REVIEW

Epigenetics in C. elegans: Facts and Challenges

Dirk Wenzel,1 Francesca Palladino,2 and Monika Jedrusik-Bode1*

1Electron Microscopy Group 3 Epigenetics in C. elegans Group, Max Planck Institute for Biophysical Chemistry,Am Fabberg 11, 37077 Gottingen, Germany

2Laboratory of Molecular and Cellular Biology, University of Lyon, CNRS UMR5239,Ecole Normale Superieure, 69364 Lyon Cedex 07, France

Received 15 November 2010; Revised 14 April 2011; Accepted 15 April 2011

Summary: Epigenetics is defined as the study of herit-able changes in gene expression that are not accompa-nied by changes in the DNA sequence. Epigeneticmechanisms include histone post-translational modifi-cations, histone variant incorporation, non-codingRNAs, and nucleosome remodeling and exchange. Inaddition, the functional compartmentalization of the nu-cleus also contributes to epigenetic regulation of geneexpression. Studies on the molecular mechanismsunderlying epigenetic phenomena and their biologicalfunction have relied on various model systems, includ-ing yeast, plants, flies, and cultured mammalian cells.Here we will expose the reader to the current under-standing of epigenetic regulation in the roundwormC. elegans. We will review recent models of nuclear or-ganization and its impact on gene expression, the bio-logical role of enzymes modifying core histones, andthe function of chromatin-associated factors, with spe-cial emphasis on Polycomb (PcG) and Trithorax (Trx-G)group proteins. We will discuss how the C. elegansmodel has provided novel insight into mechanisms ofepigenetic regulation as well as suggest directions forfuture research. genesis 49:647–661, 2011. VVC 2011

Wiley-Liss, Inc.

Key words: core histones; post-translational modifications;chromatin; Hox; trithorax; PcG

INTRODUCTION

In metazoan development, cellular identity is estab-lished by tissue and lineage-specific transcription fac-tors acting in concert with epigenetic mechanisms. Dif-ferentiated cells also rely on epigenetic mechanisms inorder to transmit specific gene expression patterns that

distinguish the differentiated state through multiple celldivisions (Hemberger et al., 2009).

The basic subunit of chromatin is the nucleosome,composed of the highly conserved core histones H2A,H2B, H3, and H4. Epigenetic mechanisms can modifythis structure in several ways. Depending on the chro-mosomal context, core histones can be replaced byhistone variants, including H3.3, H2A.Z, or H2A.X,each of which is associated with specialized functions(Jin and Felsenfeld, 2007; Talbert and Henikoff, 2010).The flexible N- and C-terminal tails of core histonesare also subjected to a variety of post-translationalmodifications, including acetylation, methylation,phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, and biotinylation. These modifications,alone or in combination, correlate with transcrip-tional repression or activation (Jenuwein and Allis,2001). In addition, chromatin-associated factors suchas Trithorax (Trx-G) and Polycomb group (PcG) pro-teins function to maintain cellular memory of tran-scriptional states of developmentally important genesthrough many rounds of cell division. Transcriptionalregulation by PcG and Trx-G group proteins isachieved through both histone methyltransferaseactivities of individual components, as well as specific

*Correspondence to: Dr. Monika Jedrusik-Bode, Max Planck Institute for

Biophysical Chemistry, Epigenetics in C. elegans Group, Am Fassberg 11,

37077 Gottingen, Germany.

E-mail: mjedrus@gwdg.de

Contract grant sponsor: German National Funding Agency, Contract

grant number: JE 505/1-3, Contract grant sponsor: Max Planck Society

Published online 2 May 2011 in

Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/dvg.20762

' 2011 Wiley-Liss, Inc. genesis 49:647–661 (2011)

binding to and interpretation of these histone marks.PcG and Trx-G group proteins are also able to recruitadditional proteins that greatly expand their reper-toire of covalent histone modifications (Sparmann andvan Lohuizen, 2006).

Histone post-translational modifications are carriedout by a variety of complexes containing histone-modi-fying enzymes. Histone methyltransferases (HMTs), his-tone demethylases (HDMs), histone acetyltransferases(HATs), histone deacetylases (HDACs), and histone ki-nases not only modify histone tails, but also act asrecruiters of protein complexes and nucleosomeremodelers (Jenuwein and Allis, 2001). Histone tailmodifications are in turn associated with specific chro-matin-binding proteins or protein complexes (readers)that modify chromatin structure. Small non-codingRNAs, nucleosome exchange and remodeling by ATP-dependent complexes, and histone variant incorpora-tion, are additional mechanisms that can alter thedegree of chromatin compaction (Jenuwein and Allis,2001). In addition, nuclear organization has recentlyemerged as another important player in epigenetic regu-lation (Luger and Hansen, 2005).

This review summarizes recent models of nuclearcompartmentalization in the nematode C. elegans, anddescribes the different developmental roles attributedto histone-modifying enzymes, focusing on the initiationand maintenance of epigenetic memory. In particular,we will describe the molecular players involved in PcGand Trx-G regulation of Hox gene expression and lifespan determination.

Compartmentalization: from NuclearGenome to Epigenome

Higher-order chromatin organization results frominterplay between the compacted nuclear DNAsequence, DNA methylation, histone modifications,small RNAs, and chromatin remodeling. Nucleararchitecture is dynamically dependent on (1) celltype, (2) developmental timing (embryo/adult), (3)environmental challenges, and (4) changes in tran-scriptional activities correlated with the repositioningof genes from active to repressive nuclear compart-ments. In vivo observations have shown that in thecell nucleus, transcriptionally active DNA sequencesare organized into regions called euchromatin, andtranscriptionally silent DNA sequences in regionsknown as heterochromatin.

In C. elegans, heterochromatin can be identified byelectron microscopy as specific regions enriched in re-pressive marks: di- and tri-methylated core histone H3at lysine 9, and 27 (H3K9me2/3 and H3K27me3) (Fig.1a,b). In contrast, euchromatin is marked by core his-tone H3 di-methylated at lysine 4 (H3K4me2) (Fig. 1c).Therefore, C. elegans possesses an epigenetic landscape

very similar to that of flies and mammals. In C. elegans,transcriptionally silent regions marked by the repressivemark H3K9me3 have been found at the end of chromo-somes, including telomeres (Gerstein et al., 2010; Liuet al., 2011; Wirth et al., 2009). In many organisms,telomeric heterochromatin participates in gene repres-sion and recombination. Actively transcribed genesmoved to telomeric regions are reversibly silenced, aphenomenon termed telomere position effect (TPE)(Aparicio et al., 1991) or position-effect variegation(PEV) (Henikoff, 1990). Curiously, although C. eleganspossesses telomeric heterochromatin, TPE has not beendescribed so far (Wirth et al., 2009). The importance oftelomeric chromatin structure in telomere stability ishighlighted by the fact that its disruption in mammaliancells alters telomere length and increases aberrantrecombination events (Schoeftner and Blasco, 2009).Whether a similar function exists in C. elegans remainsto be established.

Additional findings have suggested a conserved rolefor RNAi in the formation of heterochromatin. In fissionyeast, endogenous siRNAs (small interfering RNAs) arespecifically implicated in RNA-induced transcriptionalsilencing (RITS), and RITS activity is required forH3K9me2 accumulation on repetitive sequences, includ-ing centromeric repeats (Hall et al., 2002; Moazed, 2009;Volpe et al., 2002; Zaratiegui et al., 2007). Mutations ofany component of the RNAi machinery results in defectsin chromosome segregation due to defects in centromerefunction. Although C. elegans chromosomes are holocen-tric and lack pericentromeric repeats, recent studiesidentified CSR-1 (a Piwi/PAZ/Argonaute protein), EKL-1(a Tudor domain protein), and DRH-3 (a DEAH/D-boxhelicase), as regulators of meiotic H3K9me2 accumula-tion on unpaired chromosomes during C. elegans meio-sis, implicating RNAi in the regulation of meiotic chroma-tin (She et al., 2009).

Several genes in the RNAi-pathway have also beenshown to be involved in RNAi-induced Transcrip-tional Gene Silencing (TGS) to influence heterochro-matin formation and silence the expression of repeti-tive transgenes in both the C. elegans germ line andsoma (Grishok et al., 2005a,b; Robert et al., 2005;Sijen and Plasterk, 2003). Repetitive transgenes in theC. elegans germ line are effectively silenced through achromatin-related mechanism dependent on the MES(maternal effect sterility) group proteins, the histonedeacetylase SIR-2.1 (sirtuin-2), the HP1-like proteinHPL-2, and linker histone HIS-24 (see Fig. 2) (Couteauet al., 2002; Jedrusik and Schulze, 2001, 2003; Kellyand Fire, 1998). Interestingly, all of these proteins areassociated with heterochromatin associated marks:MES-2 deposits H3K27me3, SIR-2.1 deacetylatesH3K9, HPL-2 recognizes H3K9me2/3, and HIS-24binds H3K27me3 (Bender et al., 2004; Couteau et al.,2002; Wirth et al., 2009). Although worms do not

648 WENZEL ET AL.

encode a conventional DNA methyltransferase tosilence DNA repeats, as observed in plants and mam-mals (Bird, 2002; Simpson et al., 1986), the presenceof H3K27me3 and H3K9me2 on repetitive transgenesmay play a function similar to DNA methylation inthis context.

To probe the existence of nuclear compartments dur-ing C. elegans development, the behavior of stably inte-grated fluorescent reporter constructs was recently ana-lyzed through live imaging in embryos and larvae (Mei-ster et al., 2010). It was shown that in embryos, largerepeat-induced heterochromatic arrays are associatedwith the nuclear envelope, while arrays carrying tissue-specific promoters are decompacted and relocated tothe nuclear lumen when activated in specific cell types(Meister et al., 2010). These findings suggest the pres-

ence of a nuclear compartmentalization similar to thatof mammals.

C. elegans Enzymes Modifying Core Histones andTheir Biological Function

Reversible modifications play a key role in definingchromatin states and in regulating transcription in a cell-specific manner. Whether the functions of individualmembers of chromatin-modifying complexes are uniqueor redundant, and to what extent the observed redun-dancy depends on temporal and spatial regulation oftheir expression, remains elusive (Jenuwein and Allis,2001). In C. elegans, many chromatin-modifying enzymes(modifiers) have been identified by genetic approaches,or through a combination of multiple sequence align-ments to visualize evolutionary relationships.

FIG. 1. Electron microscopy of somatic nuclei showing extended regions of heterochromatin (electron-dense and dispersed) as well asless-dense regions corresponding to euchromatin. Electron-dense regions are marked by H3K9me3 (a) and H3K27me3 (b) (labeling with10 nm gold) whereas less-dense regions are labeled with H3K4me2 (c) euchromatic marks. Eu-euchromatin, he- heterochromatin, nu-nucle-olus and env-envelope. Scale bar, 500 nm.

649EPIGENETICS IN C. elegans

Tables 1 and 2 list the major chromatin-modifyingenzymes identified in C. elegans and summarize their bi-ological function in conserved multiprotein complexes.As shown, most enzymes not only have specificity fortarget residues, but also differ in tissue-specificity anddevelopmental requirements, reflecting the dynamic na-ture of histone modifications. Nonetheless, althoughthe function of histone modifying enzymes in C. elegans

development has become the focus of intense studies,we still know very little about the mechanisms responsi-ble for the establishment and maintenance of specificchromatin marks, and their recognition.

During C. elegans development, important roles forchromatin modifiers have been identified in vulval cell-fate specification, lifespan determination, genome sta-bility, and embryonic, germline and foregut develop-ment (Table 1). The C. elegans genome contains 38genes predicted to encode histone lysine methyltrans-ferase (HMT) activity, based on the presence of a con-served SET domain. The first of these genes to be identi-fied through a genetic screen were mes-2 and mes-4

(Capowski et al., 1991; Garvin et al., 1998; Holdemanet al., 1998). MES-2 is the ortholog of Drosophila Poly-comb group protein Enhancer of zeste E(z), while MES-4 is a nuclear SET domain protein related to mouseNSD1 (nuclear receptor binding SET-Domain 1) (Benderet al., 2004, 2007; Holdeman et al., 1998). MES-2 physi-cally interacts with both MES-6, homologue of the Pcgroup protein Extra sex combs (Esc), and MES-3 (anovel protein), in a repressive complex with H3K27

HMT activity similar to mammalian and fly E(z)/ESCcomplexes (Bender, 2004; Paulsen et al., 1995; Xuet al., 2001). MES deficiency results in maternal-effectsterility, derepression of high copy transgenes in thegermline, and specific loss of the H3K27me3 hetero-chromatin mark from the X chromosome (Bender et al.,2004, 2006; Fong et al., 2002). In addition, the absenceof MES activity suppresses the synthetic multivulva(synMuv) phenotype associated with inactivation of alarge group of histone modifying enzymes which actredundantly in vulval cell fate specification (Cui et al.,2004, 2006b; see below).

The C. elegans vulva represents a simple system inwhich to study how chromatin factors influence spe-cific developmental pathways (Ceol and Horvitz, 2004;Cui et al., 2006a,b; Lu and Horvitz, 1998; Sternebrg andHorvitz, 1991). synMuv genes fall into three classesencoding chromatin associated factors that act redun-dantly for vulval cell fate specification. Simultaneousinactivation of synMuv genes from any two differentclasses leads to ectopic EGF/RTK/Ras signaling and theformation of ectopic vulvae (the Multivulva phenotype).synMuv genes include the homologue of mammalianclass I histone deacetylases (HDA-1), histone methyl-transferases (MET-2; MET-1), and homologues of the evo-lutionarily conserved NuRD/Mi-2 nucleosome remodel-ing and histone deacetylase complex (LET-418/Mi-2,LIN-53/RbAp48, MEP-1, and HDA1/HDAC-1; Andersenand Horvitz, 2007; Dufourcq et al., 2002; Lu and Hor-vitz, 1998; Poulin et al., 2005; Unhavaithaya et al.,

FIG. 2. Difference between heterochromatic and euchromatic domains. Extrachromosomal arrays consisting of tandem DNA repeats aremarked by repressive histone methylation marks (H3K27me3, H3K9me2/3) and silenced by the HP1-like protein HPL-2 and SIR-2.1/SIRT1(sirtuin), whereas euchromatic domains are stained with active histone methylation marks and recognized by transcription factors or remod-eling complexes.

650 WENZEL ET AL.

Table

1MajorChromatin-M

odifyingEnzymesIdentifiedin

C.elegansandTheirBiologicalF

unction

Classofhistone

modifyingenzymes

Nameofprotein

Mutantphenotype;function

References

HMT

MES-2/EzH

2(H3K27me3)

maternalsterile;requiredforchromatinorganisation

inthegerm

lineandX-chromosomesilencing

Holdemannetal.,

1998;Xuetal.,

2001;

Benderetal.,

2004

MES-4/N

SD1(H3K36me2)

maternalsterile;associateswithautosomalc

hromatin,

functionsin

epigeneticmemory

ofgerm

cells

Fongetal.,

2002;Benderetal.,

2006;

Rechtsteineretal.,

2010

MET-1/Set2pyeast(H3K9me3,

H3K36me3)

synMuv;

inhibitionofvulvalc

ellfate

AndersenandHorvitz,

2007

MET-2/SETDB1(H3K9me3,

H3K36me3)

synMuv;

inhibitionofvulvalc

ellfate

AndersenandHorvitz,

2007;

Bessleretal.,

2010.

BLMP-1/S

ETdomain-containingortholog

small,protrudingvulva(pvl)

Ceronetal.,

2007

SET-1/S

ETdomain-containingortholog

embryonic

andlarvallethal

AndersenandHorvitz,

2007

SET-3/S

MYD4,SET-4/S

UV420H1,SUV420H2,

SET-10,-14,-18,-30/SMYD1,2,3,

SET-11/EHMT1,TTLL-12/TTLL12,

SET-17/PRDM11,SET-27/SETD3,

SET-29/S

ETD4,SET-5,-6,-8,-9,-12,-13,-

15,-19,-20,-21,-24,-26,-28,-31,-32

noobviousphenotypes

AndersenandHorvitz,

2007

SET-2(H3K4me2)

slow

growth,mortalg

erm

line;regulateslifespan

XuandStrome2001;Sim

onetetal.,

2007;

Greeretal.,

2010;Xiaoetal.,

2011

SET-16/MLL(H3K4me3)

vulvald

evelopment;regulatesexpressionof

Hoxgenelin-39

Fisheretal.,2010

SET-23/SETMAR

embryonic

lethal

AndersenandHorvitz,

2007

SET-25/S

ETdomain-containingprotein

requiredfornorm

ala

xonalg

uidance,protects

genomeagainstmutations

Pothofetal.,

2003

WDR-5

(SWD-3

orTA

G-125)/WDR-5

(H3K4me3)

regulateslifespan;mortalg

erm

linephenotype,

antagonizessynMuvtranscriptionalrepressors

Fisheretal.,2010;Sim

onetetal.,

2007;

Greeretal.,

2010

ASH-2/ASH2fly(H3K4me3)

embryonic

lethalitycharacterize

dbydefects

incortical

activityandspindle

rocking;regulateslifespan

Fisheretal.,2010;Greeretal.,

2010

RBBP-5

(F21H12.1)

n/d

Fisheretal.,2010

HDM

JMJD2(forH3K9me3andH3K36me3)

regulateslifespan,increasedgerm

cellapoptosis,

activationofDNA-damagecheckpoint

Whetstineetal.,

2006

UTX-1/U

TX-1/JmjC

(H3K27me3)

vulvald

evelopment,cellcycle

arrest;regulates

expressionofHoxgenelin-39

Fisheretal.,2010;Wangetal.,

2010

TAG-279/JMJD3(H3K27me3)

abnorm

alg

onaddevelopment

Aggeretal.,

2007

C29F7.6/JMJD3

n/d

Aggeretal.,

2007

F29B9.2/JmjC

(H3K9me2and

H3K27me2)

regulateslifespan,dauerform

ation

Lin

etal.,

2010

F23D12.5;C06H2.3;F23D12.5;

F43G6.6;T26A5.5;T28F2.4;

C27F2.5;Y48B6A.11/JmjC

n/d

Aggeretal.,

2007;Klose

etal.,2006

SPR-5/LSD-1

(H3K4me2)

mortalg

erm

linephenotype

Katz

etal.,

2009

RBR-2/JARID1A(H3K4me3)

vulvald

evelopment;regulateslifespan

Christiansenetal.,

2007;Greeretal.,2010

HAT

MYS-1/TIP-60

syntheticslow

growth

phenotype;negativelyregulates

expressionofgenesrequiredforvulvalinduction

Ceola

ndHorvitz,

2004

LSY-12/M

YS-3

vulvald

evelopment,locomotion,egg-laying,fecundity

Sarinetal.,

2007

NuA4/M

YS-4

regulatesRassignalingandspecifiescellfates

Ceola

ndHorvitz,

2004

CBP-1/C

BP/p300

requiredduringembryogenesisfordifferentiation

ofallnon-neuronalsomaticcelltypes

Eastburn

DJ,HanM,2005

651EPIGENETICS IN C. elegans

2002; von Zalewsky et al., 2000). HDA-1 is sumoylated inC. elegans, and both SUMO and the E2SUMO ligase UBC9are also members of the synMuv group, suggesting thatSUMOylation may regulate the activity of the NuRD/Mi-2complex in vulval cell fate specification (Poulin et al.,2005). Additionally, HDA-1 physically interacts with TRA-4, the C. elegans homolog of the human proto-oncopro-tein and transcriptional repressor PLZF, to promotefemale development by repressing the transcription ofmale-specific genes (Grote and Conradt, 2006). Interest-ingly, in these studies TRA-4 was also found to act as asynMuv gene to influence vulval cell fate specification.

Deletion of many synMuv genes results in tempera-ture sensitive developmental abnormalities, suggestingthat a temperature sensitive process is involved in chro-matin remodeling (Cui et al., 2006b; Petrella et al.,2011). For example, the C. elegans HP1 homolog hpl-2,a synMuv gene at permissive temperatures, shows ahighly penetrant multivulval (Muv) phenotype and ste-rility at 258C, the non-permissive temperature (Cous-tham et al., 2006; Couteau et al., 2002).

Until recently, it was unclear whether enzymes antag-onizing histone methylation are present in C. elegans.

The first histone demethylases (HDM) to be describedwere SPR-5, ortholog of the human LSD1 H3K4 demethyl-ase, and UTX-1, homolog of the mammalian Jumonji-C(JmjC) domain H3K27me2/3 demethylase UTX (Table 1)(Agger et al., 2007; Katz et al., 2009). Histone demethy-lation catalyzed by LSD1 relies on an oxidative reactionin which flavin is a cofactor, while JmjC-domain contain-ing enzymes require Fe(II) and a-ketoglutarate as cofac-tors (Klose et al., 2006; Lee et al., 2005; Metzger et al.,2005; Shi et al., 2004; Tsukada et al., 2006). JmjC-domain enzymes can remove all three histone lysine-methylation states, whereas LSD-1 removes only mono-and dimethyl lysines.

spr-5 loss of function suppresses the egg-laying defec-tive (egl) phenotype of mutations in sel-12/presenilinand derepresses expression of the second C. elegans

presenilin, hop-1. In binding assays and yeast two-hybrid experiments, SPR-5 interacts with SPR-1, the C.

elegans ortholog of CoREST (Eimer et al., 2002; Lakow-ski et al., 2003) (Table 2). A recent study demonstratedthat SPR-5 deficiency results in progressive sterility over20–30 generations (the mortal germline phenotype,mrt), suggesting an important function in the mainte-nance of germline immortality. This progressive sterilitycorrelates with the accumulation of H3K4me2 in germ-cell precursors, and the misregulation of spermatogene-sis genes (Katz et al., 2009). Mutation of another HDM,JMJD-2, causes increased germ cell apoptosis, consistentwith the activation of a DNA-damage checkpoint. Thisphenotype correlates with higher levels of H3K9me3 inthe germline and H3K36me3 on the X chromosome dur-ing meiotic prophase (Whetstine et al., 2006). Defi-ciency of the JMJD3 homolog F18E9.5 leads to defects in

Table

1Continued

Classofhistone

modifyingenzymes

Nameofprotein

Mutantphenotype;function

References

TAF-1-TAFII250(TBP)

embryonic

andlarvald

evelopment

Walkeretal.,

2004

MYS-2/M

OF

n/d

Shibata

etal.,

2010

TAG-235/H

AT-1

n/d

–GCN5-likesubfamily

n/d

Abo-D

alo

Betal.,

2004

HDAC

SIR

-2.1/SIRT1(H3K9)/classIII

requiredforgerm

linesilencing,longevity,

associateswithtelomeres

JedrusikandSchulze,2003;

Tissenbaum

andGuarente,2001;

Wirth

etal.,

2009

HDAC-11/classIV

decreasedgenomestability

Pothofetal.,

2003

HDA-1(G

ON-19)/H

DAC1/

RPD3yeastandfly/classI

embryonic

viability,gonadogenesisand

vulvald

evelopment

Dufourcqetal.,

2002

HDA-2/classI

genomestability

Lansetal.,

2010

HDA-3/H

D1/classI

affects

radiationsensitivity

Lansetal.,

2010

HDA-4/classII

locomotion,bodymorphology,andgrowth

Choie

tal.,

2002

HDA-6/classII

n/d

–F43G6.4/classII

n/d

–

HMT,histonemethyltransferase;HDM,histonedemethylase

;HAT,histoneacetyltransferase;HDAC,histonedeacetyltransferase.Annotationoftheproteinsfrom

C.elegansandtheir

human,flyoryeasthomologuesare

listed.

652 WENZEL ET AL.

somatic gonad development (Agger et al., 2007), whileloss of function of rbr-2, encoding an ortholog of humanJARID1A, results in vulval specification defects associatedwith increased global levels of H3K4me3 in late larvalstages and adults (Christensen et al., 2007).

In mammals, the activity of different histone modify-ing enzymes is often functionally linked to reinforce theactivation or repression of gene expression. Severalexamples of this cross talk have been observed in C. ele-

gans, showing that similar mechanisms are implicatedin the dynamic regulation of chromatin structure in ver-tebrates and invertebrates. For example, the UTX-1H3K27 demethylase and the SET-16/MLL H3K4 methyl-transferase act in the same genetic pathway to attenuateLET-60 RAS signalling and regulate egl-5 Hox geneexpression (Fisher et al., 2010). In mammalian cells,UTX physically associates with MLL (Cho et al., 2007;Lee et al., 2007), suggesting that UTX-1 and SET-16 mayphysically interact to modulate LET-60 RAS signalling in

C. elegans. Coordinate regulation of HMT and HDMactivities has also been shown in the case of Hox generegulation during ES cell differentiation (Christensenet al., 2007) and in neuronal development (Jepsenet al., 2007). Whether the activity of histone-modifyingenzymes is also coordinately regulated to influence Hoxgene expression in C. elegans is an area of future inves-tigation. Interestingly, a recent study has shown thatcoordinate regulation can also be mediated by a singleenzyme (Lin et al., 2010). The C. elegans KDM7A his-tone demethylase can demethylate both H3K9me2 andH3K27me2, and activate gene expression by interactionof its PHD domain with H3K4me3.

Collectively, most of the enzymes modifying chroma-tin are evolutionary conserved (Tables 1 and 2). There-fore, novel insight from C. elegans on how histonemodifiers and readers contribute to cellular functionwill help us to better understand the underlying molec-ular mechanisms in mammalian systems and in physio-

Table 2Conservation of NuRD, CoREST, Sin3, DRM, and SET1/COMPASS Components and Their Function in C. elegans and Mammals

Complex C. elegans function Complex components Mammalian function Complex components

Mi2/NuRD vulva specification(Ahringer, 2000,von Zalewski 2000)

HDA-1RBA-1LIN-53LET-418 CHD-3MEP-1

important componentsof developmental signalingpathways, potentially withcancer relevance (Xue et al.,1998; Denslow andWade, 2006)

HDAC1, HDAC2RbAp46, RbAp48RbAp38Mi-2alfa, Mi-2beta––

CoREST repressor of the presenilingene hop-1 (Lakowskiet al., 2003)

HDA-1SPR-1SPR-5SPR-3SPR-4DIN-1 (putative

ortholoque)

regulate neuronal geneexpression and neuronalstem cell fate(Lakowski et al., 2006)

HDAC1HDAC2CoRESTLSD1––SHARP

Sin3 formation of malesensory rays(Choy et al., 2007)

HDA-1RBA-1LIN-53SIN-3MAB-21

epigenetic reprogramming(Farias et al., 2010)

HDAC1, HDAC2RbAp46, RbAp48RbAp38Sin3AMab21L1, Mab21L2

NuA4 vulva specification(Ceol et al., 2004)

MYS-1TRR-1EPC-1

essential for cell cycleprogression, gene- specificregulation and DNArepair (Doyon et al., 2004)

HATTRRAPE(Pc)

DRM vulva specification(Harrison et al., 2006)

EFL-1DPL-1LIN-53LIN-35LIN-37LIN-9LIN-52LIN-54

cell proliferation (van denHeuvel and Dyson 2008 )

E2FDPRbAp38RbMip40Mip130–Mip120

SET-1/COMPASS germline immortality,longevity (Simonet et al.,2007; Greer et al.,2010; Li and Kelly,2011; Xiao et al., 2011)

SET-12SET-16ASH-2RBBP-5WDR-5.1WDR-5.2WDR-5.3CFP-1WDR-82DPY-30

essential for vertebratedevelopment, regulationof Hox gene expression(Wysocka et al., 2005)

SET1MLL1Ash2LRbBP5WDR5––CxxC1/Cfp-1WDR82hDPY30

653EPIGENETICS IN C. elegans

logical processes leading to disease. Future studiesshould shed light on how histone-modifying enzymesregulate the binding of effector proteins to chromatin ina developmental context. Additionally, it will be inter-esting to determine which non-histone substrates arealso modified by these same enzymes.

Histone Modification Patterns and UnusualReaders in C. elegans

In C. elegans, the distribution of H3K36me3(enriched in gene bodies; exons preferentially markedrelative to introns), H4K4me3 (enriched near transcrip-tion start sites) and H3K27me3 (enriched in transcrip-tionally silent regions) is similar to that observed inother organisms (Gerstein et al., 2010; Kolasinska-Zwierz et al., 2009). In addition, H3K36me3 on exonswas found to be dependent on transcription, and waspresent at lower levels on alternatively spliced exons,suggesting a link to splicing. This link between H3K36methylation and splicing may be conserved in mammals(Luco and Misteli, 2011).

Despite the presence of similarities in histone modifi-cation patterns in different species, recent results suggestthat some patterns may vary from one organism to theother (Allis et al., 2006). In C. elegans H3K9 methylation,a repressive mark, is not concentrated at a single regionon each chromosome, as in flies and mammals, but occu-pies broad domains on chromosome arms (Liu et al.,2011). This distribution is consistent with the absence ofcytological heterochromatin and the holocentric natureof C. elegans chromosomes, and may play a role in nu-clear organization through an interaction with the LEM-2protein (Ikegami et al., 2010). Striking novel patterns ofhistone modifications associated with their respectivebinding proteins (readers) may also exist in C. elegans.For example, the linker histone variant HIS-24 recognizesH3K27me3 and is post-translationally modified at lysine14, in contrast to vertebrate H1 which is modified at ly-sine 26 (Kuzmichev et al., 2004; Wirth et al., 2009; M.J-Bunpublished data). Another example is provided by theMBT domain protein LIN-61. While MBT proteins fromDrosophila and mammals recognize mono- and di-meth-ylated lysines on histone H3 and H4 (Bonasio et al.,2010), LIN-61 prefers H3K9me2/3 residues (Koester-Eiserfunke and Fischle, 2011; M.J-B unpublished data).

Histone-Modifying Enzymes Help to MaintainGermline Continuity

Recent data has begun to shed light on how epige-netic mechanisms contribute to the maintenance of cellu-lar identity in multi-cellular organism (cellular memory)(Fisher, 2002). In C. elegans, the H3K4 demethylase SPR-5/LSD1 and the H3K36 methyltransferase MES-4 are impli-cated in distinct mechanisms of epigenetic programmingbetween generations (Katz et al., 2009; Rechtsteiner

et al., 2010). SPR-5/LSD-1 contributes to germline immor-tality by reprogramming epigenetic memory of primordialgerm cells (PGCs) (Katz et al., 2009). Accumulation ofH3K4me2 in PGCs in the absence of SPR-5 demethylaseactivity correlates with misregulation of genes requiredfor spermatogenesis, and with a mortal germline pheno-type (mrt) characterized by progressive sterility. Theseresults suggest that SPR-5/LSD-1 is required to eraseH3K4me2 in the germline and prevent inappropriatetransmission of transcriptional states (Katz et al., 2009).Interestingly, absence of the SET-2 H3K4 HMT is also asso-ciated with a mortal germline phenotype (Xiao et al.,2011), suggesting that the H3K4 methylation state in gen-eral plays an important role in germline stability.

MES-4 has been shown to transmit memory of geneexpression from one generation of germ cells to the nextby maintaining H3K36 methylation of germline-expressed loci in embryos. Genome-wide analysisshowed that in early embryos, MES-4 binds to the all fiveautosomes, where it is abundantly present on the mosthighly transcribed genes, but is under-represented on theX chromosome. Interestingly, the researchers found thatonly genes previously expressed in the maternal germline are bound by MES-4 in a Pol II independent manner,suggesting a mechanism unrelated to ongoing transcrip-tion (Rechtsteiner et al., 2010). Many of these genes areknown to be involved in meiosis (syp-2, zim-2, him-8)and P-granule assembly (pgl-1, -3, glh-2, -4), consistentwith an important role for MES-4 in imparting epigeneticmemory of the transcriptional state of PGCs.

Chromatin regulators are also involved in the mainte-nance of somatic cell fate. synMuv genes antagonizegermline fate in somatic cells, and in their absence germ-line-specific genes are ectopically expressed in the soma(Ceron et al., 2007, Wang et al., 2005; Petrella et al.,2011). Interestingly, MES-4 is required for somatic cellsto acquire germ cell fate in the absence of synMuv activ-ity (Wang et al., 2005) (see Fig. 3). Somewhat surpris-ingly, recent results show that transformation of germcells into somatic cells only requires CHE-1, a transcrip-tion factor that specifies the fate of somatic cells, andloss of the chromatin regulatory factor LIN-53 (Tursunet al., 2011). Further investigation of the important roleplayed by chromatin factors in regulating the germline-soma distinction will shed light on the processesinvolved in germline/soma identity and how germline/soma specific gene expression patterns are controlled.

The Molecular Complexity of PcG

PcG proteins are involved in many aspects of meta-zoan development, including the regulation of Hoxgene clusters, cell cycle progression, X inactivation, celldifferentiation and genomic imprinting (Schuetten-gruber and Cavalli, 2009; Sparmann and van Lohuizen,2006). Ubiquitination of lysine 119 of histone H2A

654 WENZEL ET AL.

(H2AK119ub), and trimethylation of histone H3 lysine27 (H3K27me3) are indicative of Polycomb group(PcG) activity and are regulated by two major PolycombRepressive Complexes, PRC1 and PRC2, respectively.H2AK119ub is mediated by the PRC1 componentdRing, and H3K27me3 by the PRC2 component E(z)(Cao and Zhang, 2004; Cao et al., 2002, 2005).

C. elegans possesses two Pc-related protein com-plexes: Polycomb group (MES-2/3/6) (Korf et al., 1998)and Polycomb group like (SOP-2/Suppressors of Pal-1and SOP-2-related protein 1/SOR-1). The SOP-2/SOR-1complex appears to be specific to C. elegans (Zhanget al., 2003). More recently, it has been shown thatMIG-32 (abnormal cell migration) and SPAT-3A (sup-pressor of par-two defect), encode functional homo-logues of the human PRC1 complex and are requiredfor H2A ubiquitylation (Karakuzu et al., 2009).

As already discussed, MES proteins were initially iden-tified as factors important for germline development(Capowski et al., 1991; Fong et al., 2002; Holdemanet al., 1998). Subsequently mes-2, mes-3, and mes-6

were identified in a genetic screen to identify regulatorsof male tail neurogenesis. Functional analysis showedthat mes-2, mes-3, and mes-6 act upstream of the Hoxgenes mab-5 and egl-5, and are required for formationof sensory rays in the male tail as well as migration ofspecific neurons (Ross and Zarkower, 2003). Absenceof another Pc related protein, SOP-2, was also shown toresults in homeotic transformations related to wide-spread misexpression of Hox genes (Zhang et al.,2003). However, sop-2 function appears to be inde-pendent ofmes gene activity.

MES-2 also plays a role in late embryonic develop-ment. mes-2 inactivation at this stage results in inappro-priate expression of developmental regulators as well asglobal changes in chromatin structure and organization.Tissue-specific developmental regulators whose expres-sion is upregulated in the absence of MES-2 activityinclude the MADS-box factor UNC-120/SRF (muscle),the GATA factor END-1 (intestine), and the Tbox pro-teins TBX-37 and TBX-38 (foregut) (Yuzyuk et al.,2009). This is similar to the phenomenon observed inDrosophila and mammalian ES cells, where loss ofPRC2 activity results in upregulation of important devel-opmental regulators required for maintenance of cellfate (Schuettengruber et al., 2007).

In flies, the ESC-E(z) complex binds Hox genes anddeposits H3K27me3. This mark is in turn recognized bythe PRC1 complex, thereby antagonizing Trx-G com-plex activity (Simon and Tamkun, 2002). It is unclearwhether the same interplay between E(z)/ESC andPRC1 activities exists in C. elegans. Lack of MIG-32 andSPAT-3 activity causes neuronal defects similar to thoseobserved in mes mutants, and a reduction in H2A ubiq-uitylation. However, unlike mes mutants, mig-32 andspat-3a mutant worms are fertile (Karakuzu et al.,2009). It has been suggested that in the nervous system,MIG-32 and SPAT-3 may play a subtle role together withMES-proteins, distinct from the more global role playedby MES proteins in the establishment of embryonic cellfate and germline development (Karakuzu et al., 2009).How the diverse PcG complexes are targeted to chro-matin to regulate histone modifications and mediaterepression in C. elegans is a key question whichremains to be answered.

Over the last few years, a number of studies haveimplicated RNA in the process of PcG recruitment, pri-marily in mammalian cells. A novel role for a non-codingRNA, HOTAIR, was identified in the regulation of mam-malian Hox gene transcription (Rinn et al., 2007).HOTAIR interacts with both PRC2 and LSD1 to couplehistone H3K27 methylation and H3K4 demethylation(Tsai et al., 2010). A similar mechanism may exist in theworm, where specific short noncoding RNAs (50–200bases) and lincRNAs (long, intergenic noncoding RNAs)can potentially trigger complex chromatin patterns atspecific genes in a temporally organized manner duringdevelopment (M.J-B, unpublished results).

In Drosophila, specific DNA sequences called Poly-comb response elements (PREs) serve as an assemblyplatform for the repressive function of different PcGcomplexes through DNA–protein and protein–proteininteractions (Muller and Kassis, 2006). Whether suchPREs also exist in C. elegans is still unknown. Additionalcomponents of the fly and mammalian ESC-E(z) com-plex include NURF55 and Su(Z)12 (Muller et al., 2002),which are required for binding of the PRC2 complex tonucleosomes (Nekrasov et al., 2005). C. elegans doesnot possess a Su(Z)12 homologue, but does have twoNURF55 homologues: LIN-53 and RBA-1. However, it isunknown whether either one of these two proteinsinteracts with the MES proteins. It remains possible that

FIG. 3. Cellular identity in C. elegans is the sum of interplay between Polycomb (PcG) and Trithorax (Trx-G) group proteins, as well as his-tone modifying enzymes (MES-4, SPR-5). The soma (grey) influences germline cell fate decisions and conversely the germline (black) cancontrol soma-specific gene expression.

655EPIGENETICS IN C. elegans

additional PRC2 components and recruitment factorsmay exist in C. elegans. Genome-wide studies and bio-chemical analysis, in addition to forward and reversegenetic approaches, may help to understand the molec-ular basis of PcG repression in C. elegans Hox gene reg-ulation and additional processes.

Trx-G and Its Interplay With PcG

PcG repression is antagonized by the Tritorax group(Trx-G) complex, which activates gene expression (Caoet al., 2002; Kal et al., 2000). Trx-G proteins includetwo H3K4 HMTs, MLL (mixed-lineage leukemia), ASH1,ASH2, and the WD40 protein WDR5. Mutations in lin-

59, encoding the C. elegans homologue of ASH-1, affectexpression of two C. elegans HOM-C genes required forposterior development: egl-5 an mab-5 (Chamberlinand Thomas, 2000). Additionally, LIN-59 and UNC-62 (aTALE-class Hox cofactor) were identified as regulatorsof the programmed cell death of specific cells in thedeveloping ventral nerve cord (Potts et al., 2009). InDrosophila, the antagonistic roles of PcG and Trx-Ggroup proteins in the maintenance of transcriptionalON and OFF states of HOX genes have been well char-acterized. Recent data suggests that this antagonismextends to other processes, including longevity control(Siebold et al., 2010). e(z) and esc mutations in fliesincrease life span, resistance to oxidative stress, andstarvation. Furthermore, mutations in Trx-G genes par-tially suppress the increased longevity and stress resist-ance phenotypes of e(z) mutants. While in C. elegans

ash-2 inactivation extends lifespan (Greer et al., 2010),it is not known if the MES/PcG proteins play a similarrole in lifespan regulation.

Are Alterations in the Epigenome Dependenton Early Experience?

C. elegans has a relatively short life cycle (2–3 weeks)and is therefore an excellent model organism to explorewhether and how different environmental or develop-mental experiences influence the cellular memory ofgenetically identical populations. It has recently beenshown that worms that have gone through the stress-resistant dauer larval stage display distinct gene expres-sion patterns and chromatin profiles compared to adultanimals which never exited the reproductive cycle (Hallet al., 2010). These effects depend on the activity ofchromatin-associated proteins, suggesting that chroma-tin remodeling plays a pivotal role in the establishmentof post-dauer expression profiles (Hall et al., 2010).These genomic alterations may contribute to pheno-typic differences among animals exposed to differentenvironmental or developmental experiences, and there-fore influence the phenotypic variation of geneticallyidentical animals. It would be of great interest to betterunderstand the molecular mechanism involved in the

‘‘epigenetically imprinted experience’’ resulting in globalchanges in the adult organism, as well as to establishwhether these mechanisms are evolutionary conserved.

CONCLUSIONS

It is now well established that histone modifiers, theassociated histone modifications and their readers, of-ten function in a concerted way to contribute to chro-matin function. Nonetheless, we are now only begin-ning to understand how the transition from repressedto active chromatin states is achieved, how normal cellsuse dynamic chromatin modifications to establish epige-netic homeostasis, how environmental cues are readand translated through epigenetic mechanisms, andhow chromatin states are established and maintainedfrom one generation to the next.

Information obtained from genome-wide histonemodification profiling, combined with the power ofgenetic approaches in C. elegans, will undoubtedly pro-vide novel insight into the underlying molecular mecha-nisms and the functional importance of epigenetic regu-lation. Given the high degree of conservation betweenchromatin modifiers across species, the informationderived from these studies will likely contribute to abetter understanding of epigenetic regulation in normaldevelopment and longevity. C. elegans itself can be animportant model organism to test different drug targetsinhibiting the function of chromatin modifyingenzymes, and thereby impact research on cancer thera-peutics as well as contribute important information rel-evant to stem-cell technology and cancer.

ACKNOWLEDGMENTS

The authors thank Maja Studencka and Helena Mileticfor critically reading the manuscript.

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