[1st Edition] 9780128122211, 9780128122204

Table of contents :
Content:
Half Title PagePage i
Series PagePage ii
Title PagePage iii
Copyright PagePage iv
ContributorsPages ix-xi
Chapter One - Molecular Mechanisms of Noncanonical AutophagyPages 1-23N. Dupont, A.C. Nascimbeni, E. Morel, P. Codogno
Chapter Two - Epigenetic Control of Gene Expression in MaizePages 25-48J. Huang, J.S. Lynn, L. Schulte, S. Vendramin, K. McGinnis
Chapter Three - Mitochondria in Multiple Sclerosis: Molecular Mechanisms of PathogenesisPages 49-103S. Patergnani, V. Fossati, M. Bonora, C. Giorgi, S. Marchi, S. Missiroli, T. Rusielewicz, M.R. Wieckowski, P. Pinton
Chapter Four - Molecular Regulation of the Spindle Assembly Checkpoint by Kinases and PhosphatasesPages 105-161G. Manic, F. Corradi, A. Sistigu, S. Siteni, I. Vitale
Chapter Five - BH3-Only Proteins in Health and DiseasePages 163-196J.A. Glab, G.W. Mbogo, H. Puthalakath
Chapter Six - The Multifaceted Contributions of Chromatin to HIV-1 Integration, Transcription, and LatencyPages 197-252E. De Crignis, T. Mahmoudi
Chapter Seven - The Inflammatory Signal Adaptor RIPK3: Functions Beyond NecroptosisPages 253-275K. Moriwaki, F.K.-M. Chan
IndexPages 277-288

Citation preview

VOLUME THREE HUNDRED AND TWENTY EIGHT

INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY

International Review of Cell and Molecular Biology Series Editors GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER JONATHAN JARVIK LORENZO GALLUZZI

1949—1988 1949—1984 1967—2016 1984—1992 1993—1995 2016—

Editorial Advisory Board KEITH BURRIDGE AARON CIECHANOVER SANDRA DEMARIA SILVIA FINNEMANN KWANG JEON

CARLOS LOPEZ-OTIN WALLACE MARSHALL SHIGEKAZU NAGATA MOSHE OREN ANNE SIMONSEN

VOLUME THREE HUNDRED AND TWENTY EIGHT

INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY

Edited by

LORENZO GALLUZZI Department of Radiation Oncology Weill Cornell Medical College New York, New York

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom First edition 2017 Copyright © 2017 Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-812220-4 ISSN: 1937-6448 For information on all Academic Press publications visit our website at https://www.elsevier.com/

Publisher: Zoe Kruze Acquisition Editor: Alex White Editorial Project Manager: Fenton Coulthurst Production Project Manager: Magesh Kumar Mahalingam Designer: Mark Rogers Typeset by Thomson Digital

CONTRIBUTORS M. Bonora Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy F.K.-M. Chan Department of Pathology, Immunology and Microbiology Program, University of Massachusetts Medical School, Worcester, MA, United States P. Codogno Institut Necker-Enfant Malades (INEM), INSERM, Universite´ Paris Descartes-Sorbonne Paris Cite´, Paris, France F. Corradi Department of Biology, University of Rome “Tor Vergata”, Rome, Italy E. De Crignis Department of Biochemistry, Erasmus University Medical Centre, Rotterdam, The Netherlands N. Dupont Institut Necker-Enfant Malades (INEM), INSERM, Universite´ Paris Descartes-Sorbonne Paris Cite´, Paris, France V. Fossati The New York Stem Cell Foundation Research Institute, New York, NY, United States C. Giorgi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy J.A. Glab Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Kingsbury Drive, Melbourne, VIC, Australia J. Huang Department of Biological Science, Florida State University, Tallahassee, FL, United States J.S. Lynn Department of Biological Science, Florida State University, Tallahassee, FL, United States

ix

x

Contributors

T. Mahmoudi Department of Biochemistry, Erasmus University Medical Centre, Rotterdam, The Netherlands G. Manic Regina Elena National Cancer Institute, Rome, Italy S. Marchi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy G.W. Mbogo Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Kingsbury Drive, Melbourne, VIC, Australia K. McGinnis Department of Biological Science, Florida State University, Tallahassee, FL, United States S. Missiroli Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy E. Morel Institut Necker-Enfant Malades (INEM), INSERM, Universite´ Paris Descartes-Sorbonne Paris Cite´, Paris, France K. Moriwaki Department of Pathology, Immunology and Microbiology Program, University of Massachusetts Medical School, Worcester, MA, United States A.C. Nascimbeni Institut Necker-Enfant Malades (INEM), INSERM, Universite´ Paris Descartes-Sorbonne Paris Cite´, Paris, France S. Patergnani Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy P. Pinton Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy H. Puthalakath Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Kingsbury Drive, Melbourne, VIC, Australia

Contributors

T. Rusielewicz The New York Stem Cell Foundation Research Institute, New York, NY, United States L. Schulte Department of Biological Science, Florida State University, Tallahassee, FL, United States A. Sistigu Regina Elena National Cancer Institute, Rome, Italy S. Siteni Regina Elena National Cancer Institute; Department of Biology, University of Rome “Roma Tre”, Rome, Italy S. Vendramin Department of Biological Science, Florida State University, Tallahassee, FL, United States I. Vitale Regina Elena National Cancer Institute; Department of Biology, University of Rome “Tor Vergata”, Rome, Italy M.R. Wieckowski Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland

xi

CHAPTER ONE

Molecular Mechanisms of Noncanonical Autophagy N. Dupont, A.C. Nascimbeni, E. Morel, P. Codogno* Institut Necker-Enfant Malades (INEM), INSERM, Universite´ Paris Descartes-Sorbonne Paris Cite´, Paris, France

*Corresponding author. E-mail address: [email protected]

Contents 1. Introduction 2. The Canonical Autophagic Pathway 2.1 The Core ATG Machinery and the Formation of Autophagosomes 2.2 Maturation of Autophagosomes 3. Noncanonical Autophagy 3.1 ULK1-Independent Autophagy 3.2 Beclin 1-Independent and VPS34-Independent Autophagy 3.3 VPS34/VPS15-Independent Autophagy 3.4 Autophagy Independent of Ubiquitin-Like Conjugation Actors 4. Conclusions Acknowledgments References

2 3 5 6 7 7 8 13 14 16 17 17

Abstract Macroautophagy is a lysosomal catabolic process that maintains the homeostasis of eukaryotic cells, tissues, and organisms. Macroautophagy plays important physiological roles during development and aging processes and also contributes to immune responses. The process of macroautophagy is compromised in diseases, such as cancer, neurodegenerative disorders, and diabetes. The autophagosome, the double-membrane-bound organelle that sequesters cytoplasmic material to initiate macroautophagy, is formed by the hierarchical recruitment of about 15 autophagyrelated (ATG) proteins and associated proteins, such as DFCP1, AMBRA1, the class III phosphatidyl-inositol 3-kinase VPS34, and p150/VPS15. Evidence suggests that in addition to the canonical pathway, noncanonical pathways that do not require the entire repertoire of ATGs can also result in formation of autophagosomes. Here we will discuss recent discoveries concerning the molecular regulation of these noncanonical forms of macroautophagy and their potential roles in cellular responses to stressful situations.

International Review of Cell and Molecular Biology, Volume 328 ISSN 1937-6448 http://dx.doi.org/10.1016/bs.ircmb.2016.08.001

© 2017 Elsevier Inc. All rights reserved.

1

2

N. Dupont et al.

1. INTRODUCTION Autophagy refers to catabolic processes by which eukaryotic cells recycle their own constituents by a vacuolar transport of cargo to the lysosomes (Boya et al., 2013). Macroautophagy (hereafter referred to as autophagy) starts with the formation of a double-membrane-bound vacuole named the autophagosome that unselectively or selectively sequesters fractions of the cytoplasm. Autophagosomes deliver cargo to the lysosomes either directly or after fusion with endosomal compartments. Autophagy is induced in response to a variety of extracellular and intracellular stress situations, such as nutrient shortage, hypoxia, the production of reactive oxygen species, or the accumulation of protein aggregates. Autophagy is necessary during development and in adult organisms maintains tissue homeostasis and innate and adaptive immunity (Kroemer et al., 2010; Mehrpour et al., 2010; Mizushima and Komatsu, 2011). Alterations in autophagy are observed in pathologies, such as cancer, neurodegenerative diseases, type II diabetes, and chronic inflammatory diseases (Choi et al., 2013; Jiang and Mizushima, 2014). The discovery of autophagy-related (ATG) genes in yeast was a milestone in our understanding of the molecular aspects and the physiological functions of autophagy (Ohsumi, 2014; Yang and Klionsky, 2010). About 40 ATG proteins have been characterized in yeast. Eighteen ATG proteins constitute the core machinery of the autophagosome (Mizushima et al., 2011; Nakatogawa et al., 2009). These core ATG proteins were conserved during the evolution and act in different modules during the assembly and the elongation of a membrane called phagophore to form the autophagosome. During selective autophagy, autophagy receptors recognized by the core machinery sequester the targeted protein, organelle, protein aggregate, bacteria, or virus into the autophagosome. Recently, noncanonical autophagy pathways, that result in formation of functional autophagosomes, that include only some of the core ATG machinery have been discovered (Codogno et al., 2011). The existence of these alternative pathways underscores the complexity of the molecular aspects of autophagy and raises the possibility that different stimuli induce subtle molecular changes in autophagy. Although our understanding of noncanonical autophagy is still in its childhood, it is important to summarize and discuss the knowledge on this topic. Here, we discuss the differences between canonical and noncanonical

Molecular Mechanisms of Noncanonical Autophagy

3

forms of autophagy. We will not consider in this review other noncanonical pathways involving ATG proteins, such as the recruitment of ATG to the phagosomal membrane emanating from the plasma membrane or the formation of autophagy-like structures that fuse with the plasma membrane. Readers interested in these topics can refer to recent reviews (Bestebroer et al., 2013; Boya et al., 2013; Munz, 2015; Subramani and Malhotra, 2013).

2. THE CANONICAL AUTOPHAGIC PATHWAY Before discussing noncanonical autophagy, we first need to briefly define the canonical autophagy pathway. For readers who want to have a more complete view of the biogenesis of autophagosomes, many excellent recent reviews on this topic can be consulted (Abada and Elazar, 2014; Carlsson and Simonsen, 2015; Kawabata and Yoshimori, 2015; Lamb et al., 2013; Roberts and Ktistakis, 2013; Sanchez-Wandelmer et al., 2015; Shibutani and Yoshimori, 2014). The formation of an autophagosome can be subdivided into different phases that involve five functional modules (Fig. 1). Two complexes are involved in the initiation of autophagy: the ULK1 (unc-51 like autophagy activating kinase 1) complex (ULK1 is the mammalian homolog of yeast ATG1), phosphatidylinositol 3-kinase complex I (which contains PIK3C3/VPS34 or class III phosphatidylinositol 3-kinase and Beclin 1, the mammalian homolog of the yeast ATG6). Phosphatidylinositol 3-phosphate (PtdIns(3)P), which is produced by the enzymatic activity of VPS34, recruits WD repeat domain of phosphoinositide-interacting proteins WIPI1 and WIPI2 (homologs of yeast ATG18) to the phagophore and also recruits DFCP1 (double FYVE-domain-containing protein 1) to the endoplasmic reticulum (ER) membrane, which is the site of autophagosome formation known as the omegasome (Axe et al., 2008). The two PtdIns(3)P-binding proteins WIPI1/2 and DFCP1 characterize the third functional cassette. One of the functions of WIPI2 is to control the transport of the multimembrane-spanning ATG9 from the phagophore to a peripheral endosome/Golgi localization (Zavodszky et al., 2013). The ATG9 protein is the fourth functional module. In yeast, the fusion of ATG9-containing vesicles is important in the very early stages of autophagy (Yamamoto et al., 2012). In mammalian cells, the trafficking of ATG9 to the phagophore is also an early event that occurs soon after autophagy induction (Orsi et al., 2012). The last functional module consists of the two ubiquitin-like conjugation systems: ATG12–ATG5 and the

4

N. Dupont et al.

Initiation

Elongation

Signaling

Maturation

LC3

ATG12 ATG7 ATG5 ATG12

ATG101 ULK1 ATG13

ULK complex

ATG3-ATG12

LC3-PE

Conjugation systems

Ambra1 Beclin-1 ATG14L

PIK3C3 complex

ATG3

ATG5 ATG12 -ATG16L1

FIP200

VPS34

LC3

ATG10

WIPI-1/2

VPS15

Lysosome

PI3P PI5P

PIKfyve

ATG9L vesicles

Phagophore

Autophagosome

Lysosome

Figure 1 ATG proteins involved in the early steps of mammalian macroautophagy. Macroautophagy (or simply autophagy) involves the formation of double-membranebound organelles called autophagosomes in which cargoes are sequestered and then degraded after fusion with the lysosomes. Briefly, there are three important stages in macroautophagy: initiation, which involves the nucleation step that results in phagophore formation; elongation, which involves the closure step that results in the autophagosome; and maturation, in which the autophagosome fuses with the lysosome. The different modules that constitute the ATG protein core machinery involved in the first two steps of autophagosome formation are delimited by the gray dotted lines. Upon autophagy stimulation, the ULK complex (consisting of ULK1, FIP200, ATG13, and ATG101 shown in yellow) activates the class III phosphatidylinositol-3-kinase (PIK3C3) complex I (in blue), consisting of the core structure (Beclin 1, VPS15, and VPS34), and two regulators (ATG14L and AMBRA1). This induction allows the production of PI3P (blue lines) on the omegasome to promote the recruitment of the two ubiquitin-like conjugation systems (in orange) through the WIPI proteins (in green). The ATG12–ATG5–ATG16L1 complex acts as an E3-like enzyme to generate the PE-conjugated form of LC3 (LC3-II), the well-known marker of the macroautophagic pathway. The early stages of phagophore formation depend on vesicular transport of the transmembrane-bound ATG9. Different noncanonical autophagy pathways also lead to the formation of functional autophagosomes but require only certain of the core machinery factors. Examples are ULK1- and VPS34-independent pathways. The role of PtdIns(5)P produced by the phosphatidylinositol 5-kinase PIKfyve has also been documented to support the autophagosome formation in a PtdIns(3)P-independent manner. Finally, noncanonical autophagy can also require a nonclassical ubiquitin-like conjugate (e.g., ATG12–ATG3 conjugate).

Molecular Mechanisms of Noncanonical Autophagy

5

MAP1LC3 (microtubule-associated protein 1 light chain 3)–PE (phosphatidylethanolamine) conjugate (or LC3-II). The conjugate is the covalent link between the C-terminus of LC3 (the mammalian homolog of yeast ATG8) and the polar head of PE. The ubiquitin-like conjugation systems are involved in the elongation and closure of the autophagosomal membrane (Kabeya et al., 2000; Mizushima et al., 1998).

2.1 The Core ATG Machinery and the Formation of Autophagosomes Autophagy is initiated by the activation of the ULK1 complex, which contains a serine/threonine kinase ULK1 or ULK2, ATG13, FIP200 (a 200-kDa focal adhesion kinase family interacting protein), and ATG101. Once autophagy has been induced, this complex localizes at the site of phagophore formation to regulate the nucleation machinery (Wirth et al., 2013). Phagophore nucleation is highly dependent on the production of PtdIns(3)P by VPS34. VPS34 is a part of the core phosphatidylinositol 3-kinase complex I that also contains adaptor protein VPS15, Beclin 1, and ATG14 (Levine et al., 2015). Several proteins, such as AMBRA1 (activating molecule in Beclin 1-regulated autophagy) or Bcl-2, can activate or inhibit the production of PtdIns(3)P by the phosphatidylinositol 3-kinase complex I (Wirth et al., 2013). ULK1 activates the phosphatidylinositol 3-kinase complex I by phosphorylating Beclin 1 and AMBRA1 (Di Bartolomeo et al., 2010; Russell et al., 2013). In turn, AMBRA1 interacts with the E3-ligase TRAF6 (TNF receptor-associated factor 6) to induce the ubiquitination of ULK1, thus increasing its stability and functional efficiency (Nazio et al., 2013). ULK1 can also modulate the activity of other modules of the ATG core machinery by controlling the vesicular transport of ATG9 (Zavodszky et al., 2013) and by interacting with ATG8 homologs via a LC3-interacting region (LIR motif) (Kraft et al., 2012). Another component of the ULK1 complex, FIP200, interacts with ATG16L1 (Gammoh et al., 2013; Nishimura et al., 2013), which is an element in the ubiquitin-like cassette of the ATG core machinery. The production of PtdIns(3)P in the phagophore membrane allows the recruitment of WIPI1 and WIPI2 (Polson et al., 2010). Recently WIPI2b [an isoform of WIPI2 functional in autophagy (Muller and ProikasCezanne, 2015)] was shown to interact with ATG16L1 (Dooley et al., 2014). These results suggest WIPI2b and ATG16L1 contribute to the expansion and the closure of the vesicle in concert with the two ubiquitin-like conjugation systems, resulting in the ATG12–ATG5–ATG16L complex,

6

N. Dupont et al.

and the formation of the PE conjugate of microtubule-associated protein LC3. Interestingly different domains of ATG16L1 interact with FIP200 and WIPI2b suggesting that ATG16L1 can recruit both proteins at the same time. Autophagosome formation may occur at different membranes. During starvation, the phagophore is nucleated at the omegasome, a subdomain of the ER characterized by its Ω shape and by the presence of the PtdIns(3)P binding protein DFCP1 (Axe et al., 2008). The ER-mitochondrial membrane plays a pivotal role in the recruitment of ATG proteins through the t-SNARE (target membrane—soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) protein syntaxin 17 and the ER membrane protein VMP1 (vacuole membrane protein 1) (Hamasaki et al., 2013; Koyama-Honda et al., 2013; Molejon et al., 2013). The plasma membrane has also been shown to be engaged during autophagosome formation. The t-SNARE protein, VAMP7 (vesicle-associated membrane protein 7) and its partner v(vesicle)-SNARE (a complex of syntaxin 7, syntaxin 8, and Vti1b) regulate the homotypic fusion of ATG16L1-positive vesicles after internalization from the plasma membrane (Moreau et al., 2011). It has also been suggested that these vesicles are precursor elements of the phagophore (Ravikumar et al., 2010). The transmembrane protein ATG9 is also involved in the nucleation of the phagophore membrane; it cycles between different compartments and the phagophore (Orsi et al., 2012). The ER exit site and the ERGIC (ER-Golgi intermediate compartment) compartment have also been shown to play major roles in early events of autophagosome biogenesis (Ge et al., 2013; Stadel et al., 2015). More detailed discussion on the membrane origin in the autophagic pathway can be found in recent reviews (Abada and Elazar, 2014; Carlsson and Simonsen, 2015; Kawabata and Yoshimori, 2015; Lamb et al., 2013; Roberts and Ktistakis, 2013; Sanchez-Wandelmer et al., 2015; Shibutani and Yoshimori, 2014).

2.2 Maturation of Autophagosomes Autophagosome maturation and final fusion with the lysosome occurs in the vicinity of the centrosome and depends on several lysosomal membrane proteins, such as the small GTPase Rab7 (Ras-related protein 7) and the transmembrane lysosome-associated membrane protein 2 (LAMP2) (Eskelinen, 2005). These fusion events are also dependent on SNAREs. VAMP3 contributes to the fusion of multivesicular bodies with autophagosomes to form amphisomes (Fader et al., 2009). Recently, syntaxin 17 has been shown to be targeted by autophagosomes to control fusion with endosomes/lysosomes (Itakura et al., 2012). Interestingly ATG14L binds

Molecular Mechanisms of Noncanonical Autophagy

7

syntaxin 17 and stabilizes the syntaxin 17-SNAP29 (synaptosomal-associated protein 29) complex (Diao et al., 2015). This interaction primes the interaction with VAMP8 to control autophagosome–endosome fusion. In contrast to its role in autophagosome biogenesis, the role of ATG14L in autophagosome maturation requires its homooligomerization (Diao et al., 2015). Syntaxin 17 and ATG14L are involved in the early and late stages of autophagosome formation (Hamasaki et al., 2013). This is only one of the examples in which the same actors have been demonstrated to be involved in different steps of the autophagic pathway. The phosphorylation of LC3 at position Thr50 by the Hippo kinase STK3/STK4 (serine/ threonine-protein kinase 3/serine/threonine-protein kinase 4) (Wilkinson et al., 2015) and the activity of the ER calcium pump CaP60A/SERCA are also critical for the fusion of autophagosomes with lysosomes (Mauvezin et al., 2015). The multivalent adaptor protein PLEKHM1 (pleckstrin homology domain-containing family M member 1), which interacts with Rab7, LC3, and the HOPS (homotypic fusion and protein sorting) complex, is a central hub for both the maturation of autophagosomes and the progression of endocytosis (McEwan et al., 2015). The reformation of lysosomes through a process called autophagosome-lysosome reformation (ALR) is fundamental to lysosomal identity (Yu et al., 2010). Phosphatidylinositol 3-phopshate and VPS34, which are necessary in the early stage of autophagy, are important regulators of ALR with the protein UVRAG as a partner (Munson et al., 2015).

3. NONCANONICAL AUTOPHAGY Noncanonical autophagy does not require all of the ATG proteins to build-up the double-membrane-bound autophagosome. There is evidence for several types of noncanonical autophagy; each will be discussed in the following sections.

3.1 ULK1-Independent Autophagy A form of autophagy that bypasses the canonical ULK1 initiation step has been reported to occur in response to ammonia or to glucose deprivation (Cheong et al., 2011). Interestingly, Gammoh et al. (2013) identified a FIP200-binding domain (FBD) in ATG16L1 adjacent but distinct from the WIPI2b binding site (Dooley et al., 2014). This domain is required for amino acid starvation-induced autophagy (ULK-dependent autophagy) but

8

N. Dupont et al.

is not required for glucose deprivation-induced autophagy (which is ULK independent). These results suggest that ATG16L1 can integrate upstream signals independently of the ULK1 complex. Probably other ATG16L1 domains in addition to the FBD domain are also involved in the signaling because ATG16L2, an ATG16L1 homolog in mammalian cells, does not have an FBD domain and is unable to support autophagy initiation (Gammoh et al., 2013). The C-terminal WD40 repeats of ATG16L1 are not required for amino acid starvation-dependent autophagy. However, this region is required for selective forms of autophagy (Lassen et al., 2014). Recently Stork and coworkers reported that a ULK1/2 binding-deficient ATG13 partially restores amino acid-induced and serum starvation-induced autophagy in ATG 13-deficient cells (Alers et al., 2011). These results suggest that ULK1independent autophagy can occur in the absence of amino acids either mediated by a low-affinity interaction between ULK1 and the truncated form of ATG13 or by an ULK1-dependent but ULK1-ATG13-independent event. Although amino acid-dependent autophagy is impaired in ULK1/2deficient cells (Cheong et al., 2011; McAlpine et al., 2013), some LC3-II formation is present in the absence of amino acids (McAlpine et al., 2013). Overall these data challenge the view of the absolute requirement of the ULK1/2 complex to initiate autophagy.

3.2 Beclin 1-Independent and VPS34-Independent Autophagy Beclin 1 and VPS34 can form complexes with different partners. These complexes promote the induction of autophagy (in concert with ATG14) or the maturation of autophagosomes (in concert with UVRAG and Rubicon) (Funderburk et al., 2010; Wirth et al., 2013). However, the Beclin 1–VPS34 complex is not obligatory under all circumstances (Codogno et al., 2011; Proikas-Cezanne and Codogno, 2011). Noncanonical Beclin 1-independent autophagy occurs after cells have been treated with proapoptotic compounds (Table 1). The first evidence for Beclin 1-independent autophagy was shown in the context of neuronal cell death induced by the neurotoxin 1-methyl4-phenylpyridinium (Zhu et al., 2007). Treatment of human breast cancer cells with the polyphenol resveratrol also induces Beclin 1-independent autophagy (Scarlatti et al., 2008). Furthermore, Z18, a compound that targets the BH3 binding groove of Bcl-XL and Bcl-2, causes Beclin 1-independent autophagosome formation in HeLa cells (Tian et al., 2010), and other proapoptotic compounds, such as staurosporine, MK801, and

Inducers

ATGs bypassed

Methods used and inhibitorsa

Cell types

References

Ammonia (metabolic intermediate) Glucose deprivation

ULK1, ULK2

DKO Ulk1/2 (WB LC3)

Mouse embryonic fibroblasts

ULK1, ULK2

Mouse embryonic fibroblasts

Carbonyl cyanide m-chlorophenylhydrazone (CCCP)

FIP200, ATG13, ATG14, Beclin 1 PIK3C3

DKO Ulk1/2 (WB LC3, IFLC3) KO Fip200, KD ATG13, ATG14, Beclin 1 (WB LC3, IFLC3)

Mouse embryonic fibroblasts, epithelial cells (HeLa), glioblastoma cells (U251)

Cheong et al. (2011) Cheong et al. (2011) Chen et al. (2013)

WM (WB LC3, IF LC3)

Mouse embryonic fibroblasts Epithelial cells (HeLa) and human breast cancer cells (U2OS) Mouse embryonic fibroblasts, epithelial cells (HeLa) Human breast cancer cells (U2OS, MCF7)

Glucose deprivationb Unsaturated fatty acids

Beclin 1, VPS34

KD, WM, 3-MA (WB LC3, IF LC3)

Glucose deprivation

VPS34

Resveratrol

Beclin 1, VPS34

KD, KO, WM (WB LC3, IF LC3, EM) KD, 3-MA, WM (WB LC3, IF LC3)

1-Methyl-4-phenylpyridinium (MPP)

Beclin 1

KD, 3-MA (WB LC3, IF LC3)

Neurons (SH-SY5Y)

McAlpine et al. (2013) Niso-Santano et al. (2015)

Molecular Mechanisms of Noncanonical Autophagy

Table 1 Inducers of noncanonical autophagy.

Vicinanza et al. (2015) Scarlatti et al. (2008); Mauthe et al. (2011) Zhu et al. (2007) (Continued ) 9

10

Table 1 Inducers of noncanonical autophagy.—cont'd. ATGs bypassed

Methods used and inhibitorsa

Z18 Staurosporin

Beclin 1, VPS34 Beclin 1

KD, 3-MA (WB LC3, IF LC3) KD (WB LC3, IF LC3)

MK801

Beclin 1

KD (WB LC3, IF LC3)

Primary cortical neurons

Gossypol

Beclin 1

KD (WB LC3, IF LC3)

Arsenic trioxide

Beclin 1

KD (WB LC3, IF LC3)

Human epithelial cells (HeLa cells and not MCF7) Ovarian cells (HEY cells)

Etoposide

Beclin 1

KD (WB LC3, IF LC3)

Primary cortical neurons

α-Hemolysin

Beclin 1

Recombinant capsid protein VP1 1,3-Dibutyl-2-thiooxoimidazolidine-4,5-dione (C1) 3-Methylcyclopentylidene-[4(40 -chlorophenyl)thiazol-2-yl] hydrazone (CPTH6)

Beclin 1

KD, 3-MA, WM (IF LC3) KD (WB LC3, EM)

Beclin 1

KD, 3-MA (WB LC3)

Beclin 1

KD (WB LC3, IF LC3)

Chinese hamster ovary fibroblast (CHO) Murine macrophage cells (RAW264.7) Colorectal carcinoma cells (HCT116) Human lung carcinoma cells (H1299)

Cell types

References

Human epithelial cells (HeLa)

Tian et al. (2010)

Primary cortical neurons

Grishchuk et al. (2011) Grishchuk et al. (2011) Gao et al. (2010) Smith et al. (2010) Grishchuk et al. (2011) Mestre et al. (2010) Liao et al. (2013) Wong et al. (2010) Ragazzoni et al. (2013) N. Dupont et al.

Inducers

Beclin 1

Etoposide, staurosporine

AG5, ATG7, ATG9, ATG12, ATG16 ATG9

Amino acid and growth factor deprivation a

ATG7

KD, 3-MA, WM (WB LC3, IF LC3) KD ATG7 (WB LC3, WB p62) KO Atg5, Atg7; KD ATG9, ATG12, ATG16 (EM, proteolysis) KO, KD (WB LC3, IF LC3)

Human hepatocarcinoma cells (HepG2) Cardiac myocytes

Liu et al. (2013)

Mouse embryonic fibroblasts, erythroid cells

Nishida et al. (2009); Honda et al. (2014)

Mouse embryonic fibroblasts, human embryonic kidney cells (HEK)

Orsi et al. (2012)

Yan et al. (2013)

DKO, Double knockout; EM, electron microscopy; IF, immunofluorescence (or fluorescence); KD, knockdown; KO, knockout; 3-MA, 3-methyladenine; WB, Western blot; WM, wortmannin. b These authors demonstrated a partial inhibition of autophagy in ULK1/2 DKO MEFs upon glucose starvation.

Molecular Mechanisms of Noncanonical Autophagy

Epoxomicin, lactacystin, bortezomib, MG132 Raclopride

11

12

N. Dupont et al.

etoposide, induce Beclin 1-independent autophagy in primary cortical neurons (Grishchuk et al., 2011). These studies suggest that it might become feasible to use prodeath compounds that induce noncanonical autophagy for cancer therapy when the functions of canonical autophagy proteins are compromised. Beclin 1-independent autophagy has also been observed in settings unrelated to cell death, such as during differentiation (Arsov et al., 2011), bacterial toxin uptake (Mestre et al., 2010), and viral infection (Berryman et al., 2012). Interestingly cis-unsaturated fatty acids (oleate and arachidonate) trigger autophagy that is independent of Beclin 1 and VPS34, whereas saturated fatty acids (palmitate and stearate) cause autophagy that is dependent on these two factors (Niso-Santano et al., 2015). The existence of Beclin 1-independent autophagy induced by unsaturated fatty acids has been confirmed in mice, in the nematode Caenorhabditis elegans, and in the yeast Saccharomyces cerevisiae. Whether the stimulation of noncanonical autophagy by cis-unsaturated fatty acids underlies the beneficial effects of these nutrients on obesity, atherosclerosis, and neurodegenerative disease is an intriguing possibility. BAG3 (Bcl-2 associated athanogene 3), a cochaperone of HSP70 (70-kDa heat shock protein) family, is engaged in Beclin 1-independent autophagy in response to proteasome inhibition (Liu et al., 2013) and in estrogen receptor-mediated autophagy in breast cancer cells (Felzen et al., 2015). A role of BAG3 in the regulation of LC3 translation has been recently reported (Rodriguez et al., 2016). How this is related to the role of BAG3 in noncanonical autophagy remains to be elucidated. It may be that BAG3 is a bona-fide signature for Beclin 1-independent autophagy (Liu et al., 2013). Interestingly, Beclin 1-independent autophagy is not synonymous with pathways that exclude the VPS34–WIPI–ATG5-LC3 route. Exposure of human tumor cells to arsenic trioxide (Smith et al., 2010) or gossypol promotes autophagy that is VPS34-dependent, but Beclin 1-independent. The autophagy activated by these inducers involves WIPI-1 under some circumstances (Gao et al., 2010). Recently, resveratrol was found to promote WIPI-1-dependent LC3 lipidation in the absence of induced phagophore formation, indicating that different membrane sites may be used during noncanonical autophagosome formation (Mauthe et al., 2011). In fact, WIPI-1 specifically localizes to both the plasma membrane and the ER in response to the induction of autophagy, indicating that the WIPI–ATG5–LC3 pathway can function at several different membrane sites.

13

Molecular Mechanisms of Noncanonical Autophagy

Table 2 In vivo detection of noncanonical autophagy by the use of Atg-knockout mouse models. KO In vivo autophagosome model formation References

Vps15 Vps34 Vps34 Vps34 Vps34 Vps34 Atg5

Muscle Mature sensory neurons T lymphocytes T lymphocytes Heart and liver T lymphocytes Brain, liver, heart, erythrocytes in embryos

Nemazanyy et al. (2013) Zhou et al. (2010) McLeod et al. (2011) Willinger and Flavell (2012) Jaber et al. (2012) Parekh et al. (2013) Nishida et al. (2009); Honda et al. (2014); Ma et al. (2015)

3.3 VPS34/VPS15-Independent Autophagy Recent studies suggest that autophagy can be independent of VPS34 and p150/VPS15 (Table 2). In Vps34/ sensory neurons, autophagosomes and LC3-II production are observed (Zhou et al., 2010). In the same mouse model, autophagy was observed in T lymphocytes (McLeod et al., 2011). However, in other genetic mouse models in which Vps34 is ablated, autophagy is absent or only minimally observed (Jaber et al., 2012; Willinger and Flavell, 2012). A recent report shows that VPS15-deficient mouse skeletal muscle is capable of forming LC3-positive autophagosomes (Nemazanyy et al., 2013). It is possible that PtdIns(3)P is required in these situations. This would suggest that this lipid is produced either by the degradation of PtdIns (3,4)P2 or PtdIns(3,4,5)P3 or by the activity of another phosphatidylinositol 3-kinase (Dall’Armi et al., 2013). In fact, class II phosphatidylinositol 3-kinase (PIK3C2) has been shown to contribute to the pool of PtdIns(3)P involved in autophagosome biogenesis (Devereaux et al., 2013). The formation of PtdIns(3)P by PIK3C2 would explain why autophagy is sometimes insensitive to classical PIK3 inhibitors, such as 3-methyladenine and wortmannin (Rubinsztein et al., 2012). In autophagy induced by glucose starvation, PtdIns(3)P is not required to initiate autophagy (McAlpine et al., 2013; Vicinanza et al., 2015). Recently, the role of PtdIns(5)P produced by the phosphatidylinositol 5-kinase PIKfyve in support of autophagosome formation in the absence of PtdIns(3)P has been documented (Vicinanza et al., 2015). Whether PtdIns(5)P-dependent autophagy requires Beclin 1 is not known. It

14

N. Dupont et al.

remains to be investigated whether DFCP1, similarly to WIPI2b, can bind to PtdIns(3)P and PtdIns(5)P. The fact that PtdIns(5)P makes up a minor fraction of the cellular phosphoinositide suggests that autophagy induction is dependent on subtle molecular and cellular conditions.

3.4 Autophagy Independent of Ubiquitin-Like Conjugation Actors Although the formation of LC3-II is not observed in ATG5/ cells, transition electron microscopy reveals the presence of autophagosomes and amphisomes (preautolysosomal vacuoles that are formed by autophagosome–endosome fusion) in ATG5/ cells treated with etoposide over a prolonged period of time (Nishida et al., 2009). Based on the unusual lamination of the membrane forming the phagophores and autophagosomes in this context, the trans-Golgi network seems to be the membrane source for this form of alternative autophagy, which is initiated by ULK1 and VPS34 complexes in a manner similar to canonical autophagy (Codogno et al., 2011). In contrast to the canonical pathway, the elongation of the initial autophagosomal membrane does not require ATG9 or the ATG proteins of the ubiquitin-like conjugation system (ATG7, ATG5, and LC3). Instead, the monomeric GTPase Rab9, which is involved in vesicular trafficking between the trans-Golgi network and late endosomes, is required to elongate the initial autophagosomal membrane through fusion events with Rab9positive vesicles. The resulting noncanonical, double-membrane autophagosome can mature and fuse with the lysosomal compartment to deliver cargo for degradation. The form of autophagy that is independent of the ubiquitin-like conjugation systems has been observed in various cell types and embryonic tissues (Codogno et al., 2011) and plays a role in the removal of mitochondria during erythrocyte maturation and mitophagy in vivo (Nishida et al., 2009). However, in erythroblasts, mitochondria are also degraded by autophagy in a manner dependent on ATG5 and ATG7 (Mortensen et al., 2011). These results suggest that both canonical autophagy and noncanonical autophagy may contribute to the elimination of mitochondria during erythroblast differentiation. Interestingly, ULK1-dependent and ATG5/ ATG7-independent autophagy is the major pathway that eliminates mitochondria from fetal erythroblasts (Honda et al., 2014). ATG5-independent autophagy also functions in mitochondrial clearance during induced

Molecular Mechanisms of Noncanonical Autophagy

15

pluripotent stem cell (iPSC) reprogramming (Ma et al., 2015). Since this metabolic reprogramming is similar to that observed during tumorigenesis, noncanonical autophagy may be involved in pathophysiological situations. Recently, a form of autophagy that is independent of ATG3 and ATG7 has been reported in the programmed reduction of cell size during intestinal cell death in Drosophila (Chang et al., 2013). This noncanonical autophagy involves ATG8 and Uba1 (Ubiquitin-like modifier-activating enzyme 1), the E1 enzyme that catalyzes the first step in ubiquitination. Uba1 is not a substitute for the E1 activity of ATG7 in the conjugation of ATG8 to PE (ATG3 functions as the E2-conjugating enzyme in the formation of ATG8-PE). As Chang et al. propose, Uba1 could function at a different stage of the autophagy pathway (Chang et al., 2013). The mechanism by which ATG8 is recruited to the autophagosomal membrane remains to be elucidated. Evidence indicates that noncanonical forms of autophagy exist in different phyla, suggesting that noncanonical forms of autophagy did not in fact emerge recently in the course of evolution. Interestingly, the intracellular bacterium Brucellaabortus requires a vacuole with autophagic features during replication (Starr et al., 2012). The formation of this compartment is dependent on ULK1 and components of the Beclin 1 complex (including ATG14L1 and a VPS34 activity) but is independent of ATG4B, ATG5, ATG7, and LC3B, suggesting that ATG5-independent autophagy or autophagic-like processes can be subverted by microorganisms. Noncanonical autophagy may also involve nonclassical ubiquitin-like conjugates. Recently ATG3, the E2-like enzyme involved in LC3 lipidation during autophagy, has been shown to be an ATG12 conjugation target (Radoshevich et al., 2010). The same group reported that the ATG12–ATG3 conjugate interacts with the ESCRT (endosomal sorting complex required for transport)-associated protein Alix to control Alixdependent functions, such as multivesicular body distribution, exosome biogenesis and virus budding, and facilitation of basal autophagy, but not starvation-induced autophagy (Murrow et al., 2015). In basal autophagy, the ATG12–ATG3 conjugate contributes to the maturation of autophagosomes. This role is distinct from the role of ATG3 and ATG12 during autophagosome formation; however, the mechanism by which the ATG12–ATG3 conjugate contributes to basal autophagy remains to be elucidated.

16

N. Dupont et al.

4. CONCLUSIONS Noncanonical autophagy pathways and structures have the same function as canonical autophagy: these pathways specifically or nonspecifically sequester a portion of the cytoplasm and compartmentalize pathogens. In many cases, material sequestered by noncanonical autophagy is ultimately degraded in the lysosomal compartment. Noncanonical autophagy, which requires only a subset of ATG proteins, has been observed in various settings. It is not yet clear how some ATG modules can be bypassed to form a functional autophagosome. Recent evidence notably suggests that PtdIns (3)P can be produced in the absence of Beclin 1 and VPS34 by the activity of PIK3C2 (Devereaux et al., 2013). This provides a rational for the fact that the PtdIns(3)P effector WIPI1/2 is necessary for initiation of LC3 lipidation in noncanonical autophagy in many settings (Codogno et al., 2011). Alternatively, other phosphoinositides, such as PtdIns(5)P, can initiate autophagy. Evidence indicates that components of ubiquitin-like systems can recruit effectors that bypass the conventional route for membrane elongation and sealing. Since the LC3 family is large in mammals, a careful investigation of the roles of different members of the LC3/GABARAP family must be done when a bypass of LC3 is suspected. A growing body of evidence shows that GABARAP family members can substitute for LC3B. One important question is whether noncanonical forms of autophagy have specific functions in cell physiology or pathological situations. From the literature available on Beclin 1-independent autophagy, it is difficult to identify a single function corresponding to this form of noncanonical autophagy. For example, Beclin 1-independent autophagy has been reported in contexts, such as survival, death, and proliferation, and immune cell development (Codogno et al., 2011; Proikas-Cezanne and Codogno, 2011). Beclin 1-independent autophagy is the most commonly reported noncanonical form of autophagy. Beclin 1 is a key autophagy protein with a large interactome (Behrends et al., 2010; He and Levine, 2010). It is involved in both the formation and maturation of autophagosomes, depending on the interacting partners (Funderburk et al., 2010; Wirth et al., 2013). Moreover, Beclin 1 is targeted by many viruses to block autophagy at different stages. Beclin 1-independent autophagy could, therefore, be an evolutionary adaptation that prevents the blockade of autophagy by an invading virus, which would otherwise compromise cell survival.

Molecular Mechanisms of Noncanonical Autophagy

17

Another intriguing question involves the relationship between noncanonical autophagy and selective forms of autophagy, such as mitophagy, which specifically sequesters mitochondria (Youle and Narendra, 2011). ATG5- and ATG7-independent autophagy is known to be involved in the clearance of mitochondria during erythroid maturation (Nishida et al., 2009), and determining the roles of the different ATG proteins in the various forms of selective autophagy may provide a way to identify the molecular signatures of autophagy that lie beyond the selective recognition of cargo (Weidberg et al., 2011). Another important question is whether both noncanonical autophagy and canonical autophagy can be activated in a coordinated manner in the same cell in response to a stressful situation. Being able to distinguish between noncanonical autophagic pathways and the canonical autophagic pathway by means of specific markers would make a significant contribution to the molecular understanding of how autophagy is regulated and functions.

ACKNOWLEDGMENTS A.C.N. and N.D. are supported by fellowships from the “Association pour la Recherche sur le Cancer” (ARC). Studies in Patrice Codogno’s laboratory are supported by institutional funding from INSERM (Institut National de la Sante´ et de la Recherche Me´dicale), CNRS (Centre National de la Recherche Scientifique), University Paris-Descartes, and grants from ANR (French National Research Agency), and INCa (French National Cancer Institute). Conflict of interest: The authors declare that they have no conflict of interest.

REFERENCES Abada, A., Elazar, Z., 2014. Getting ready for building: signaling and autophagosome biogenesis. EMBO Rep. 15, 839–852. Alers, S., Loffler, A.S., Paasch, F., Dieterle, A.M., Keppeler, H., Lauber, K., Campbell, D.G., Fehrenbacher, B., Schaller, M., Wesselborg, S., Stork, B., 2011. Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction. Autophagy 7, 1423–1433. Arsov, I., Adebayo, A., Kucerova-Levisohn, M., Haye, J., MacNeil, M., Papavasiliou, F.N., Yue, Z., Ortiz, B.D., 2011. A role for autophagic protein beclin 1 early in lymphocyte development. J. Immunol. 186, 2201–2209. Axe, E.L., Walker, S.A., Manifava, M., Chandra, P., Roderick, H.L., Habermann, A., Griffiths, G., Ktistakis, N.T., 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701. Behrends, C., Sowa, M.E., Gygi, S.P., Harper, J.W., 2010. Network organization of the human autophagy system. Nature 466, 68–76. Berryman, S., Brooks, E., Burman, A., Hawes, P., Roberts, R., Netherton, C., Monaghan, P., Whelband, M., Cottam, E., Elazar, Z., Jackson, T., Wileman, T., 2012. Foot-and-mouth disease virus induces autophagosomes during cell entry via a class III phosphatidylinositol 3-kinase-independent pathway. J. Virol. 86, 12940–12953.

18

N. Dupont et al.

Bestebroer, J., V’Kovski, P., Mauthe, M., Reggiori, F., 2013. Hidden behind autophagy: the unconventional roles of ATG proteins. Traffic 14, 1029–1041. Boya, P., Reggiori, F., Codogno, P., 2013. Emerging regulation and functions of autophagy. Nat. Cell Biol. 15, 713–720. Carlsson, S.R., Simonsen, A., 2015. Membrane dynamics in autophagosome biogenesis. J. Cell Sci. 128, 193–205. Chang, T.K., Shravage, B.V., Hayes, S.D., Powers, C.M., Simin, R.T., Wade Harper, J., Baehrecke, E.H., 2013. Uba1 functions in Atg7- and Atg3-independent autophagy. Nat. Cell Biol. 15, 1067–1078. Chen, D., Chen, X., Li, M., Zhang, H., Ding, W.X., Yin, X.M., 2013. CCCP-induced LC3 lipidation depends on Atg9 whereas FIP200/Atg13 and Beclin 1/Atg14 are dispensable. Biochem. Biophys. Res. Commun. 432, 226–230. Cheong, H., Lindsten, T., Wu, J., Lu, C., Thompson, C.B., 2011. Ammonia-induced autophagy is independent of ULK1/ULK2 kinases. Proc. Natl. Acad. Sci. USA 108, 11121–11126. Choi, A.M., Ryter, S.W., Levine, B., 2013. Autophagy in human health and disease. N. Engl. J. Med. 368, 651–662. Codogno, P., Mehrpour, M., Proikas-Cezanne, T., 2011. Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat. Rev. Mol. Cell Biol. 13, 7–12. Dall’Armi, C., Devereaux, K.A., Di Paolo, G., 2013. The role of lipids in the control of autophagy. Curr. Biol. 23, R33–R45. Devereaux, K., Dall’Armi, C., Alcazar-Roman, A., Ogasawara, Y., Zhou, X., Wang, F., Yamamoto, A., De Camilli, P., Di Paolo, G., 2013. Regulation of mammalian autophagy by class II and III PI 3-kinases through PI3P synthesis. PloS ONE 8, e76405. Di Bartolomeo, S., Corazzari, M., Nazio, F., Oliverio, S., Lisi, G., Antonioli, M., Pagliarini, V., Matteoni, S., Fuoco, C., Giunta, L., D’Amelio, M., Nardacci, R., Romagnoli, A., Piacentini, M., Cecconi, F., Fimia, G.M., 2010. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J. Cell Biol. 191, 155–168. Diao, J., Liu, R., Rong, Y., Zhao, M., Zhang, J., Lai, Y., Zhou, Q., Wilz, L.M., Li, J., Vivona, S., Pfuetzner, R.A., Brunger, A.T., Zhong, Q., 2015. ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature 520, 563–566. Dooley, H.C., Razi, M., Polson, H.E., Girardin, S.E., Wilson, M.I., Tooze, S.A., 2014. WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. Mol. Cell 55, 238–252. Eskelinen, E.L., 2005. Maturation of autophagic vacuoles in mammalian cells. Autophagy 1, 1–10. Fader, C.M., Sanchez, D.G., Mestre, M.B., Colombo, M.I., 2009. TI-VAMP/VAMP7 and VAMP3/cellubrevin: two v-SNARE proteins involved in specific steps of the autophagy/multivesicular body pathways. Biochim. Biophys. Acta 1793, 1901–1916. Felzen, V., Hiebel, C., Koziollek-Drechsler, I., Reissig, S., Wolfrum, U., Kogel, D., Brandts, C., Behl, C., Morawe, T., 2015. Estrogen receptor alpha regulates non-canonical autophagy that provides stress resistance to neuroblastoma and breast cancer cells and involves BAG3 function. Cell Death Dis. 6, e1812. Funderburk, S.F., Wang, Q.J., Yue, Z., 2010. The Beclin 1-VPS34 complex—at the crossroads of autophagy and beyond. Trends Cell Biol. 20, 355–362. Gammoh, N., Florey, O., Overholtzer, M., Jiang, X., 2013. Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy. Nat. Struct. Mol. Biol. 20, 144–149.

Molecular Mechanisms of Noncanonical Autophagy

19

Gao, P., Bauvy, C., Souquere, S., Tonelli, G., Liu, L., Zhu, Y., Qiao, Z., Bakula, D., Proikas-Cezanne, T., Pierron, G., Codogno, P., Chen, Q., Mehrpour, M., 2010. The Bcl-2 homology domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-independent cytoprotective autophagy in cancer cells. J. Biol. Chem. 285, 25570–25581. Ge, L., Melville, D., Zhang, M., Schekman, R., 2013. The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. eLife 2, e00947. Grishchuk, Y., Ginet, V., Truttmann, A.C., Clarke, P.G., Puyal, J., 2011. Beclin 1-independent autophagy contributes to apoptosis in cortical neurons. Autophagy 7, 1115–1131. Hamasaki, M., Furuta, N., Matsuda, A., Nezu, A., Yamamoto, A., Fujita, N., Oomori, H., Noda, T., Haraguchi, T., Hiraoka, Y., Amano, A., Yoshimori, T., 2013. Autophagosomes form at ER-mitochondria contact sites. Nature 495, 389–393. He, C., Levine, B., 2010. The Beclin 1 interactome. Curr. Opin. Cell Biol. 22, 140–149. Honda, S., Arakawa, S., Nishida, Y., Yamaguchi, H., Ishii, E., Shimizu, S., 2014. Ulk1mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes. Nat. Commun. 5, 4004. Itakura, E., Kishi-Itakura, C., Mizushima, N., 2012. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151, 1256–1269. Jaber, N., Dou, Z., Chen, J.S., Catanzaro, J., Jiang, Y.P., Ballou, L.M., Selinger, E., Ouyang, X., Lin, R.Z., Zhang, J., Zong, W.X., 2012. Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function. Proc. Natl. Acad. Sci. USA 109, 2003–2008. Jiang, P., Mizushima, N., 2014. Autophagy and human diseases. Cell Res. 24, 69–79. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., Yoshimori, T., 2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728. Kawabata, T., Yoshimori, T., 2015. Beyond starvation: an update on the autophagic machinery and its functions. J. Mol. Cell. Cardiol. 95, 2–10. Koyama-Honda, I., Itakura, E., Fujiwara, T.K., Mizushima, N., 2013. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy 9, 1491–1499. Kraft, C., Kijanska, M., Kalie, E., Siergiejuk, E., Lee, S.S., Semplicio, G., Stoffel, I., Brezovich, A., Verma, M., Hansmann, I., Ammerer, G., Hofmann, K., Tooze, S., Peter, M., 2012. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 31, 3691–3703. Kroemer, G., Marino, G., Levine, B., 2010. Autophagy and the integrated stress response. Mol. Cell 40, 280–293. Lamb, C.A., Yoshimori, T., Tooze, S.A., 2013. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774. Lassen, K.G., Kuballa, P., Conway, K.L., Patel, K.K., Becker, C.E., Peloquin, J.M., Villablanca, E.J., Norman, J.M., Liu, T.C., Heath, R.J., Becker, M.L., Fagbami, L., Horn, H., Mercer, J., Yilmaz, O.H., Jaffe, J.D., Shamji, A.F., Bhan, A.K., Carr, S.A., Daly, M.J., Virgin, H.W., Schreiber, S.L., Stappenbeck, T.S., Xavier, R.J., 2014. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc. Natl. Acad. Sci. USA 111, 7741–7746. Levine, B., Liu, R., Dong, X., Zhong, Q., 2015. Beclin orthologs: integrative hubs of cell signaling, membrane trafficking, and physiology. Trends Cell Biol. 25, 533–544. Liao, C.C., Ho, M.Y., Liang, S.M., Liang, C.M., 2013. Recombinant protein rVP1 upregulates BECN1-independent autophagy, MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration. Autophagy 9, 5–19.

20

N. Dupont et al.

Liu, B.Q., Du, Z.X., Zong, Z.H., Li, C., Li, N., Zhang, Q., Kong, D.H., Wang, H.Q., 2013. BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells. Autophagy 9, 905–916. Ma, T., Li, J., Xu, Y., Yu, C., Xu, T., Wang, H., Liu, K., Cao, N., Nie, B.M., Zhu, S.Y., Xu, S., Li, K., Wei, W.G., Wu, Y., Guan, K.L., Ding, S., 2015. Atg5-independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming. Nat. Cell Biol. 17, 1379–1387. Mauthe, M., Jacob, A., Freiberger, S., Hentschel, K., Stierhof, Y.D., Codogno, P., ProikasCezanne, T., 2011. Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation. Autophagy 7, 1448–1461. Mauvezin, C., Nagy, P., Juhasz, G., Neufeld, T.P., 2015. Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification. Nat. Commun. 6, 7007. McAlpine, F., Williamson, L.E., Tooze, S.A., Chan, E.Y., 2013. Regulation of nutrientsensitive autophagy by uncoordinated 51-like kinases 1 and 2. Autophagy 9, 361–373. McEwan, D.G., Popovic, D., Gubas, A., Terawaki, S., Suzuki, H., Stadel, D., Coxon, F.P., Miranda de Stegmann, D., Bhogaraju, S., Maddi, K., Kirchof, A., Gatti, E., Helfrich, M. H., Wakatsuki, S., Behrends, C., Pierre, P., Dikic, I., 2015. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol. Cell 57, 39–54. McLeod, I.X., Zhou, X., Li, Q.J., Wang, F., He, Y.W., 2011. The class III kinase Vps34 promotes T lymphocyte survival through regulating IL-7Ralpha surface expression. J. Immunol. 187, 5051–5061. Mehrpour, M., Esclatine, A., Beau, I., Codogno, P., 2010. Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview. Am. J. Physiol. Cell Physiol. 298, C776–C785. Mestre, M.B., Fader, C.M., Sola, C., Colombo, M.I., 2010. Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells. Autophagy 6, 110–125. Mizushima, N., Komatsu, M., 2011. Autophagy: renovation of cells and tissues. Cell 147, 728–741. Mizushima, N., Sugita, H., Yoshimori, T., Ohsumi, Y., 1998. A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy. J. Biol. Chem. 273, 33889–33892. Mizushima, N., Yoshimori, T., Ohsumi, Y., 2011. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132. Molejon, M.I., Ropolo, A., Re, A.L., Boggio, V., Vaccaro, M.I., 2013. The VMP1-Beclin 1 interaction regulates autophagy induction. Sci. Rep. 3, 1055. Moreau, K., Ravikumar, B., Renna, M., Puri, C., Rubinsztein, D.C., 2011. Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303–317. Mortensen, M., Soilleux, E.J., Djordjevic, G., Tripp, R., Lutteropp, M., Sadighi-Akha, E., Stranks, A.J., Glanville, J., Knight, S., Jacobsen, S.E., Kranc, K.R., Simon, A.K., 2011. The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J. Exp. Med. 208, 455–467. Muller, A.J., Proikas-Cezanne, T., 2015. Function of human WIPI proteins in autophagosomal rejuvenation of endomembranes? FEBS Lett. 589, 1546–1551. Munson, M.J., Allen, G.F., Toth, R., Campbell, D.G., Lucocq, J.M., Ganley, I.G., 2015. mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival. EMBO J. 34, 2272–2290. Munz, C., 2015. The different autophagic roads by which phagosomes travel to lysosomes. EMBO J. 34, 2391–2392. Murrow, L., Malhotra, R., Debnath, J., 2015. ATG12-ATG3 interacts with Alix to promote basal autophagic flux and late endosome function. Nat. Cell Biol. 17, 300–310.

Molecular Mechanisms of Noncanonical Autophagy

21

Nakatogawa, H., Suzuki, K., Kamada, Y., Ohsumi, Y., 2009. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell Biol. 10, 458–467. Nazio, F., Strappazzon, F., Antonioli, M., Bielli, P., Cianfanelli, V., Bordi, M., Gretzmeier, C., Dengjel, J., Piacentini, M., Fimia, G.M., Cecconi, F., 2013. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol. 15, 406–416. Nemazanyy, I., Blaauw, B., Paolini, C., Caillaud, C., Protasi, F., Mueller, A., ProikasCezanne, T., Russell, R.C., Guan, K.L., Nishino, I., Sandri, M., Pende, M., Panasyuk, G., 2013. Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease. EMBO Mol. Med. 5, 870–890. Nishida, Y., Arakawa, S., Fujitani, K., Yamaguchi, H., Mizuta, T., Kanaseki, T., Komatsu, M., Otsu, K., Tsujimoto, Y., Shimizu, S., 2009. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461, 654–658. Nishimura, T., Kaizuka, T., Cadwell, K., Sahani, M.H., Saitoh, T., Akira, S., Virgin, H.W., Mizushima, N., 2013. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep. 14, 284–291. Niso-Santano, M., Malik, S.A., Pietrocola, F., Bravo-San Pedro, J.M., Marino, G., Cianfanelli, V., Ben-Younes, A., Troncoso, R., Markaki, M., Sica, V., Izzo, V., Chaba, K., Bauvy, C., Dupont, N., Kepp, O., Rockenfeller, P., Wolinski, H., Madeo, F., Lavandero, S., Codogno, P., Harper, F., Pierron, G., Tavernarakis, N., Cecconi, F., Maiuri, M.C., Galluzzi, L., Kroemer, G., 2015. Unsaturated fatty acids induce noncanonical autophagy. EMBO J. 34, 1025–1041. Ohsumi, Y., 2014. Historical landmarks of autophagy research. Cell Res. 24, 9–23. Orsi, A., Razi, M., Dooley, H.C., Robinson, D., Weston, A.E., Collinson, L.M., Tooze, S.A., 2012. Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol. Biol. Cell. 23, 1860–1873. Parekh, V.V., Wu, L., Boyd, K.L., Williams, J.A., Gaddy, J.A., Olivares-Villagomez, D., Cover, T.L., Zong, W.X., Zhang, J., Van Kaer, L., 2013. Impaired autophagy, defective T cell homeostasis, and a wasting syndrome in mice with a T cell-specific deletion of Vps34. J. Immunol. 190, 5086–5101. Polson, H.E., de Lartigue, J., Rigden, D.J., Reedijk, M., Urbe, S., Clague, M.J., Tooze, S.A., 2010. Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. Autophagy 6, 506–522. Proikas-Cezanne, T., Codogno, P., 2011. Beclin 1 or not Beclin 1. Autophagy 7, 671–672. Radoshevich, L., Murrow, L., Chen, N., Fernandez, E., Roy, S., Fung, C., Debnath, J., 2010. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell 142, 590–600. Ragazzoni, Y., Desideri, M., Gabellini, C., De Luca, T., Carradori, S., Secci, D., Nescatelli, R., Candiloro, A., Condello, M., Meschini, S., Del Bufalo, D., Trisciuoglio, D., 2013. The thiazole derivative CPTH6 impairs autophagy. Cell Death Dis. 4, e524. Ravikumar, B., Moreau, K., Jahreiss, L., Puri, C., Rubinsztein, D.C., 2010. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat. Cell Biol. 12, 747–757. Roberts, R., Ktistakis, N.T., 2013. Omegasomes: PI3P platforms that manufacture autophagosomes. Essays Biochem. 55, 17–27. Rodriguez, A.E., Lopez-Crisosto, C., Pena-Oyarzun, D., Salas, D., Parra, V., Quiroga, C., Morawe, T., Chiong, M., Behl, C., Lavandero, S., 2016. BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism. Autophagy 12, 287–296. Rubinsztein, D.C., Codogno, P., Levine, B., 2012. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat. Rev. Drug Discov. 11, 709–730.

22

N. Dupont et al.

Russell, R.C., Tian, Y., Yuan, H., Park, H.W., Chang, Y.Y., Kim, J., Kim, H., Neufeld, T.P., Dillin, A., Guan, K.L., 2013. ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase. Nat. Cell Biol. 15, 741–750. Sanchez-Wandelmer, J., Ktistakis, N.T., Reggiori, F., 2015. ERES: sites for autophagosome biogenesis and maturation? J. Cell Sci. 128, 185–192. Scarlatti, F., Maffei, R., Beau, I., Codogno, P., Ghidoni, R., 2008. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells. Cell Death Differ. 15, 1318–1329. Shibutani, S.T., Yoshimori, T., 2014. A current perspective of autophagosome biogenesis. Cell Res. 24, 58–68. Smith, D.M., Patel, S., Raffoul, F., Haller, E., Mills, G.B., Nanjundan, M., 2010. Arsenic trioxide induces a beclin-1-independent autophagic pathway via modulation of SnoN/SkiL expression in ovarian carcinoma cells. Cell Death Differ. 17, 1867–1881. Stadel, D., Millarte, V., Tillmann, K.D., Huber, J., Tamin-Yecheskel, B.C., Akutsu, M., Demishtein, A., Ben-Zeev, B., Anikster, Y., Perez, F., Dotsch, V., Elazar, Z., Rogov, V., Farhan, H., Behrends, C., 2015. TECPR2 cooperates with LC3C to regulate COPIIdependent ER export. Mol. Cell 60, 89–104. Starr, T., Child, R., Wehrly, T.D., Hansen, B., Hwang, S., Lopez-Otin, C., Virgin, H.W., Celli, J., 2012. Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle. Cell Host Microbe 11, 33–45. Subramani, S., Malhotra, V., 2013. Non-autophagic roles of autophagy-related proteins. EMBO Rep. 14, 143–151. Tian, S., Lin, J., Jun Zhou, J., Wang, X., Li, Y., Ren, X., Yu, W., Zhong, W., Xiao, J., Sheng, F., Chen, Y., Jin, C., Li, S., Zheng, Z., Xia, B., 2010. Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound, Z18. Autophagy 6, 1032–1041. Vicinanza, M., Korolchuk, V.I., Ashkenazi, A., Puri, C., Menzies, F.M., Clarke, J.H., Rubinsztein, D.C., 2015. PI(5)P regulates autophagosome biogenesis. Mol. Cell 57, 219–234. Weidberg, H., Shvets, E., Elazar, Z., 2011. Biogenesis and cargo selectivity of autophagosomes. Annu. Rev. Biochem. 80, 125–156. Wilkinson, D.S., Jariwala, J.S., Anderson, E., Mitra, K., Meisenhelder, J., Chang, J.T., Ideker, T., Hunter, T., Nizet, V., Dillin, A., Hansen, M., 2015. Phosphorylation of LC3 by the Hippo kinases STK3/STK4 is essential for autophagy. Mol. Cell 57, 55–68. Willinger, T., Flavell, R.A., 2012. Canonical autophagy dependent on the class III phosphoinositide-3 kinase Vps34 is required for naive T-cell homeostasis. Proc. Natl. Acad. Sci. USA 109, 8670–8675. Wirth, M., Joachim, J., Tooze, S.A., 2013. Autophagosome formation-The role of ULK1 and Beclin1-PI3KC3 complexes in setting the stage. Semin. Cancer Biol. 25, 301–309. Wong, C.H., Iskandar, K.B., Yadav, S.K., Hirpara, J.L., Loh, T., Pervaiz, S., 2010. Simultaneous induction of non-canonical autophagy and apoptosis in cancer cells by ROS-dependent ERK and JNK activation. PLoS ONE 5, e9996. Yamamoto, H., Kakuta, S., Watanabe, T.M., Kitamura, A., Sekito, T., Kondo-Kakuta, C., Ichikawa, R., Kinjo, M., Ohsumi, Y., 2012. Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J. Cell Biol. 198, 219–233. Yan, H., Li, W.L., Xu, J.J., Zhu, S.Q., Long, X., Che, J.P., 2013. D2 dopamine receptor ant agonist raclopride induces non-canonical autophagy in cardiac myocytes. J. Cell. Biochem. 114, 103–110. Yang, Z., Klionsky, D.J., 2010. Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814–822. Youle, R.J., Narendra, D.P., 2011. Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14.

Molecular Mechanisms of Noncanonical Autophagy

23

Yu, L., McPhee, C.K., Zheng, L., Mardones, G.A., Rong, Y., Peng, J., Mi, N., Zhao, Y., Liu, Z., Wan, F., Hailey, D.W., Oorschot, V., Klumperman, J., Baehrecke, E.H., Lenardo, M.J., 2010. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946. Zavodszky, E., Vicinanza, M., Rubinsztein, D.C., 2013. Biology and trafficking of ATG9 and ATG16L1, two proteins that regulate autophagosome formation. FEBS Lett. 587, 1988–1996. Zhou, X., Wang, L., Hasegawa, H., Amin, P., Han, B.X., Kaneko, S., He, Y., Wang, F., 2010. Deletion of PIK3C3/Vps34 in sensory neurons causes rapid neurodegeneration by disrupting the endosomal but not the autophagic pathway. Proc. Natl. Acad. Sci. USA 107, 9424–9429. Zhu, J.H., Horbinski, C., Guo, F., Watkins, S., Uchiyama, Y., Chu, C.T., 2007. Regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl4-phenylpyridinium-induced cell death. Am. J. Pathol. 170, 75–86.

CHAPTER TWO

Epigenetic Control of Gene Expression in Maize J. Huanga, J.S. Lynna, L. Schultea, S. Vendramina, K. McGinnis* Department of Biological Science, Florida State University, Tallahassee, FL, United States

*Corresponding author E-mail address: [email protected]

Contents 1. Introduction 2. McClintock’s Bequest: Transposable Elements Form the Foundation of Epigenetics and Come Back Into Focus in the “Omics” Era 3. The Battle Between two Genomes: Maternal and Paternal Influences on Gene Expression 4. A Puzzling and Illuminating Phenomenon: Paramutation as a Model to Study Transcriptional Silencing in Maize 5. Putting it all Together: Mutants Replace the Mystery With Mechanism 5.1 MOP1 5.2 MOP2 5.3 MOP3 5.4 RMR1 5.5 RMR2 5.6 Unstable for Orange 1 (Ufo) 5.7 Transgene Reactivated (Tgr) 6. Location, Location, Location, and Know Your Neighbors: Real Estate Rules Apply to Genomes for Cytosine Methylation and Gene Expression 7. Cultivating Diversity: Differentially Methylated Regions Coincide With Inherited Phenotypic Diversity 8. Having the Right Tool for the Job: Diversified “RNA-Directed DNA-Methylation” May Have Emerged in Conjunction With the Complex Maize Genome 9. Maize Epigenetics in the Postgenomics Era References

26 26 31 33 34 35 36 37 38 38 38 39 39 41 42 43 44

Abstract Epigenetic gene regulation is important for proper development and gene expression in eukaryotes. Maize has a large and complex genome that includes abundant

a

These authors contributed equally to this work.

International Review of Cell and Molecular Biology, Volume 328 ISSN 1937-6448 http://dx.doi.org/10.1016/bs.ircmb.2016.08.002

© 2017 Elsevier Inc. All rights reserved.

25

26

J. Huang et al.

repetitive sequences which are frequently silenced by epigenetic mechanisms, making it an ideal organism to study epigenetic gene regulation. Epigenetic modifications are chromosome-bound, heritable changes to the genome that do not affect the DNA sequence, and can include DNA methylation, histone modification, and RNA processing. Our appreciation and understanding of epigenetic regulation has grown with the field since its inception ∼65 years ago. Early examples of epigenetic regulation were often associated with transposable elements, starting with McClintock’s early work in the 1950s. The observation of other intriguing phenotypes segregating in non-Mendelian ratios in the 1950s provided material for genetic screens that allowed for mechanistic studies of epigenetic regulation that have come to fruition within the past 20 years. The relationship between epigenetic mechanisms and genome organization has become clear with the application of new technologies to characterize maize epigenomes. Our understanding of epigenetic control of gene expression now encompasses the context of genes relative to DNA methylation, chromatin structure, and transposable element content.

1. INTRODUCTION Maize (Zea mays) has been a model organism for genetics and molecular biology for more than a 100 years. It is well studied in genetic mapping, cytogenetics, and epigenetics (Freeling and Walbot, 1996). As with any other multicellular eukaryote, normal growth, development, and environmental response in maize is dependent upon a carefully orchestrated program of gene expression. This program is regulated by a number of overlapping and interdependent mechanisms, including genetic and epigenetic pathways. Maize is an excellent model for studying epigenetic regulation of gene expression, because it has a large genome with a high percentage of transposable elements (TEs), it exhibits known types of epigenetic regulation of gene expression, and there are many reported epigenetic phenomena to focus mechanistic studies on. This review considers the role that epigenetic regulatory mechanisms play in influencing gene expression in maize, starting with the earliest examples through recent studies that take advantage of major technological advances in genomics.

2. McCLINTOCK’S BEQUEST: TRANSPOSABLE ELEMENTS FORM THE FOUNDATION OF EPIGENETICS AND COME BACK INTO FOCUS IN THE “OMICS” ERA Mechanistic studies of epigenetic regulation in Arabidopsis thaliana have substantially advanced the field in recent years. A tradition of careful

Epigenetic Control of Gene Expression in Maize

27

observation and thorough genetic analysis in maize has contributed greatly to the field, as well, providing well documented epigenetic phenomena associated with these mechanisms. In recent years, epigenomic analysis of maize lines has illustrated the impact of these mechanisms in large, repetitive genomes, and revealed important similarities and differences with observations in smaller genomes (Li et al., 2014). Plant epigenetics benefited greatly from the pioneering work of Dr. Barbara McClintock and other maize geneticists. By using pigmentation markers in maize that affect kernel color, Dr. Barbara McClintock discovered that a locus on chromosome 9, which she named Dissociation (Ds) could transpose or move to other locations and change kernel phenotype (McClintock, 1950, 1983). Her discoveries and interpretations were among the most important in the 20th century and demonstrated the powerful contributions to science that can be made using maize as a genetic research model. McClintock’s description of “jumping genes,” now known as TEs, contradicted the long-term assumption that genes remain in fixed locations on chromosomes, and her work was not immediately appreciated. The concept of mobile genetic elements was also controversial because active transposons would be anticipated to be very harmful to host genomes, casting some doubt on their existence or prevalence. In spite of their destructive potential, transposons are abundant in most genomes and make up 85% of maize genome (Schnable et al., 2009). McClintock’s work led to the discovery of transposon silencing, an epigenetic mechanism to minimize transposition and genomic disruption by TEs. Molecular characterization of some families of maize TEs revealed a high level of DNA methylation and silencing at transcriptional level, whereas TE activation is often associated with hypomethylation (Chomet et al., 1987; Chandler and Walbot, 1986). One example is the Suppressor-mutator (Spm) transposons first identified by McClintock (1957). After it was cloned (Masson et al., 1987), subsequent studies showed that the 0.2 kb promoter region and the 0.35 kb downstream control region (DCR) next to the promoter are critical for the Spm regulation. When Spm is inactive, the promoter and DCR are methylated; when Spm is activated, these two regions are demethylated (Schla¨ppi et al., 1994). It has also been shown that the DCR is required for promoter de novo methylation and prevents activation of Spm by an upstream enhancer (Schla¨ppi et al., 1994; Raina et al., 1993). Similar correlations between DNA methylation and transposon activity were observed for activator and mutator TEs (Chomet et al., 1987; Chandler and Walbot, 1986).

28

J. Huang et al.

These discoveries were initial indications of the importance of epigenetic mechanisms in transposon silencing. An early indication of the relationship between epigenetic regulation of TEs and gene expression was discovered shortly thereafter. Epigenetic silencing of transposons can mediate suppressible mutations caused by TEs, allowing plants with the same TE-associated alleles to exhibit disparate phenotypes. The suppressible hcf106-mum1 allele has a nonantonomous Mu1 element in the promoter of high chlorophyll £uorescence106 (hcf106) gene which is required for chloroplast development (Martienssen et al., 1990). The mutant shows a lethal pale phenotype because of chloroplast development defects. However, the mutant phenotype is only exhibited if an active autonomous MuDR element is present in the genome. Without MuDR, the Mu1 element inserted in the promoter will be highly methylated, and an outward reading promoter at the end of Mu1’s terminal inverted repeat will transcribe the functional hcf106 transcript (Barkan and Martienssen, 1991). On the contrary, in the presence of an active autonomous MuDR, the Mu1 element stays unmethylated and no hcf106 mRNA accumulates, potentially due to transcriptional interference between the hcf106 gene promoter and the promoter in the terminal inverted repeat region of Mu1 element (Martienssen, 1996). Consistent with a heritable, epigenetic regulatory event, methylation status was correlated with phenotype in clonal sectors, suggesting that cell methylation status in the shoot apical meristem was inherited through cell divisions, along with gene expression level and associated phenotype (Martienssen and Baron, 1994). One important pathway to silence transposons in plants is RNA-directed DNA methylation (RdDM; Fig. 1), which involves RNA polymerase IV transcription of TE regions to generate single-stranded RNA (sRNA) which serve as a substrate for an RNA-dependent RNA polymerase (RDR2) to generate double-stranded RNA(dsRNA) [reviewed by Matzke and Mosher (2014); Matzke et al. (2015)]. Some dsRNAs can also be produced from retrotransposons in nested arrays (Meyers et al., 2001) or natural antisense transcription. Once dsRNAs are synthesized, they act as substrates for dicerlike proteins to generate small RNAs that can, in turn, trigger transcriptional or posttranscriptional silencing. A naturally occurring trigger of heritable transposon silencing in maize is the Mu killer (Muk) locus (Slotkin et al., 2005). Muk includes an inverted duplication of a partial MuDR element (Fig. 2). RNAs transcribed from this locus can form hairpin structures and then be cut into 24 nt siRNAs by dicer-like proteins, which subsequently trigger RdDM and silence MuDR

Epigenetic Control of Gene Expression in Maize

29

Figure 1 A schematic overview of the RNA-directed DNA methylation (RdDM) pathway in maize. The gene identification numbers are designated in this legend for proteins with confirmed subunit composition as described by proteomic data (Haag et al., 2014). Canonical activity of this pathway is thought to include RNA polymerase IV (Pol IV) transcription at target loci to generate ssRNAs. MOP3 (gene identification number: GRMZM2G007681) and MOP2 (gene identification number: GRMZM2G054225) are the two largest subunits of Pol IV in maize. An RNA-dependent RNA polymerase, MOP1 (gene identification number: GRMZM2G042443), associates with Pol IV and transforms the ssRNA into a double-stranded RNA (dsRNA). ZmDCL3 cleaves the dsRNA molecule to form a single 24 nt small interfering RNA (siRNA) molecule. An argonaute protein, like AGO121 (gene identification number: GRMZM2G347402), interacts with the siRNA and may mediate interactions between the single-stranded siRNA, the scaffolding RNA from Pol V, and chromatin modifying proteins. RNA polymerase V (Pol V), which includes either MOP2a (gene identification number: GRMZM2G054225), b (gene identification number: GRMZM2G427031), or c (gene identification number: GRMZM2G133512) transcribes a single-stranded RNA molecule at the target locus to scaffold regulatory proteins.

elements in the same genome. Two maize proteins that interact with PolIV, required to maintain repression1 (RMR1) and mediator of paramutation1 (MOP1), are required for MuDR silencing establishment (Hale et al., 2009) by Muk and maintenance of silencing, respectively (Woodhouse et al., 2006; Lisch et al., 2002). It is possible that MOP1 is required for maintenance, but not establishment, of silencing because transcription of the Muk locus does not required RNA-dependent RNA polymerase activity to generate a dsRNA (Fig. 2). TEs may also play an important role in modifying gene expression related to stress response through epigenetic regulation. In maize, a comparison between temperate, drought sensitive B73 and tropical, drought resistant CIMBL55 by genome-wide association study identified an indel that

30

J. Huang et al.

Figure 2 A model for MuDR silencing by MuK. (A) MuDR has terminal inverted repeats at each end of the element (TIRA and TIRB). A fragment of mudrAis present in MuK (red/black arrow). (B) MuK includes an inverted repeat fragment (red/black arrows), which includes a deletion derivtative of mudrA. (C). A model for silencing is proposed based upon the genetic structure of MuK and MuDR, and the required transacting factors for silencing of MuDR by MuK (Woodhouse et al., 2006). The inverted repeat structure of MuK provides self-complementarity in the ssRNA that allows the RNA to interact and form a dsRNA. The dsRNA can act as a substrate for RNAses, which generate siRNAs that can mediate methylation and silencing of MuDR. Establishment of silencing requires RMR1, most likely in association with other proteins. Maintenance of silencing requires MOP1, likely in association with a MOP1:Pol IV complex.

appeared to regulate gene expression of a critical regulator of drought tolerance genes (Mao et al., 2015). An 82-bp miniature inverted repeat TE (MITE) was present in promoter of ZmNAC111 of B73 plants and absent in CIMBL55. Cytosine methylation in the CHH sequence context (where H is equivalent to A, T, or C) was greatly increased within the promoter of the gene in B73, particularly in the region of the MITE. In the plants with a highly methylated promoter, there was also significantly reduced expression and increased repressive histone marks (Mao et al., 2015;,Fig. 3). In this example, presence of a TE is associated with transcriptional repression. Conversely, TE insertions within and nearby genes can be associated with increased expression, perhaps though the generation of novel cis-acting enhancer elements (Makarevitch et al., 2015; Mao et al., 2015). Emerging evidence for the repurposing of TEs for controlling expression of nearby genes suggests that this genomic function may be common

Epigenetic Control of Gene Expression in Maize

31

Figure 3 ZmNAC111 encodes a transcription factor that controls the expression of some drought stress response genes. In the drought tolerant tropical maize line (CIMBL55), ZmNAC111 is expressed. In the drought sensitive temperate maize line (B73), a miniature inverted repeat transposable element (MITE) indel exists within the promoter region of the ZmNAC111 gene which is subject to methylation (M) and associated with low levels of ZmNAC111 expression (Mao et al., 2015).

among plants (Grandbastien et al., 2005; Naito et al., 2009; Pecinka et al., 2010; Mao et al., 2015; Makarevitch et al., 2015). In maize, it appears that specific TE families are activated upon stress-specific stimuli and control the increased expression of specific genes. This TE-associate stress responsiveness is generally conserved between inbred genotypes (Makarevitch et al., 2015). There may be other, indirect mechanisms by which TEs influence expression of distal genes, perhaps in association with changes in chromatin structure. The collective impact of TEs on gene expression by all mechanisms likely have implications in plant adaptation and evolution [reviewed by Lisch (2013)]. The discovery of the Muk locus and the trans-acting requirements of TE silencing provide insight into the epigenetic mechanisms of silencing of TEs. Intriguingly, there are also many protein coding genes that are subject to the same regulatory elements as TEs but do not seem to be influenced by neighboring TEs. A subset of these epigenetically regulated genes is silenced in association with paramutation or genomic imprinting.

3. THE BATTLE BETWEEN TWO GENOMES: MATERNAL AND PATERNAL INFLUENCES ON GENE EXPRESSION Genomic imprinting is an example of a parent-of-origin effect on gene expression [reviewed by Ferguson-Smith (2011); Alleman and

32

J. Huang et al.

Doctor (2000); Gehring (2013)]. Genomic imprinting was initially described for the r1 gene in maize (Kermicle, 1970). Later studies in mammals improved the mechanistic understanding of genomic imprinting (McGrath and Solter, 1984; Surani et al., 1984). Chromosomal clusters and specific control sequences like the imprinting control regions (ICRs) in mammals (Sutcliffe et al., 1994; Wutz et al., 1997) have not yet been identified in plants, but imprinting in plants appears to involve DNA methylation and histone modifications as it does in mammals (Choi et al., 2002; Danilevskaya et al., 2003; Haun et al., 2007; Gutie´rrez-Marcos et al., 2006; Haun and Springer, 2008). In plants, the first example of an imprinted gene was red1(r1) gene, which is also subject to paramutation (Chandler et al., 2000; Brink et al., 1996). The r1 locus has a complex structure for some alleles, and R1 expression leads to an accumulation of pigmentation in aleurone cells of the endosperm. Two epigenetically regulated alleles are R-r:standard, which produces fully red seeds, and r-g which produces colorless seeds (Kermicle, 1970). When R-r: standard is used as a maternal parent in a cross with r-g, the F1 offspring show the same red kernel phenotype as R-r:standard; however, when R-r:standard is used as a paternal parent in a cross with r-g, the F1 offspring show a mottled kernel phenotype due to paternal imprinting and partial silencing of R-r: standard. So far, there are at least 11 imprinted genes in maize that have been studied on an individual basis [reviewed by Raissig et al. (2011)]. Although the specific pattern may vary, differentially methylated regions have been associated with several of these imprinted genes (Jahnke and Scholten, 2009; Gutie´rrez-Marcos et al., 2006; Hermon et al., 2007; Haun et al., 2007). Genome-wide survey suggests that as many as 179 genes may be imprinted in maize, and the relationship between gene expression and DNA methylation is ambiguous for many of these genes (Waters et al., 2011, 2013; Wang et al., 2015). In the early development of maize seeds after fertilization, small RNAs are found to be parentally imprinted within the endosperm and might also influence expression of other imprinted genes, suggesting that siRNAs may play an important role in determining expression of critical genes in the developing zygote via parent-of-origin related mechanisms (Xin et al., 2014). The seed is a significant stage of plant development both agriculturally and biologically, meaning that epigenetic regulation is of interest in basic and applied research.

Epigenetic Control of Gene Expression in Maize

33

4. A PUZZLING AND ILLUMINATING PHENOMENON: PARAMUTATION AS A MODEL TO STUDY TRANSCRIPTIONAL SILENCING IN MAIZE Another example of epigenetic regulation of gene expression is paramutation, the trans-homolog interaction between two alleles that can induce a heritable change in gene expression. This phenomena has been most extensively studied in maize, including the earliest known examples described more than 60 years ago (Brink, 1956; Coe, 1959). Since these original descriptions of paramutation, maize research has provided a useful model to study paramutation and allowed scientists to describe the properties of paramutation and propose mechanistic models. Paramutation was first discovered at the red1 (r1) locus in maize (Brink, 1956), and at booster1 (b1) shortly thereafter (Coe, 1959). Both r1 and b1 encode basic helix-loop-helix transcription factors involved in the anthocyanin synthesis pathway, such that R1 and B1 plants exhibit visible pigment accumulation. A gene encoding another transcriptional factor, purple plant1 (pl1) was also shown to be subject to paramutation (Hollick et al., 1995). All three transcription factors induce gene expression and lead to pigmentation in plants, but each acts predominantly in distinct tissues. The activity of B1 is highest in the stalk and internode (Patterson et al., 1993; Dorweiler et al., 2000), R1 is most active in the aleurone layer of seeds (Brink and Mikula, 1958), and PL1 is active in the plant body and anthers (Hollick et al., 1995, 2005). Additional examples of paramutation in maize have been described at pericarp color1 (p1) and low phytic acid1 (lpa1) (Sidorenko and Peterson, 2001; Pilu et al., 2009). Paramutation at B1 is one of the most studied examples of paramutation (Coe, 1959). Similar to other examples, B1 paramutation involves two alleles, which are the paramutable, highly expressed B-I allele and the paramutagenic, transcriptionally repressed B’ allele. Because B1 activates anthocyanin synthesis, B-I plants exhibit a deeply pigmented (red) stalk phenotype, while B’ plants have a speckled, pale red pigment phenotype. Crosses between homozygous B-I and B’ plants result in F1 plants phenotypically similar to B’/B’ plants. If the F1 is crossed again with a homozygous B-I plant, the resulting progeny will again all be phenotypically similar to B’, because the original B-I allele has been paramutated to B’. Crosses between either B-I or B’ and a neutral allele, such as B-Peru or b, result in

34

J. Huang et al.

progeny with a B-I or B’ phenotype because the neutral alleles have no paramutability or paramutagenicity and are recessive to the other two alleles. Despite significant phenotypic differences, there are no genetic differences between B-I and B’ (Patterson et al., 1993). However, there are DNA methylation and chromatin changes between the B’ and B-I state (Stam et al., 2002a,b; Louwers et al., 2009), and these differences are transmitted with the expression changes (Patterson et al., 1993). Most of the chromatin differences between B-I and B’ are detectable at a 6-kb region residing 100 kb upstream of b1 transcriptional start site, at a locus that is necessary and sufficient for paramutation (Stam et al., 2002a,b; Louwers et al., 2009; Belele et al., 2013). This region is composed of seven tandem repeats, each of which is 853 bp. Both B’ and B-I allele have seven repeats while neutral alleles have one repeat. This region also functions as an enhancer element for B1 transcription (Stam et al., 2002a,b; Belele et al., 2013). In addition to being a fascinating phenomenon, loci exhibiting paramutation have provided phenotypes that proved useful in forward genetics screens to identify many trans-factors required for paramutation-associated gene silencing.

5. PUTTING IT ALL TOGETHER: MUTANTS REPLACE THE MYSTERY WITH MECHANISM Using genetic screens for release of silencing at b1 and pl1, respectively, the mediator of paramutation (mop) and required for maintenance of repression (rmr) mutants were isolated (Dorweiler et al., 2000; Hollick and Chandler, 2001; Hollick et al., 2005; McGinnis et al., 2006; Hale et al., 2007; Sidorenko and Chandler, 2008; Sidorenko et al., 2009; Stonaker et al., 2009; Barbour et al., 2012). Additional analysis revealed that trans-acting factors identified in these screens were also required for transcriptional silencing of a transgene (BTG-silent), and for the silencing associated promoter methylation (McGinnis et al., 2006). BTG-silent was exploited in a separate forward genetic screen, which resulted in the transgenereactivated(tgr) mutants (Madzima et al., 2011). Unstable factor orange1 (Ufo1) was originally identified based upon its unstable pigmentation phenotype (Styles and Ceska, 1977; Styles et al., 1987), and then later shown to be required for paramutation and silencing at b1 and p1 (Sekhon et al., 2012). Since their initial discovery, many of these genes have been cloned, and their molecular

35

Epigenetic Control of Gene Expression in Maize

Table 1 Maize mutants defective in epigenetic silencing. Most similar Arabidopsis Putative protein protein function Gene

Mediator of RDR2 paramutation1 (MOP1) MOP2/Required to NRPD2/ maintain repression 7 NRPE2a (RMR7) RMR1

CLSY1

RMR2

—

MOP3/RMR6

NRPD1

RNA-dependent RNA polymerase DNA-dependent RNA polymerase

SNF2-like chromatin remodeler — DNA-dependent RNA polymerase

References

Dorweiler et al. (2000); Alleman et al. (2006) Sidorenko et al. (2009) Stonaker et al. (2009) Hale et al. (2007, 2009) Barbour et al. (2012) Hollick et al. (2005) Sloan et al. (2014)

identity and phenotypes provide insight into epigenetic mechanisms for gene silencing and paramutation (Table 1). Despite the fact that these genes were identified through multiple genetic screens, all of the mutants exhibit phenotypes associated with epigenetic regulation of gene expression. Many of the cloned genes are highly similar to proteins in other organisms known or predicted to produce or respond to noncoding RNAs to regulate gene expression. The identities of the cloned genes along with the phenotypes associated with each mutant indicate an important role of RNA-directed gene silencing in genome regulation and gene expression profiles. Many of these mutants have pleoiotropic phenotypes, suggesting that the proteins they encode act as epigenetic regulators that have genomewide function not limited to paramutation. These mutants provide important tools for understanding how siRNA-mediated regulation of gene expression supports growth, development, and environmental responses in plants.

5.1 MOP1 Mop1 is required for paramutation in b1, r1, and pl1 genes (Dorweiler et al., 2000) and encodes a predicted RNA-dependent RNA polymerase similar to AtRDR2 (Alleman et al., 2006) that physically interacts with Pol IV

36

J. Huang et al.

(Ream et al., 2009). MOP1 interacts with Pol IV in maize (Haag et al., 2014). Mutations in Mop1 can result in the reduction of 24 nt siRNA and the release of silencing of some classes of TEs (Jia et al., 2009). Additionally, mop1 mutants exhibit variable, pleiotropic developmental phenotypes observed as small and unhealthy plants with late inflorescence development (Dorweiler et al., 2000). These developmental phenotypes resemble epimutations in unlinked genes that are similar to other known chromatin mutants, and suggest that MOP1 may function to regulate the expression of genes at multiple loci in the maize genome. However, misregulation of gene expression in mop1-1 plants may only be partially caused by the direct effects of a loss of MOP1 function, because there is not a strong and consistent association between MOP1-dependent siRNAs and changes in gene expression (Labonne et al., 2013; Madzima et al., 2014). Genome wide methylation profiles suggest that MOP1 may regulate transcription of genes in close proximity to certain genomic contexts that are associated with boundaries between heterochromatic, highly repetitive regions and gene rich regions on maize chromosome arms (Li et al., 2015a,b). Evidence of downregulation of 19 stress responsive genes in maize mop1-1 mutants has been shown (Madzima et al., 2014), and might indicate the presence of a possible transcriptional regulatory pathway in response to stress at some loci. Collectively, this provides evidence that MOP1 is part of an essential regulatory pathway that broadly influences gene expression in the maize genome.

5.2 MOP2 Mop2 is one of three maize genes that encode a conserved protein that is homologous to NRPD2/E2 (Sidorenko et al., 2009), which is incorporated as the second largest subunit of Arabidopsis RNA Polymerase IVand V (Ream et al., 2009). In maize, all three NRPD2/E2 proteins appear to interact with some Pol IV and V complexes (Haag et al., 2014). Mop2 is allelic with rmr7 (Stonaker et al., 2009). Mop2-1 is a dominant mutation with respect to preventing paramutation at B1, but is recessive in the release of previously silenced alleles (Sidorenko et al., 2009). Two alleles of Mop2 were isolated from an EMS mutagenesis, including Mop2-1 with a G → A nucleotide change in exon 7, a highly conserved domain among polymerases. The Mop2-2 allele has a single nucleotide change in exon 6 affecting a conserved domain, different from that affected in Mop2-1. MOP2 is required for some, but not all examples of transgene-induced silencing when the transgene is

Epigenetic Control of Gene Expression in Maize

37

anticipated to generate the precursor molecule for siRNA biogenesis. Such a transgene is anticipated to bypass the function of Pol IV and MOP1, and under these conditions a requirement for MOP2 is a likely indication of a requirement for Pol V (Sloan et al., 2014). MOP2 is also differentially required for paramutation at B1, Pl1, P1, and R1, suggesting that the functional dependence on MOP2-containing polymerase complexes may vary between these different examples of paramutation (Sidorenko et al., 2009). The mop2-1 mutant has been useful in generating models to explain interactions between Pol II, Pol IV, and Pol V in transcriptional silencing. Although rmr7 is allelic to mop2, rmr7 mutants do not exhibit identical phenotypes to mop2 mutants. Notably, rmr7 mutants are recessive, unlike Mop2-1, and do not exhibit the developmental phenotypes reported for Mop2 mutants. rmr7 mutants appear to be deficient in establishment but not in meiotically-transmittable maintenance of B and Pl silencing (Stonaker et al., 2009). This may be evidence for allele specific effects, with Mop2-1 having more pleiotropic phenotypes associated with a dominant negative interaction with multiple types of polymerase complexes. Additional comparative analysis will be required with the different alleles to fully understand these differences. Like mop1-1,Mop2-1 homozygous plants exhibit a global reduction in 24 nt siRNAs, and variable, pleiotropic developmental phenotypes. This suggests a role for MOP2 in regulating expression of multiple genes.

5.3 MOP3 Mop3 (Sloan et al., 2014) is allelic to Rmr6 and encodes the largest subunit of RNA Polymerase IV. MOP3/RMR6 is required for acquisition of meiotically heritable paramutant states at the pl1, b1, and r1 loci in maize (Hollick et al., 2005). While rmr6-1 and rmr6-2 mutant plants exhibit delayed flowering, flowering abnormalities, and short stature (Parkinson et al., 2007), mop3 plants do not consistently exhibit these phenotypes (Sloan et al., 2014). This may be an allele specific effect, as rmr6-2 appears to be a null allele (Hollick et al., 2005) and mop3-1 is a recessive allele with a G → A mutation within the acceptor site of producing a premature stop codon. Like MOP1, RMR6 appears to be associated with the maintenance of gene expression patterns at a wide range of genes in the maize genome, including abiotic stress responsive genes (Lunardon et al., 2016). Also like MOP1, it is likely that loss of RMR6 triggers both primary and secondary responses in the genome, or has noncanonical gene regulatory effects because

38

J. Huang et al.

changes in gene expression are not always associated with the anticipated hallmarks of RdDM-like regulation.

5.4 RMR1 The RMR screen identified factors involved with maintaining specific chromatin structures that inhibit pl1 transcription, which leads to lightly pigmented plants due to Pl1-regulation of anthocyanin biosynthetic genes. Rmr6 and Rmr7 are allelic with other mutants and have already been discussed, but Rmr1 and Rmr2 were uniquely identified by this screen. Unlike rmr6, mutations in rmr1 and rmr2 do not cause gross developmental abnormalities and do not seem to be involved in developmental gene control mechanisms; only gene silencing phenotypes have been reported for these mutants (Hollick and Chandler, 2001; McGinnis et al., 2006). Rmr1 encodes an SNF2 domain-containing protein CLASSY 4-like protein and affects methylation of cytosines and the accumulation of sRNAs of a proximal transposon at the Pl1-Rhoades allele (Hale et al., 2007). It has been reported previously that alteration of Pl1-Rhoades RNA transcript levels are independent of its transcription rate, suggesting that this pathway acts downstream of transcriptional regulation at this locus. rmr1 mutant alleles include four recessive, EMS-induced mutations, which result in darkly pigmented plants due to a loss of Pl1 repression (Hale et al., 2007).

5.5 RMR2 RMR2 (Barbour et al., 2012) is required to maintain silencing of Pl and for establishment of paramutation at Pl-Rh. The protein is not required for establishment of r1 paramutation. While in silico approaches indicate some structural similarities with known DNA-binding proteins in humans and mycobacterium, high confidence protein domains or orthologous proteins were not identified for this protein. Consistent with a role in RNA-mediated gene silencing, homozygous rmr2-1 mutants have reduced 24 nt siRNAs, and exhibit hypomethylation at Pl, but the molecular function of RMR2 has not yet been reported.

5.6 Unstable for Orange 1 (Ufo) Ufo1 encodes a modifier that has yet to be cloned. The dominant Ufo1-1 allele is associated with the upregulation of P1, a loss of cytosine methylation from P1-wr and an increase in phlobaphene pigmentation in plant organs like silk, tassel, husk, and leaf sheath (Chopra et al., 2003). Ufo1 induced phenotypes

Epigenetic Control of Gene Expression in Maize

39

present incomplete penetrance and have poor expressivity as seen in the variable phenotype of some of the progeny carrying the mutation (Sekhon et al., 2012). Ufo1 appear to act in a tissue-specific manner and to remodel the chromatin structure of Pl-wr and possibly other loci (Chopra et al., 2003).

5.7 Transgene Reactivated (Tgr) MOP1, RMR1, and RMR2 are required for transcriptional silencing of a transgene that includes the B1 coding region (McGinnis et al., 2006). A forward genetic screen of EMS mutagenized M2s based on reactivation of BTG-silent allowed for the identification of multiple genes (Tgr1,Tgr2,Tgr3, Tgr4,Tgr6,Tgr9, and Tgr11) required for transcriptional silencing (Madzima et al., 2011). Further analysis of these mutants indicates that Tgr1 resides on chromosome 1 and is linked to Rmr6 (Madzima et al., 2014), while other mutations from this screen appear to represent alleles of uncloned genes in maize. The results of this screen, along with the results discussed for other maize mutants, indicate that transcriptional silencing mechanisms may be shared between paramutation, spontaneous silencing of introduced transgenes, and developmentally relevant gene expression patterns in maize. In combination with modern genomic analysis techniques, the mutants described earlier have been subject to genome wide studies of epigenetic regulation in maize. These and other genomic studies are leading to an enhanced understanding of genome wide control of expression patterns in maize.

6. LOCATION, LOCATION, LOCATION, AND KNOW YOUR NEIGHBORS: REAL ESTATE RULES APPLY TO GENOMES FOR CYTOSINE METHYLATION AND GENE EXPRESSION Differential gene expression is often associated with methylation of DNA, and until recently the prevailing idea was that increased cytosine methylation nearby and within genes correlated with reduced gene expression. Further analysis has indicated that the relationship between gene expression and DNA methylation is slightly more complex, and is context dependent. With the advent of modern DNA sequencing platforms, combining classical enzymatic characterization of methylated bases through bisulfite conversion with high-throughput DNA sequencing allows for genome-wide analysis of cytosine methylation at single base pair resolution. Many genome wide studies of cytosine methylation have been published in maize in recent years

40

J. Huang et al.

(Eichten et al., 2011; Gent et al., 2013; Ding et al., 2014; West et al., 2014; Li et al., 2015a,b; Lu et al., 2015; Sun et al., 2015; Wang et al., 2015). These studies have indicated that there is not a simple relationship between gene silencing and cytosine methylation, although some consistent patterns can be detected. In maize embryos and endosperm nine days after pollination, increased expression levels of protein coding genes, pseudogenes, and TEs are correlated with lower levels of methylation for CG and CHG (where H is A, T, or C) nucleotide contexts at the transcription start site and transcription termination site and is consistent between tissue types (Lu et al., 2015). Conversely, genome-wide studies in maize found that increased CG methylation or low CHG/CHH methylation within the body of protein coding genes is correlated with higher expression (Lu et al., 2015; West et al., 2014). Interestingly, isolated methylated CHH sequences within regions of otherwise nonmethylated bases proximal to the transcription start site within 2-kb upstream of protein coding genes is also often associated with increased transcription (Gent et al., 2013; Lu et al., 2015), suggesting a link between “CHH islands” and transcriptional regulation, perhaps involving the establishment of permissive chromatin states for transcription. Additionally, genome-wide surveys identified a local sequence effect on methylation where the +1 and +2 positions after the methylated cytosine in a CHH context was twofold more likely to be methylated when followed by an adenine as compared to when followed by another cytosine (Lu et al., 2015). This is in contrast with TEs and pseudogenes, where higher levels of methylation are more consistently negatively correlated with expression (Lu et al., 2015). This could be indicative of distinct mechanisms or context-dependent responses. The distinction between repeat rich and gene rich contexts is further illustrated when TEs near genes are compared to those embedded in gene-poor regions of the maize genome (Fig. 4). TEs within 1 kb of genes have higher levels of CHH methylation and are enriched in 24 nt siRNA compared to genome wide averages, suggesting that regulation by RdDM attracts the RdDM machinery to near-gene TEs independent of transposon type (Gent et al., 2013). In contrast, deep intergenic TEs that are in gene poor regions have a lower abundance of 24 nt siRNA and reduced CHH methylation levels suggesting that they are regulated by different mechanisms (Gent et al., 2013;,Fig. 4). The dependence on RdDM of CHH methylation appears to be enriched when transposon insertion occurs near or within a gene to maintain the repression of the introduced TE (Li et al., 2015a,b; Lu et al., 2015). The specific influence of TEs on the

Epigenetic Control of Gene Expression in Maize

41

Figure 4 Context-dependent DNA methylation at boundaries between transposable elements (TEs) and genes. Eukaryotic chromosomes include euchromatin (white) and heterochromatin (red). (A) CHH methylation is enriched in regions where genes and TEs occur within 2 kb of each other. CHH methylation in these regions appears to be dependent on the RdDM machinery and is proposed to function in the establishment and maintenance of chromatin structure that is appropriate for transcription in genic regions and silencing in TE-rich regions. (B) In contrast, TEs in gene-poor regions have high levels of CHG and CG methylation, including the regions between TEs, which may demarcate a different type of chromatin structure.

expression of neighboring genes appears to vary with the type of transposable element and is also related to chromatin context (Eichten et al., 2012).

7. CULTIVATING DIVERSITY: DIFFERENTIALLY METHYLATED REGIONS COINCIDE WITH INHERITED PHENOTYPIC DIVERSITY Considering the association between context-dependent DNA methylation, DMRs might be expected to be associated with epigenotypic variation between haplotypes. Whole-genome bisulfite sequencing data were

42

J. Huang et al.

generated from the third leaves of five distinct elite inbred maize haplotypes (B73, Mo17, CML332, Oh43, and Tx303) and many genetically identical regions that were differentially expressed between haplotypes were also found to be differentially methylated (Li et al., 2015a). The influence of DMRs on expression is more pronounced for genes that are expressed in one genotype but not the other (on/off) rather than for quantitative expression (