fish carrying a bacteriophage λ cll transgene target. Proceedings of the National Academy of Sciences 2000, 97:12655-12660. [14] Barnett LB, Tyl RW, Shane BS, Shelby MD, Lewis SE: Transmission of Mutations in the lacl Transgene to the Offspring of ENU-Treated Big Blue® Mice. Environmental and Molecular Mutagenesis 2002:251-257. [15] Winn RN, Majeske AJ, Jagoe CH, Glenn TC, Smith MH, Norris MB: Transgenic medaka λ as a new model for germ cell mutagenesis. Environ. Mol. Mutagen. 2008, 49:173-184. [16] Bunton TE: Use of non-mammalian species in bioassays for carcinogenicity. In Data on Genetic Effects in Carcinogenic Hazard Evaluation. Volume 146. Edited by McGregor DB, Rice JM, Venitt S. Lyon: IARC Scientific Publications; 1999: 151-184 [17] Furutani-Seiki M, Wittbrodt J: Medaka and zebrafish, an evolutionary twin study. Mechanisms of Development 2004, 121:629-637. [18] Kasahara M, Naruse K, Sasaki S, Nakatani Y, Qu W, Ahsan B, Yamada T, Nagayasu Y, Doi K, Kasai Y, et al: The medaka draft genome and insights into vertebrate genome evolution. Nature 2007, 447:714-719. [19] Taniguchi Y, Takeda S, Furutani-Seiki M, Kamei Y, Todo T, Sasado T, Deguchi T, Kondoh H, Mudde J, Yamazoe M, et al: Generation of medaka gene knockout models by target-selected mutagenesis. Genome Biology 2006, 7:R116:111-114. [20] Hinton DE, Hardman RC, Kullman SW, Law JM, Schmale MC, Walter RB, N. WR, Yoder JA: Aquatic Animal Models of Human Disease: Selected Papers and Recommendations from the 4th Conference. Comparative Biochemistry and Physiology Part C 2009:Available Online. [21] Shima A, Shimada A: Development of a possible nonmammalian test system for radiation-induced germ-cell mutagenesis using a fish, the Japanese medaka, (Oryzias latipes). Proceedings of the National Academy of Sciences of the USA 1991, 88:25452549. [22] Shima A, Shimada A: The medaka as a models for studying germ-cell mutagenesis and genomic instability. Marine Biotechnology 2001, 3:S162-S167. [23] Shimada A, Shima A: Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish. Mutation Research 2004, 552:33-42. [24] Winn RN, Kling H, Norris MB: Antimutagenicity of green tea polyphenols in the liver of transgenic medaka. Environmental and Molecular Mutagenesis 2005, 46:88-95. [25] Hobbie KR, DeAngelo AB, King LC, Winn RN, Law JM: Toward a molecular equivalent dose: Use of the medaka model in comparative risk assessment. comparative Biochemistry and Physiology, Part C 2009, 149:141-151. [26] Broussard GW, Norris MB, Schwind AR, Fournie JW, Winn RN, Kent ML, Ennis DG: Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes). Comparative Biochemistry and Physiology, Part C 2009, 149 152–160. [27] Winn RN, Norris MB, Turner J, Kimlin M: Assessment of lambda transgenic fish as a new model of UV mutagenesis. Transactions on Ecology and the Environment 2005, 85:205-213. [28] Geter DR, Winn RN, Fournie JW, Norris MB, DeAngelo AB, Hawkins WE: MX [3chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone], a drinking water carcinogen,

Genome Instability and Transgenerational Effects, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Germ Cell-Mediated Mutagenesis

[29]

[30]

[31]

[32]

[33] [34] [35] [36]

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[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

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does not induce mutations in the liver of cll transgenic medaka. Journal of Toxicology and Environmental Health 2004, 67:373-383. Winn RN, Norris MB, Lothenbach D, Flynn K, Hammermeister D, Whiteman F, Sheedy B, Johnson R: Sub-chronic exposure to 1,1-dichloropropene induces framshift mutations in lambda transgenic medaka. Mutation Research 2006, 595:52-59. McElroy A, Bogler A, Weisbaum D, Norris M, Mendelman L, Setlow R, Winn R: Uptake, metabolism, mutant frequencies and mutational spectra in lambda transgenic medaka embryos exposed to benzo(a)pyrene dosed sediments. Marine Environmental Research 2006, 62:S273-S277. Cachot J, Law M, Pottier D, Peluhet L, Norris M, Budzinski H, Winn R: Characterization of toxic effects of sediment-associated organic pollutants using λ transgenic medaka. Environmental Science and Technology 2007, 41. Winn RN: Mutagenesis: Insights from lambda-transgenic medaka. In The Physiology of Fishes. 3rd edition. Edited by Evans DH, Claiborne JB. Boca Raton: Taylor and Francis; 2006: 501-522 Russell LB: Effects of male germ-cell stage on the frequency, nature, and spectrum of induced specific-locus mutations in the mouse. Genetica 2004, 122:25-36. Albanese R: Mammalian male germ cell cytogenetics. Mutagenesis 1987, 2:79-85. Norris MB: Isolated spermatozoa as indicators of mutations transmitted to progeny. University of Georgia, Warnell School of Foresty and Natural Resources 2008. Gresham C, Norris M, Winn R: Mutagen-exposed female germ cells mediate delayed mutagenesis in early stage embryos. Environmental and Molecular Mutagenesis 2008, 49:P20 Winn RN, Norris MB: Analysis of mutations in λ transgenic medaka using the cll mutation assay. In Techniques in Aquatic Toxicology 2nd edition. 2nd ed. edition. Edited by Ostrander GK. New York: CRC Press; 2005: 705-734 Jakubczak JL, Merlina G, French JE, Muller WJ, Paul B, Adhya S, Garges S: Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage l transgene target. Proc. Natl. Acad. Sci. USA 1996, 93:9073-9078. Lamatsch DK, Steinlein C, Schmid M, Schartl M: Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa. Cytometry 2000, 39:91-95. Heddle JA, Dean S, Nohmi T, Boerrigter M, Casciano DA, Douglas GR, Glickman BW, Gorelick NJ, Mirsalis JC, Martus H, et al: In vivo transgenic mutation assays. Environ Mol Mutagen 2000, 35:253-259. Walter R, B., Rolig RL, Kozak KA, McEntire BB, Morizot DC, Nairn RS: Cloning and gene map assignment of the Xiphophorous DNA ligase 1 gene. Mol. Biol. Evol. 1993, 10:1227-1238. Postlethwait JH, Amores A, Force A, Yan YL: The zebrafish genome. Methods Cell Biol 1999, 60:149-163. Postlethwait JH, Yan Y-L, Gates MA, Horne S, Amores A, Brownlie A, Donovan A, Egan ES, Force A, Gong Z, et al: Vertebrate genome evolution and the zebrafish gene map. Nature Genetics 1998, 18:345-349. David WM, Mitchell DL, Walter RB: DNA repair in hybrid fish of the genus Xiphophorus. Comp Biochem Physiol, C Toxicol. Pharmacol. 2004, 138:301-309.

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[45] Hall JG: Review and hypotheses: somatic mosaicism: observations related to clinical genetics. Am. J. Hum. Genet. 1988, 43:355–363. [46] Favor J, Neuhauser-Klaus A, Ehling UH: The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics in F1 mice derived from postspermatogonial treatment with ethylnitrosourea. Mutation Research 1990, 229:105-114. [47] Russell LB, Selby PB, Halle Ev, Sheridan W, Valcovic L: The mouse specific-locus test with agents other than ratiations: interpretation of data and recommendations for future work. Mutat. Res. 1981, 86:329-354. [48] Niwa O, Fan YJ, Numoto M, Kamiya K, Kominami R: Induction of a germline mutation at a hypervariable mouse minisatellite locus by 252Cf radiation. J. Radiat. Res. 1996, 37:217-224. [49] Niwa O: Induced genomic instability in irradiated germ cells and in the offspring; reconciling discrepancies among the human and animal studies. Oncogene 2003, 22:7078-7086. [50] Heddle JA, Shaver-Walker P, Tao KS, Zhang XB: Treatment protocols for transgenic mutation assay in vivo. Mutagenesis 1995, 10:467-470. [51] Watson DE, Cunningham ML, Tindall KR: Spontaneous and ENU-induced mutation spectra at the cll locus in Big Blue Rat2 embryonic firoblasts. Mutagenesis 1998, 13:487-497. [52] Morgan WF: Non-targeted and delayed effects of exposure to ionizing radiation. I. Radiation-induced genomic instability and bystander effects in vitro. Radiat. Res. 2003:567-580. [53] Dubrova YE, Plumb M, Brown DW, Boulton E, Goodhead D, Jefferys AJ: Induction of minisatellite mutation in the mouse germline by low-dose exposure to y-radiation and fission neutrons. Mutat. Res. 2000, 43:17-24. [54] Barber R, Plumb MA, Boulton E, Roux I, Dubrova YE: Elevated mutation rates in the germ line of first- and second- generation offspring of irradiated male mice. Proc. Natl. Acad. Sci. USA 2002, 99:6877-6882. [55] Winn R, Norris M: Germ cells mediate untargeted mutagenesis in the maternal genome of progeny. Environmental and Molecular Mutagenesis 2008, 49:S26. [56] Kane DA, Kimmel CB: The zebrafish midblastula transition. Development 1993, 119:447-456. [57] Aizawa K, Shimada A, Naruse K, Mitani H, Shima A: The medaka midblastula translition as revealed by the expression of the paternal genome Gene Expression Patterns 2003, 3:43-47. [58] Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T: DNA Repair and Mutagenesis. 2 edn. Washington, D.C.: ASM Press; 2006. [59] Little JW, Mount DW: The SOS regulatory system of Escherichia coli. . Cell 1982, 29:11-22. [60] Walker GC: Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiology Reviews 1984, 48:60-93. [61] Roilides E, Munson PJ, Levine AS, Dixon K: Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin c-treated monkey cells. Mol. Cell Biol. 1988, 8:3943-3946.

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[62] Dubrova YE, Hickenbotham P, Glen C, Monger K, Wong H, Barber R: Paternal exposure to ethynitrosoursea results in transgenerational genomic instability in mice Environ. Mol. Mutagen 2008, 49:308-311. [63] Yauk CL, Polyzos A, Rowan-Carroll A, Kortubash I, Williams A, Kovalchuk O: Tandem repeat mutation, global DNA methylation, and regulation of DNA methyltransferases in cultured mouse embryonic fibroblast cells chronically exposed to chemicals with different modes of action. Environ. Mol. Mutagen 2008, 49:26-35. [64] Yauk C, Polyzos A, Rowan-Carroll A, Somers CM, Godschalk RW, Van Schooten FJ, Berndt ML, Pogribny IP, Koturbash I, Williams A, et al: Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location. Proc. Natl. Acad. Sci. 2008, 105:605-610. [65] Dubrova YE: Radiation-induced mutation at tandem repeat DNA loci in the mouse germline: Spectra and doubling doses. Radiat. Res. 2005, 163:200-207. [66] Dubrova YE, Bersimbaev RI, Djansugurova LB, Tankimanova MK, Manyrbaeva Z, Mustonen R, Lindholm C, Hulten M, Salomaa S: Nuclear weapons tests human germline mutation rates Science 2002, 295:1037. [67] Dubrova YE, Grant G, Chumak AA, Stezhka VA, Karakasian AN: Elevated minisatellite mutation rate in the post-chernobyl families from Ukraine. Am. J. Hum. Genet. 2002, 71:801-809. [68] Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Neumann R, Neil DL, Jefferys AJ: Human minisatellite mutation rate after the Chernobyl accident. Nature 1996, 380:683-686. [69] Dubrova YE, Nesterov VN, Krouchinsky NG, Ostapenko VA, Vergnaud G, Giraudeau F, Buard J, Jefferys AJ: Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident Mutat. Res. 1997, 381:267-278. [70] Dubrova YE, Ploshchanskaya OG, Kozionova OS, Akleyev AV: Minisatellite germline mutation rate in the Techa River population. Mutat. Res. 2006, 602:74-82. [71] Dubrova YE, Plumb M, Brown J, Fennelly J, Bois P, Goodhead D, Jefferys AJ: Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation. Proc. Natl. Acad. Sci. 1998, 95:6251-6255. [72] Dubrova YE, Plumb M, Brown J, Jefferys AJ: Radiation-induced germline instability at minisatellite loci. Int. J. Radiat. Biol. 1998, 74:689-696. [73] Dubrova YE, Plumb M, Gutierrez B, Boulton E, Jeffreys AJ: Genome stability-transgenerational mutation by radiation. Nature 2000, 405:37. [74] Yatagai F, Kurobe T, Nohmi T, Masumura K-i, Tsukada T, Yamaguchi H, KasaiEguchi K, Fukunishi N: Heavy-ion-induced mutations in the gpt Delta transgene mouse: Effect of p53 gene knockout. Environmental and Molecular Mutagenesis 2002:216-225. [75] Somers CM, Sharma R, Quinn JS, Boreham DR: Gamma radiation-induced heritable mutation at repetitive DNA loci outbred mice Mutat. Res. 2004, 568:69-78. [76] Vilarino-Guell C, Smith AG, Dubrova YE: Germline mutation induction at mouse repeat DNA loci by chemical mutagens. Mutat. Res. 2003, 526:63-73. [77] Somers CM, Yauk CL, White PA, Parfett CL, Quinn JS: Air pollution induces heritable DNA mutations. Proc. Natl. Acad. Sci. USA 2002, 99:15904-15907.

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[78] Yauk CL, Berndt ML, Williams A, Rowan-Carroll A, Douglas GR, Stampfli MR: Mainstream tobacco smoke causes paternal germ-line DNA mutation. Cancer Res. 2007, 67:5103-5106. [79] Wyrobek AJ, Mulvihill JJ, Wassom JS, Malling HV, Shelby MD, Lewis SE, Witt KL, Preston RJ, Perreault SD, Allen JW, et al: Assessing Human Germ-Cell Mutagenesis in the Postgenome Era: A Celebration of the Legacy of William Lawson (Bill) Russell. Environ. Mol. Mutagen 2007, 48:71-95. [80] Lewis SE, Aitken RJ: DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res. 2005, 322:33–41. [81] Aitken RJ: The human spermatozoon - a cell in crisis? The Amoroso Lecture. J. Reprod. Fertil. 1999, 115:1-7. [82] Aitken RJ, Koopman P, Lewis SE: Seeds of concern. Nature 2004, 432:48-52.

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In: Genome Instability and Transgenerational Effects ISBN: 978-1-60876-831-8 Editors: I. Kovalchuk, O. Kovalchuk, pp. 225-262 © 2010 Nova Science Publishers, Inc.

Chapter 12

TRANSGENERATIONAL GENOMIC INSTABILITY IN MAMMALS Carmel Mothersill and Colin Seymour Medical Physics and Applied Radiation Sciences Department, McMaster University, Hamilton, Ontario, L8S 4K1, Canada

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ABSTRACT The field of transgenerational genomic instability in mammals, including humans, is a confusing area of contradictory results, generalizations based on limited data sets and confusion about terminology. This chapter will focus on radiation-induced effects on genome stability, with some discussion of the literature on chemical-induced effects where relevant. The old and recent radiation literature will be summarized, and controversial issues will be highlighted. The relevance of non-targeted effects, such as bystander signaling and stress-induced instability, will also be discussed. An emphasis will be placed on the recent data from the authors‘ laboratory concerning medium- and blood-borne signals, which appear to regulate behaviors of lower hierarchical levels to achieve coordination of tissue, organism and ecosystem response at higher levels of organization. The hypothesis is put forward that such signal-mediated coordination represents a natural response to stress such as radiation exposure, which enables an appropriate reaction to ensure an optimal outcome for irradiated organisms. Data sources will be reviewed, and limitations of these studies will be discussed. Most of the discussion will relate to experimental studies in (mainly) rodents, but available data on wild mammals (e.g., post Chernobyl) will also be discussed.

Keywords: radiation, non-targeted effects, genomic instability, bystander effects, low dose effects, transgenerational genomic instability.

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ABBREVIATIONS ROS – Reactive Oxygen Species NOS – Nitric oxide species SKY – Spectral Karyotype MALDI TOF - Matrix-Assisted Laser Desorption / Ionisation - Time Of Flight SELDI TOF (MS) - Surface-Enhanced Laser Desorption / Ionisation – Time Of Flight (mass spectrometry) chIP - Chromatin immunoprecipitation NMR - Nuclear Magnetic Resonance

INTRODUCTION

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Terminology This field can be confusing; therefore some definitions of terms used in this chapter are presented in Text Box 1. These are by no means definitive but are attributable to various researchers in the area and serve to explain what is meant by some of the more unfamiliar terms used in the literature. Much of the controversy in the field is probably caused by authors using the same term to mean different things. Such examples are highlighted in the literature review. The key points to bear in mind are that the frequency of events described as transgenerational – whether somatic or germ-line – is too high to be accounted for by specific gene mutations, and it requires an acceptance of cellular tolerance for an elevated mutation rate resulting in an increased frequency of mutation at all loci not just the one being measured. Often the terms mutation rate and mutation frequency are used interchangeably in the radiation genomic instability literature, but the mutation frequency is the number of events per cell or per given organism, whereas the mutation rate is the number of events per cell division or per single genome copy. Therefore, in terms of delayed genomic instability, the rate concerns the tolerance of the system for accumulating mutations, while the frequency refers to the frank occurrence of measured mutations at delayed time points. This is discussed eloquently in [1] and is also formalized in Niwa‘s paper concerning the components needed to get transgenerational instability [2]. This increased tolerance for mutations is not the same as a ―mutator phenotype‖ (see Text Box 1), although transgenerational instability is often described as being due to this [3-5]. However, a mutator phenotype presupposes a mutation which induces this phenotype and the non-clonal nature of the somatic data at least make this implausible. Mutator phenotypes may emerge from induced epigenetic instabilities - for example, gene amplification or increased frequency of specific mutations can result from induction of a mutator phenotype, but the initial phenomenon is clearly epigenetic and may involve non-targeted events. A similar confusion arises concerning what is meant by true germ line instability. Skinner and colleagues discuss this in a series of papers [see e.g. 6-7] and conclude that for an effect to be transgenerational in vivo, it must be measured in the F3 generation, if exposure was applied to a pregnant animal, because the F1 and stem cells for the F2 generation are already present. If exposure is applied to an adult pre-conception or to male sperm, then the F2 generation is necessary since germ cells of the F1 generation are

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present in the exposed adult. Much of the literature reports effects of irradiated parents on their offspring (i.e., the F1 generation). F1-F3 definitions are given in the Text Box 1.

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Text Box 1. Definitions as used in this chapter for some important terms Radiation-induced bystander effect (RIBE): An effect induced in an unirradiated cell which is in receipt of signals from an irradiated cell. The signaling factors are unknown. Transmission of signals can be through gap junctions or via medium transfer. Radiation-induced genomic instability (RIGI): detection of non-clonal high frequency mutations or chromosomal aberrations in distant progeny of cells which were irradiated. For somatic RIGI progeny, at least 10 generations post irradiation are usually measured, although detection of lethal chromosomal aberrations are measured in clonal progeny at earlier times. For germ line RIGI, the F2 (after adult non-pregnant irradiation) or F3 (after irradiation of pregnant females) progeny must be measured. Clonal progeny: direct descendents of an irradiated cell (somatic or germ line) Non-clonal events: Damage detected in progeny which is not consistently present in previous generations of the colony. This is the key factor which suggests an epigenetic cause of RIGI. Mutator Phenotype: A term used to define a change in mutation tolerance of a system which accepts more mutations without sending the cell into a death pathway. Two possible mechanisms are: either a gene mutation occurs in a stabilizing gene (low frequency of initial events) or a phenotypic change such as increased oxidative stress maintains the cells in a hypermutable state (high frequency of initial events). The term introduces environmental factors, including biochemical and physiological properties that also interact with the genotype. Previously, mutation had been thought to involve only the genotype. Germ line transmission: Refers to transmission to future sexual progeny of mutations or epigenetic changes which occur in the reproductive stem cells. F0-F3 generations: F1 refers to sexual progeny of the exposed individual (F0). The reproductive cells of F1 progeny are present in the exposed individual and are therefore themselves exposed. F2 progeny are the offspring of the F1 generation. Where a pregnant individual is exposed as F0, then the foetus is F1 and the stem cells of the F2 progeny are present in the F1 foetus and are also exposed. In this case, true transgenerational effects shouuild only be accepted in the next (F3) generation.

Background to Transgenerational Cellular Instability Studies The field of transgenerational instability has arisen out of attempts to explain highfrequency occurrences of mutations (lethal or non-lethal) and chromosomal damage in progeny of irradiated somatic and germ line cells. A key finding is non-clonal chromosomal aberrations in the clonal surviving progeny which are not compatible with continued reproduction of the cell lineage. Such aberrations must have arisen subsequent to exposure in descendants of the exposed cells [8-11]. Similar phenomena showing an increased frequency of reproductive cell death, lethal mutations and apoptosis in progeny of exposed cells are incompatible with the death caused by the initial radiation exposure. Rather, some processes have been set in motion by radiation exposure leading to high-frequency transmissible

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damage in the progeny of cells which survived the initial exposure to produce apparently normal progeny [12-14]. Figure 1 shows a schematic diagram of the old and new paradigms describing how ionizing radiation is through to produce long-term effects in progeny. The key to the argument that these effects cannot be explained using the classical radiation/mutation theory [15,16] is that there is no evidence for an increasing effect (the frequency of events) with dose (deposition of energy). Low doses are very effective at inducing genomic instability, and the effect saturates at very low doses (below an acute dose of 5 mGy) To many people ―transgenerational‖ studies are limited to germ line transmission (defined in Text Box 1) of effects, but much of the somatic cell literature is relevant in that it points to mechanisms which may be important in the generation of these effects. Key issues are that while manifestations of transgenerational events are generally measured as mutations or other DNA-centered endpoints, the frequency and transmissibility of the damage cannot be explained by conventional mutation theories [17,18] leading to the current conclusion that epigenetic and other non-targeted mechanisms are involved [19-22]. Ionising radiation is a particularly useful tool for studying transmissible instability because the initial causing agent (in this case, the ionizing track) leaves no residue, so that all subsequent events can be cleanly attributable to a mechanism which is not due to low-level residual doses of the inducing agent. By inference though it can be concluded that chemicals induce these effects too; and heavy metals, estrogen mimetics and various organics have been shown to produce delayed death and chromosomal aberrations in the distant progeny of apparently healthy survivors [23-25]. Text box 2 shows the types of cellular effects which have been detected in vitro in the distant somatic progeny of irradiated cells. ―Distant‖ means at least 10-15 cell generations post-insult meaning that the progeny resulted from the cells which survived the initial irradiation and were able to reproduce normally. For a comprehensive discussion of this phenomenon, see [26-28]. Many studies have taken the basic in vitro cell work further and have demonstrated these effects in vivo and ―ex vivo‖ in rodents and in humans [29-31]. Figures 2a and Figure 2b show examples of our approach, where irradiation took place in vivo, and long-term effects were measured in explant or primary cultures derived from in vivo exposed tissues. The key findings from these studies were that instability could be induced in vivo in a variety of tissues and that the progeny cells again demonstrated high frequencies of the endpoints (usually deleterious ones) being measured, even following very low doses of radiation [32]. This leads to an increasing concern about detrimental health effects of low doses of radiation which might not be apparent from conventional mutational assays [27, 3335]

Germ Line Transmission if Instability to Progeny Data in this area come from three main sources: the first one is experimental studies of research groups of Nomura, Streffer, Dubrova and others who use mouse strains [36-38]. A second source is from radioecological studies of wild populations of mammals many years (and generations) after nuclear accidents such as the Chernobyl reactor explosion in 1986 [3941]. A further source is follow-up studies of atomic bomb survivors, nuclear workers and other cohorts of irradiated human populations [42-44]. There are several good reviews of these fields, but some key observations and controversies are outlined here.

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Text Box 2. Types of cellular effects detected in vitro and in vivo Endpoints measured in distant progeny of irradiated cells •Apoptosis and other forms of cell death •over expression of early response proteins and signaling proteins •Oxidative stress •Proliferation •Genomic instability •Cytogenetic abnormalities •Transformation

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Characteristics of delayed effects syndrome in vivo and in vitro ØA genome-wide process induced at very high (epigenetic) frequencies Ø Response may saturate at low doses Ø Genetic, morphological and functional abnormalities Ø Persists over many cell generations (indefinitely ?) Ø Expression influenced by cell type & genetic factors Ø Expression in vivo but significant variability Ø Lesions tend to have characteristics of ―spontaneous‖ abnormalities Ø Inter-cellular mechanisms implicated Ø Mechanisms involving oxidative stress / free radicals implicated

The major papers are summarized in Table 1 where reference sources, measured endpoints, animals that were used, life stage at the time of exposure as well as types of dose/radiation and main findings are presented. It is apparent from the Table that general conclusions are hard to identify, but some obvious limitations of the studies are presented below. Limitations of experimental studies: the key issue here is the dependence on mouse strains which are extensively in-bred, often with carcinogenic genotypes. While the data are models of experimental design, they are of questionable relevance to human health. However, they can answer very precise questions about mechanisms and dose responses. They also do show incontrovertible evidence for transmission of instability through the germline irrespective of whether irradiation took place in utero or in adult mice prior to mating. The key findings are that irradiation of male mice prior to mating does produce instability in their offspring, which appears to involve a permanent reprogramming of the genome [6, 45]. Genomic imprinting is a suggested mechanism involving silencing of certain genes in a way which is transmitted to distant generations [6, 46], although a mechanism involving radiationinduced genomic instability leading to a higher than normal tolerance for mutations has also been suggested [2, 17, 47]. The characteristic pattern of point mutations distributed throughout the genome provides compelling evidence for the latter view [1, 48]. Lord and colleagues [49-51] and Nomura and colleagues [36, 52, 53] have also reported that irradiation of parents can lead to a higher predisposition in progeny for chemically induced carcinogenesis. These experiments are again done using single strains of mice, but they do point to the complexity of the field and to the importance of environmental factors (such as chemical or lifestyle factors) in modulating the expression of effects in F2 and F3 or more distant progeny.

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Table 1. Summary of Papers Dealing With Transgeneration Radiation Effects This table gives the main points from papers in the field, comparing dose, endpoint used and life stage. References for this table are listed as a footnote to the table and are not in the main reference list Paper

Major Point

Endpoint Used

Time after exposure when endpoint measured

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

Effects were determined by the amount of radioactivity in the offspring. -Expanded simple tandem repeat mutation data - Single-stranded and double-stranded DNA breaks - Chromosome aberrations in bone marrow cells - Embryonic lethality

10 day feeding plus reproduction

Algae food radio labelled with 0.56 uM of Se

Adult exposed. F1 and F2 generations examined

Se

- 8 weeks (in offspring)

CBA/Ca mice – 2 Gy

- Offspring measured after 8 weeks

Acute xrays

Exposed until captured.

external ray and internal  irradiation by 137Cs and 134Cs

Experimental animal studies [127]

[128]

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[41]

Examined trangenerational retention and maternal transfer of selenium in 3 consecutive generations of Daphnia magna. Concluded that dietary-derived SE was more efficiently transferred to the F1 then the maternally derived Se transferred to F2 - Irradiated mice produce offspring that have higher mutation rates in expanded simple tandem repeats, which is correlated with a higher mutation rate at protein-coding genes (hprt in this case). Also, the mice had a higher increase in single-stranded and double-stranded breaks - The bank vole was examined in areas affected by the Chernobyl accident. Bank voles from 5 different areas were studied. It was found that there was a dosedependent increase in chromosome aberrations. Ten years after the Chernobyl accident, the chromosome aberration rate was still high and the frequency of embryonic losses increased.

BALB/c mice – 1 Gy The bank voles were captured 5 and 10 years after the accident. 2-4 months old animals were studied for chromosome aberrations and embryonic losses. Pregnant females were studied for embryo mortality

Soil contained 88500 kBq/m2 137Cs, 4-5650 kBq/m2 134 Cs, 5-5790 kBq/m2 106Ru, 017200 kBq/m2 144Ce

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Paper

Major Point

Endpoint Used

Time after exposure when endpoint measured

[129]

- NIH NCr outbred Swiss male mice were fasted one or six times 1 to 4 weeks before mating. The offspring were found to have lowered average serum glucose. In some cases there were changes in the corticosterone and insulin-like growth factor-1

- Sera analyzed for glucose, corticosterone and insulin-like growth factor-1

- 4 to 10 weeks

[130]

Male and female mice were exposed to x-rays to observe DNA methylation changes in liver, spleen and lung tissues. As the dose was increased from 0Gy to 5 Gy, hypomethylation was seen in the female liver, and female and male spleen (but more pronounced in female). No changes were observed in the lung tissues. In another experiment, the mice were chronically exposed, receiving a total dose of 5Gy over 10 days. In the group sacrificed 6h after irradiation, no methylation changes in tissues were found. One month after irradiation however, changes were seen in the lung tissue for both acute and chronic exposures. When looking at DNA damage there was a strong correlation between DNA damage and DNA methylation. Worked with mice to create NC hyprids by exposing the mouse pronuclei at the one-cell stage to a different cytoplamsic environment. This was done by transferring them to eggs of a different genotype. When comparing the NC hybrids to control mice, there were a number of differences in protein spots. These differences in protein spots are transmitted to the offspring through the germline.

- DNA methylation changes - DNA damage in the exposed tissue

- Experiment 1: Dosedependence analysis – 6h after irradiation - Experiment 2: Immediate and delayed effects – half of mice 6h after last irradiation, other half of mice 1 month after last irradiation

[131]

Protein expression patterns in heart and brain

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

8 week-old males fasted for 1-4 weeks and mated. 4 and 10 week-old offspring were analyzed

Experiment 1: Dose-dependence analysis – 0.5Gy, 1Gy, 2.5Gy, 5Gy Experiment 2: Immediate and delayed effects – control, acute (5Gy on day 10), 2 chronic exposure (50cGy per day for 10 days)

- 45 days old

X-rays

Table 1. (Continued) Paper

Major Point

Endpoint Used

[132]

Male transgenic mice (40 copies of a  shuttle vector per cell) that were irradiated either at 0.1Gy, 1Gy, 2Gy, and 4Gy were mated with non-transgenic females. The mutation frequency of the shuttle vector from the bone marrow of the offspring was measured, and was found to have significantly increased compared with the controls. There was found to be no significant effect of the time between mating and irradiation on the mutation frequency. - Adult male mice (NIH:NCr Swiss) were treated with Cr(III) chloride, and their sperm was tested. It was found that there was hypomethylation of two regions of the 45S rRNA gene of those mice treated. The offspring of the mice treated were found to be heavier and to have higher levels of serum T3 (a thyroid hormone) - 18 C57/B1 mice had acute exposure (50cGy) and chronic exposure (total of 50cGy). The mice exposed to the acute radiation experienced no changes in global genome methylation, whereas those chronically exposed had genome-wide hypomethylation. It was also found that the irradiated mice had no changes in their MGMT promoters (DNA repair gene), and males chronically exposed had changes in the methylation patterns of p16INKa (tumor suppressor gene) It is questionable whether or not to feed you children soy products, such as soy-based infant formulas, due to the genistein inside. Mice treated with genistein developed ovarian differentiation, which lead to multioocyte follicles (at 2 months after treatment). Those give 0.5 mg/kg, 5 mg/kg, or 25 mg/kg of genistein had reduced fertility and those given 50 mg/kg were infertile.

Mutations in a  shuttle vector

[133]

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[22]

[134]

Time after exposure when endpoint measured

Dose

0.1Gy, 1Gy, 2Gy, 4Gy

Lifestage of parent exposed and lifestage of offspring measured Lifestage of offspring measured: 10 weeks

Type of Radiation -rays

-Gene methylation in sperm of parent - levels of serum T3

-Sperm tested 2 weeks after Cr(III) chloride treatment on adult males.

1mmol/kg Cr(III) chloride

Lifestage of parent exposed: 6-8 weeks Lifestage of offspring: 10 weeks

N/A

- global genome methylation in muscle and liver tissues

- 2 hours after last treatment on day 10

chronic exposure group: 5cGy per day for 10 days; acute group: 50cGy on the

Lifestage of parents exposed: 45 days

- X-rays

- ovaries, fertility

Began monitoring right after exposure

0.5 – 50 mg/kg of genistein

Lifestage of parents exposed: 1-5 days

N/A

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Paper

Major Point

Endpoint Used

[135]

Female AVY/a mice were either given a control diet (NIH31) or a methyl donor supplemented diet. The average coat colour was found to be darker in the supplemented group. This experiment was carried out through three generations of mice, and it was found that there was no cumulative effect of the supplemented diet in further generations of the mice.

Coat colour

[136]

4279 CBA/J mice were exposed to either urethane or Xrays. The mice were mated 1, 3, and 9 weeks after treatment. Males treated with x-rays had a lower number of offspring. The number of offspring in the 3 and 9 week mating time-points was lower than those mated after 1 week. The male offspring of the urethane treated parent had a higher incidence of lung tumors. There was also an increase in the lung tumors of male offspring born to parents mated after 1 week and exposed to 2Gy x-rays. Tumors in the haematopoietic system occurred more often in females. K-ras mutations in F1 mouse lung tumors increased as the tumor progressed. Male and female C57BL/6 mice were exposed to ionizing radiation. After exposure, the mice were mating according to 4 groups: animals with maternal exposure, animals with paternal exposure, animals with combined parental exposure, and control. There was a loss of global genome methylation in the thymus tissue of the offspring in which both parents were exposed, which meant a decrease in the methyl-binding protein MeCP2. DNA methyltransferases DNMT1, DNMT3a and DNMT3b were decreased in this group. No DNMT changes were seen in spleen or liver tissues.

- Tumors - K-ras and p53tumor suppressor gene mutations

Endpoint was constantly measured for entire life after exposure

Levels of cytosine DNA methylation in somatic tissues (thymus, spleen liver) of unexposed offspring upon maternal, paternal, or combined parental exposure

- Mice mated 7 days after exposure, offspring sacrificed after 15 days

[71]

Time after exposure when endpoint measured

Dose

Either control NIH31 diet or NIH-31 supplemented with folic acid (5.0mg), vitamin B12 (0.5mg), choline chloride (5.8g), and anhydrous betaine (5.0g) Urethane group: two s.c. doses of 1mg/g body wt Two x-ray radiation groups: 1Gy and 2Gy (2 0.5Gy and 1Gy doses, respectively)

Dose to exposed group: 2.5Gy

Lifestage of parent exposed and lifestage of offspring measured Lifestage of parent exposed: 21 days old Lifestage of offspring measured: 21 days old

Type of Radiation

Lifestage of parents exposed: 9 weeks of age - Offspring were measured throughout entire life for tumors, and once dead an autopsy was performed

X-rays

Lifestage of parents exposed: sexually mature Lifestage of offspring measured: 15 days

- Ionizing radiation

N/A

Table 1. (Continued) Paper

Major Point

Endpoint Used

[10]

Male and female HLG/Zte mice exposed. Female mice were exposed to x-rays and mated with non-irradiated mice. The number of offspring with gastroschisis (hernia of the bowel) was increased with increasing radiation dose, and the effect was highest when mating took place 1 week after exposure. Males were irradiated with gamma-rays and the survival of fetuses decreased and congential malformations increased. Gastroschisis was highest after exposure during the preimplantation period, especially during zygote stage. The increase of genomic instability was found to contribute to the expression of the congenital malformation. This review goes over the effects of urethane and dioxin. Urethane, which was found in many alcoholic beverages, food and drugs, induces gene (point) mutations. It was found in one study that an increase in urethane dose increased somatic mutations in mice. Also, in Drosophila melanogaster increased x-linked recessive lethal mutations. One study involving Dioxin showed that the offspring of North Vietnamese soldiers, who were exposed to dioxins in herbicides, had increased congenital malformations. In mice, exposure to TCDD (a dioxin) lowered implantation rates and increased congenital malformations. This review explains how paternal exposure to ionizing radiation could increase the likeliness of cancer in offspring. It has been shown in mice and rats that irradiated male mice have offspring with increased chromosome aberrations. This transgenerational increases in cancer in the offspring and increased mutation rates are caused by some unknown signal that is transmitted through the sperm.

Congential malformations and genomic instability

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[36]

[1]

Congenital malformations, implantation rates, mutations

Time after exposure when endpoint measured Preconception (1-4wk, 8wk), zygote stage, preimplantation stage

Dose

N/A

In example of dioxin experiment on mice: male mice exposed to 50ng/g bw and 100ng/g bw

Female mice: 1Gy, 3Gy x-rays Male mice: 2.8 Gy gamma rays Also, in another experiment: 0.5Gy, 0.12 Gy neutrons, and 1.0Gy, 0.25Gy x-rays

Lifestage of parent exposed and lifestage of offspring measured Lifestages: Preconception (1-4wk, 8wk), zygote stage, preimplantation stage

Type of Radiation

N/A

N/A

X-rays, Gammarays, Cyclotron Neutrons

Paper

Major Point

Endpoint Used

[137]

Mutation rates at 2 expanded simple tandem repeat loci were studied in the germ line of 1st and 2nd generation offspring of inbred male mice. Showed exposure to x-rays or fission neutrons resulted in increased mutation rates in germline of both generations

DNA taken from tails PCR primers used to identify functionality— Amplification preformed in 20 ul 96-well plates on an MJ DNA Engine PTC 220.

[138]

Parental exposure can increase the rate of mutation in somatic cells and confer a predisposition to cancer in offspring suggesting that there could be an indirect effect of radiation on somatic genome stability that is transmissible through the germ line of irradiated parents. Exposure to radiation results in the destabilization of ESTR loci in germline of offspring Demonstrate that the presence or absence of Axinfu characteristic phenotype, a kinked tail, correlates with differential DNA methylation at a retrotranspoon with Axinfu and identify mutant transcripts arising adjacent to the retrotransposon LTR that are likely to be causative of the phenotype

Doesn‘t say specifically but appears to be testing DNA Methylation

N/A

Tail and kidney DNA were extracted. DNA was digested overnight with PstI either alone or with HhaI. The fragments were separated on a 2% agarose gel and analyzed by Southern transfer. Necropsy and lung nodules were counted after fixation in 10% buffered formalin. Lungs and other tissues embedded in paraffin and the sections stained with haematoxylin and eosin.

Overnight.

Length of pregnancy + 1 yr.

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[139]

[140]

The effect of benz(a)pyrene (BP) given to female mice of A strain on the 18 and 19th days of pregnancy was studies in 5 consecutive generations, Tumour multiplicity increase occurred in F1-F5 generations

Time after exposure when endpoint measured Mating occurred at 3 and 6 weeks after irradiation for the CBA/H and at 6 weeks after for the other strands.

Dose

Lifestage of parent exposed and lifestage of offspring measured Exposed as Adults Examined--???

Type of Radiation

Exposed Adult

Fission neutrons or x-rays

N/A mice were separated according to degree of kink in tail

Adult

n/a

150 mg/body

18th and 19th day of pregnancy mice exposed, 1 yr old offspring examined

Benz(a)p yrene

CBA/H and C57BL/6 males 0.4 Gy of fission neutron with 252Cf source 0.003Gy/min Also CBS/H and BALB/c males 2 and 1 Gy x-rays CBA/H males 0.5 Gy 252Cf source 0.03Gy/min

x-rays and neutron fission

Table 1. (Continued) Paper

Major Point

Endpoint Used

[4]

Determined the effects of irradiation of parental male mice on the frequency and characteristics of mitotically occurring DNA deletion events at the pun locus in the offspring.

Timing of DNA deletions

[141]

Transgenerational transmission of radiation damage of rat genome was studied on the basis of cytogenetic changes in somatic cells. With the radiation found that caused latent cytogenetic damage to the liver

[142]

The effects of organochlorine (OC) in dolphin‘s blubber Analyzed the effects of parental treatment by ethylnitrosourea (forms specific types of DNA adducts) on the transgenerational effects in the F1 offspring of exposed mice. Results suggest that transgenerational instability is not attributed to a specific sub-set of DNA lesions, and is most probably triggered by a stress-like response to a generalized DNA damage. Transplacental effect of orthoaminoasotoluol (OAAT) on the liver was studied in 3 generations of CBA mice. A high incidence of liver tumors was observed in treated mice offspring in comparison to the controls

Cell proliferation was stimulated by hepatectomy of the middle and left lateral lobes. Rats examined 30 hours after procedure. biopsy

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[143]

[144]

[145]

OC levels of male and female humans were tested and compared with those of ages 21-35 and 41-55. Lower levels of OC found in young adults but they showed the same prevalence of adverse health signs as the old generation.

DNA isolation and ESTR typing - DNA extracted from tails – ESTR probes Ms6-hm and Hm-2, then, Southern blot hybridization.

Tumors were measured, fixed and then embedded in paraffin. Sections were stained with haematoxylin and eosin. Used blood, ELISA also tested thyroid volume using a sonography

Time after exposure when endpoint measured 4 weeks after irradiation male mice were mated with females (unirradiated) Pre mating till adult.

Dose

Lifestage of parent exposed and lifestage of offspring measured Adult exposed Embryo examined

Type of Radiation

3 Gy 25 days before mating— 1/2 of offspring given another 3 Gy

Adult exposed

Gamma radiation

Dependent upon age of Dolphin Mating took place 1 and 10 weeks after exposure plus the amount of time of pregnancy until the end point

unknown

Ages 7-37

OC

150 mg/kg

Adult exposed Offspring examined and F2 generation also examined

ENU

Exposure on the 17, 18 and 19th day of pregnancy, offspring housed with mothers until lactation was finalized n/a

3 doses of 4 mg for 12mg total

Pregnant mother exposed, offspring of 3 generations examined

OAAT

n/a

Adults ages 21-35 and 41-55

OC

1 Gy for 28 days

X-ray

Paper

[146]

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[147]

[148]

Major Point

It was concluded that adverse effects in the young adults from POLL possibly did not result from their actual OC levels but very likely from their exposure to high OC levels of their mothers during their prenatal and perinatal life. Therefore showing transgenerational transmission of endocrine disruptors action. Studied the transgenerational effect of the sublethal dose of gamma radiation on the proliferactive activity of cells in the rostral migratory stream (RMS) in the brain of neonatal and young progeny of male rats. Results showed that paternal exposure to ionizing radiation induced the genome instability, which manifested itself in the progeny by alteration in proliferactive activity or slackening of cell migration in the RMS Examine Mate Preference in male and female rats whose progenitors had been treated with the antiandrogenic fungidie vinclozolin. Showed that females will not show a preference for males with exposure history but males do not show any preference. Shows that EDCs are not only transgenerational but also transpopulational. Analyzed the mutation Frequency at an ESTR locus in the germline and bone marrow of the 1st generation male offspring. There was a significant elevation in the BALB/c X BALB /c and in the male scidX female BALB/c. However the oocytes of the scid female are unable to fully support the repair of double-strand breaks induced in paternal sperm which results in the elimination of cells/embryos contain high levels of DNA damage

Endpoint Used

Time after exposure when endpoint measured

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

Labeled proliferating cells, brain removed, detected BrdU labeled nuclei.

Exposure was 25 days before mating offspring lived till the 28th day of life.

3 Gy using a 60Co source at a rate of 0.08Gy/min

2-5 month old Adult males exposed

Gamma Ray

Rats were presented with 1 control lineage and 1 EDC lineage rat of the opposite sex

n/a

n/a

Embryonic days 8-14

Vinclozoli n

Tissue samples- used protein kinase and singlemolecule polymerase to find the ESTR frequency.

5 days before mating adults were exposed and the offspring was examined at 7 weeks old

2Gy

Adult exposed Offspring examined

X-ray

Table 1. (Continued) Paper

Major Point

Endpoint Used

[149]

Evidence shows that elements of heritability are transmitted non-genomically and can have effects beyond a single generation. This may be through epigenetic mechanisms involving regulation of either imprinted or non-imprinted genes and also mechanisms related to parental physiology or behavior. ‗Developmental origins of disease‘ is a maladaptive consequence of an ancestral mechanism of developmental plasticity that may have had adaptive value in evolution of generalist species. Mutation rates at two expanded simple tandem repeat loci were studied in the germ line of the 1st and 2nd generation offspring of inbred male CBA/H, C57BL/6 and BALB/c mice exposed to either high linear energy transfer fission neutrons or low linear energy transfer x-rays. Results should that radiation-induced germline instability persists for at least two generations raises important issues of risk evaluation in humans. This paper focuses on the effects that predation causes such as morphological, behavioral and life-history adaptations. Some of these aren‘t life saving but will help complete reproduction. Exposure of Daphnia to invertebrate and vertebrate kairomones results in changes in motion, behavior, and life history and at the molecular level involves changes in heat-shock proteins (HSPs) level and the actin and tubulin cytoskeleton.

n/a

[150]

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[151]

Time after exposure when endpoint measured n/a

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

n/a

n/a

n/a

DNA isolation and ESTR typing

n/a

0.4 Gy fission neutron and 2GY and 1 Gy – x-ray

Adults exposed 3 and 6 weeks before mating

Fission neutron and x-ray

n/a

n/a

n/a

n/a

n/a

Human studies [152]

Tandemly repeated DNA loci (TRDLs) mutations in humans can sometimes be used as a biomarker. TRDL mutations have been found in people exposed to radiation from the Chernobyl accident because they were exposed to internal emitters. Largely negative results occur in humans exposed to external radiation. TRDL mutation can be used as a biomarker in mice.

- Tandemly repeated DNA loci (TRDLs) – minisatellites in humans and expanded simple tandem repeats in mice

Paper

Major Point

[153]

- Endocrine-Disrupting Chemicals (EDCs) produce a signal that causes vertebrate systems to interpret the signal as being estrogen - Effects are long term and acute - Function as epigenetic factors - Exposure can negatively affect physical and mental health. 25 adult survivors of cancer who were treated with radiation as a child, 26 partners who had not been irradiated, and 43 offspring had blood samples taken. There was an increase in aberrations in the cancer survivor group compared with the partner and offspring groups. The lower amount of aberrations in offspring show that the instability was not passed through the germline

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[154]

[155]

[156]

A review explaining Radiation-induced genomic instability, the death-inducing effect, radiation-induced bystander effects, clastogenic factors, trangenerational effects, and implications of nontargeted effects. It is possible that there is a secreted factor released by irradiated cells that affect these nonirradiated cells This study surveyed the ratio of stillbirths to livebirths of Sellafield nuclear reprocessing plant with those of the Cumbria county who did not work at the plant. There were 248 097 livebirths and 3715 still births in Cumbria between 1950 and 1989, and 130 stillbirths and 9078 livebirths of male radiation workers working at Sellafield. It was found that the father‘s total exposure from working at Sellafield has a positive correlation with the baby being stillborn.

Endpoint Used

Time after exposure when endpoint measured

Dose

Lifestage of parent exposed and lifestage of offspring measured

Type of Radiation

- Chromosome analysis of peripheral blood lymphocytes at their first in vitro division

- 7-36 years

Lifestage of parent exposed: 0.1 – 19 years Lifestage of offspring measured: 0.4-14 years (5 years old average)

Radiation used to treat cancer

N/A

N/A

- Testes: