Defining heterochromatin in C. elegans through genome-wide analysis of the heterochromatin protein 1 homolog HPL-2

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Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In most organisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavily studied model C.
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  Research Defining heterochromatin in  C. elegans   throughgenome-wide analysis of the heterochromatinprotein 1 homolog HPL-2 Jacob M. Garrigues, 1 Simone Sidoli, 2 Benjamin A. Garcia, 2 and Susan Strome 1 1 Department of Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, California 95064, USA; 2 Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania,Philadelphia, Pennsylvania 19104, USA Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In mostorganisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavilystudied model  C. elegans  , which possesses holocentric chromosomes with dispersed centromeres, we analyzed the genome-wide distribution of the heterochromatin protein 1 (HP1) ortholog HPL-2 and compared its distribution to other featurescommonly associated with heterochromatin. HPL-2 binding highly correlates with histone H3 mono- and dimethylated atlysine 9 (H3K9me1 and H3K9me2) andformsbroaddomains onautosomalarms. AlthoughHPL-2,likeother HP1 orthologs,binds H3K9me peptides in vitro, the distribution of HPL-2 in vivo appears relatively normal in mutant embryos that lackH3K9me, demonstrating that the chromosomal distribution of HPL-2 can be achieved in an H3K9me-independent manner.Consistent with HPL-2 serving roles independent of H3K9me,  hpl-2   mutant worms display more severe defects than mutantworms lacking H3K9me. HPL-2 binding is enriched for repetitive sequences, and on chromosome arms is anticorrelated withcentromeres. At the genic level, HPL-2 preferentially associates with well-expressed genes, and loss of HPL-2 results in up-regulationofsomebindingtargetsanddown-regulationofothers.Ourworkdefinesheterochromatininanimportantmodelorganism and uncovers both shared and distinctive properties of heterochromatin relative to other systems.[Supplemental material is available for this article.] Eukaryotic genomes are packaged into two general types of chro-matin: euchromatin and heterochromatin. This packaging is im-portant for the regulation of gene expression and organization of the genome. Initially, heterochromatin was cytologically definedas the condensed, dark-staining chromatin that remains visiblethroughout the cell cycle (Heitz 1928). Since then, numerousmolecularcharacteristicsofheterochromatinhavebeenidentified.These include an enrichment of repetitive DNA elements, such assatellite DNA and sequences derived from transposable elements,and enrichment of histone H3 methylated at lysine 9 (H3K9me)(Grewal and Elgin 2002). Another hallmark of heterochromatin isthe enrichment of heterochromatin protein 1 (HP1), a highlyconserved, small nonhistone protein first identified in  Drosophila (JamesandElgin1986).Heterochromatinistypicallyconcentratedat pericentric and subtelomeric regions. How heterochromatin isdistributedinanorganismwithnumerouscentromeresdistributedalong the length of each chromosome (i.e., holocentric) is notknown. This paper defines the distribution of an HP1 protein andheterochromatin in the nematode  C. elegans , which possesses hol-ocentric chromosomes and is a valuable model for genome organi-zation,chromosomesegregation,geneexpression,anddevelopment.It has been shown through a variety of methods that thechromo domain (CD) of metazoan HP1 proteins specifically rec-ognizes H3K9me2 and H3K9me3 (Bannister et al. 2001; Jacobset al. 2001; Lachner et al. 2001). Evidence that this interaction isimportant for proper HP1 protein localization comes from theobservation that loss or reduction of H3K9me results in loss orreduction of HP1 binding in vivo (Bannister et al. 2001; Lachneretal.2001;Schottaetal.2002;Seumetal.2007;Tzengetal.2007).Other interactions besides the CD-H3K9me interaction are in-volved in HP1 localization as well, as  Drosophila  SU(VAR)205 (alsoknownasHP1A)isabletoassociatewithpromoterregionsofgenesindependently of H3K9me (Figueiredo et al. 2012), and HP1AlackingitsCDisabletoassociatewithheterochromatin(SmothersandHenikoff2001).Furthermore,invitrostudieshaveshownthatmouse CBX1, CBX3, and CBX5 (also known as HP1 b , HP1 g , andHP1 a , respectively) bind the histone-fold domain of histone H3(Nielsen et al. 2001) and that fly HP1A binds DNA in a sequence-independent manner (Zhao et al. 2000). Interestingly, studies infissionyeast,flies,andmammalshavedemonstratedthattheRNAimachinery and RNA itself contribute to HP1 protein localization(Pal-Bhadra et al. 2004; Verdel et al. 2004; Maison et al. 2011).Taken together, these studies implicate interactions between HP1and methylated histone tails, histone cores, DNA, and RNA ascontributing to the recruitment and retention of HP1 at particularDNA regions in vivo. In this study, we specifically tested whetherH3K9me is required for proper HP1 localization in  C. elegans .The nematode  C. elegans  has two HP1 paralogous proteins: HP1Like (heterochromatin protein) 1 and 2 (HPL-1 and HPL-2) (Couteauetal.2002).HPL-2servesmorerolesand/ormoreimportantrolesthanHPL-1, as  hpl-2  mutants display diverse defects while  hpl-1  mutantsgenerally lack observable mutant phenotypes. HPL-2 is an importantfactor for germline health, as  hpl-2  mutants display maternal-effect   2015 Garrigues et al. This article is distributed exclusively by Cold SpringHarborLaboratoryPressforthefirstsixmonthsafterthefull-issuepublicationdate(see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it isavailable under a Creative Commons License (Attribution-NonCommercial 4.0International), as described at http://creativecommons.org/licenses/by-nc/4.0/. Corresponding author: sstrome@ucsc.edu  Article published online before print. Article, supplemental material, and pub-lication date are at http://www.genome.org/cgi/doi/10.1101/gr.180489.114. 76 Genome Research www.genome.org 25:76–88 Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/15; www.genome.org  Cold Spring Harbor Laboratory Presson April 25, 2018 - Published by genome.cshlp.orgDownloaded from   sterility at elevated temperature (25 ° C) (Coustham et al. 2006) anda reduced ability to silence exogenous ‘‘non-self’’ sequences in thegermline (Couteau et al. 2002; Robert et al. 2005; Ashe et al. 2012;Shirayama et al. 2012). HPL-2 is also important in somatic de-velopment,as hpl-2 mutantsshowlarval,somaticgonad,andvulvaldevelopmental defects (Schott et al. 2006). Comparisons of   hpl-2hpl-1  double mutants and  hpl-2  single mutants suggest that HPL-2and HPL-1 have some overlapping roles, as double mutant wormsdisplaymoreseverephenotypesthan hpl-2 alone(Schottetal.2006;Shirayama etal. 2012). Because HPL-2 is the more important of thetwo  C. elegans  HP1 homologs and is the only HP1 homolog in  C.briggsae  , a close relative of   C. elegans  (Vermaak and Malik 2009), wefocused our current study on HPL-2.Here, we show that HPL-2 binding to chromatin highly cor-relateswithH3K9me1andH3K9me2throughoutthegenomeandthat HPL-2-enriched regions form domains that are also enrichedforrepetitiveDNAelements.TheseobservationssuggestthatHPL-2indeed has functions associated with heterochromatin and thatHPL-2-enriched domains represent the distribution of hetero-chromatin in  C. elegans . Surprisingly, H3K9me is not necessary forthe normal distribution of HPL-2, as the genome-wide pattern of HPL-2 is largely unchanged in  met-2 set-25  mutant embryos, whichTowbinetal.reportedandweverifiedtolackH3K9me(Towbinetal.2012).ConsistentwithHPL-2havingrolesindependentofH3K9me, met-2 set-25  mutants display less sterility at elevated temperaturethan  hpl-2 . Interestingly, worm heterochromatin has a unique dis-tribution relative to other organisms: enrichment on the autosomal‘‘arms’’ and on the leftmost region of the X chromosome. On auto-somal arms, elevated HPL-2 levels flank centromeric chromatin,creating regions that resemble pericentric heterochromatin. HPL-2shows a bias toward association with well-expressed genes, where itseems to repress the expression of some genes it binds and promotethe expression of others. Our studies uncover both shared andunique properties of worm heterochromatin compared to other or-ganisms, and reveal how heterochromatin is distributed in an or-ganism with holocentric chromosomes. Results HPL-2 is concentrated along with H3K9 methylationon autosomal arms TodeterminethedistributionoftheHP1homologHPL-2inworms,we performed chromatin immunoprecipitation using a validatedantibody (Supplemental Fig. S1) followed by microarray analysis(ChIP-chip) in early to mid-stage embryos. We observed large do-mains of HPL-2 enrichment on autosomal arms and the leftmostregion of the X chromosome, and depletion of HPL-2 from thecentral regions of autosomes and most of the length of the X (Fig.1A). Genomic coordinates of HPL-2-enriched arms and HPL-2-de-pleted centers are defined in Supplemental Figure S2. In agreementwith low HPL-2 ChIP signal on the X, immunostaining of her-maphrodite germline nuclei for HPL-2 and H4K12ac, a histonemodification enriched on autosomes in germ nuclei (Kelly et al.2002),revealedthatHPL-2stainingislowerontheXchromosomesthan the autosomes (Fig. 1C).In addition to enrichment for HP1 proteins, other hall-marks of heterochromatin are enrichment of H3K9me and re-petitive DNA elements, as well as sparser gene density relative toeuchromatin (Richards and Elgin 2002). To determine if HPL-2-enriched arms also possess these characteristics, we comparedour HPL-2 ChIP-chip data to these features (Fig. 1A). HPL-2 hasa similar overall distribution to that of previously publishedH3K9me ChIP signal (Liu et al. 2011). Like H3K9me, HPL-2 ismore heavily enriched on pairing center arms compared tononpairing center arms (Gu and Fire 2010; Liu et al. 2011). In-terestingly, thepatternofHPL-2mostcloselyresemblesH3K9me1andH3K9me2andlesscloselyresemblesH3K9me3,withgenome-wide Pearson correlation coefficients (PCCs) of 0.72, 0.78, and0.39, respectively (Fig. 1B). Within HPL-2-enriched arms, thosePCCs are 0.68, 0.74, and 0.15, respectively (Supplemental Fig. S3).ThedistributionofHPL-2doesnotmatchthepreviouslypublishedpattern of H3K27me3 (Liu et al. 2011) (PCC  =  0.01) (Fig. 1A,B).Based on comparing the distribution of HPL-2 to standardized re-petitive DNA element densities, HPL-2-enriched arms are alsoenrichedfor repetitive elements, with the genome-wide pattern of HPL-2 modestly matching that of repetitive sequences (PCC  = 0.45)(Fig.1A,B).Thisobservationisconsistentwithpreviouswork showing chromosome arms to be enriched for repetitive elements(The  C. elegans  Sequencing Consortium 1998). To explore the re-lationshipbetweenHPL-2bindingandgenedensity,wecomparedthe distribution of HPL-2 to the number of protein-coding genebase pairs per unit length along each chromosome. Interestingly,there is no obvious relationship between HPL-2 and standardizedgenedensities(PCC = 0.01)(Fig.1A,B).AsHPL-2-enrichedarmsarealso enriched for H3K9me and repetitive DNA elements, we con-clude that these regions represent heterochromatin in  C. elegans .However, differing from what has been observed in other organ-isms, worm heterochromatin does not show a depletion of genicDNA relative to the rest of the genome. HPL-2 can associate with chromatin in an H3K9me-independent manner The genomic distribution of HPL-2 correlates well with H3K9me1and H3K9me2, and other studies, as well as our own, have shownthat HPL-2 can bind H3K9me peptides in vitro (SupplementalFig. S4; Wirth et al. 2009; Koester-Eiserfunke and Fischle 2011;Studencka et al. 2012). To test if the localization of HPL-2 and itspersistenceonchromatindependonaninteractionwithH3K9me,we performed HPL-2 ChIP-chip using extracts prepared from mu-tant embryos lacking the two H3K9 histone methyltransferasesMET-2andSET-25.Towbinetal.previouslyreportedthat met-2set-25  double-mutant embryos lack detectable H3K9me1, me2, andme3 (Towbin et al. 2012). Because a recent paper identified  C.elegans  SET-26 as another H3K9 histone methyltransferase (Greeretal.2014),werepeatedmeasurementofH3K9me1,me2,andme3levels in  met-2 set-25  double-mutant embryos by both immuno-staining and mass spectrometry (Supplemental Fig. S5A,B). Inagreement with Towbin et al., H3K9me was below the limit of detection in  met-2 set-25  mutant embryos.Surprisingly, the distribution of HPL-2 in  met-2 set-25  mutantembryos was similar to that in wild type, although dampened. Togenerate the most comparable profiles of HPL-2 in wild-type and met-2 set-25  samples, ChIP signal was normalized relative to re-gions outside of HPL-2-enriched domains and not bound by HPL-2;thoseregionswouldnotbeexpectedtodifferbetweenwildtypeand mutant. At the genomic level, the distribution of HPL-2 overthe length of each chromosome in  met-2 set-25  embryos was verysimilar to wild type (genome-wide PCC  =  0.79) but dampened (Fig.2A). Importantly, different normalization methods yielded similarresults (Supplemental Fig. S6A,B), indicating that the HPL-2 ChIP-chip signal present in  met-2 set-25  embryos is indeed substantial.Consistentwiththesefindings,HPL-2showedsimilarchromosomal Genome Research 77 www.genome.org Heterochromatin in  C. elegans   Cold Spring Harbor Laboratory Presson April 25, 2018 - Published by genome.cshlp.orgDownloaded from   Figure 1.  Features of heterochromatin are concentrated on autosomal arms and the  leftmost   region of the X chromosome in  C. elegans  . ( A )Chromosomal heatmaps depicting median  Z  -scores of ChIP-chip signal over 2-kbp regions for HPL-2, H3K9me1, H3K9me2, H3K9me3, H3K27me3, andLEM-2, along with standardized repetitive element densities and gene densities over 10-kbp regions. Red indicates enrichment, blue indicates depletion.HPL-2-enriched arms are marked by black bars. Validations of anti-HPL-2 antibody and ChIP-chip of HPL-2 from  hpl-2  mutant embryos are shown inSupplementalFigureS1.( B  )Genome-widePearsoncorrelationcoefficients(PCCs)formedian Z  -scoresofChIP-chipsignalbetweenpairsofthechromatinmarks and genomic features shown in panel  A . PCCs for regions that lie either in HPL-2-enriched arms or in chromosome centers are shown in Supple-mentalFigureS3.( C  )ImmunofluorescenceimagesshowingDNA(blue),H4K12ac(green),andHPL-2(red)stainingofwild-typehermaphroditegermlinenuclei. Arrows point to paired X chromosomes. Scale bar, 2  m m. Garrigues et al. 78 Genome Research www.genome.org  Cold Spring Harbor Laboratory Presson April 25, 2018 - Published by genome.cshlp.orgDownloaded from   staining in wild-type and  met-2 set-25  mutant nuclei (Fig. 2B). Weconclude that, despite the extensive colocalization of HPL-2 andH3K9meinwild-typecellsandtheabilityofHPL-2tobindH3K9mepeptides (Supplemental Fig. S4; Wirth et al. 2009; Koester-EiserfunkeandFischle2011;Studenckaetal.2012),H3K9meisnotessentialforHPL-2 to associate with chromatin. However, as the levels of HPL-2bound to chromatin appear to be reduced in  met-2 set-25  mutantsrelative to wild type, H3K9me may have roles in promoting therecruitment or retention of HPL-2.TheChIPanalysisdescribedaboveshowsthatH3K9meisnotnecessaryforHPL-2binding.Tocomplementthatanalysisandtestif in wild type HPL-2 preferentially associates with chromatinenriched for H3K9me, we took advantage of male germline nuclei,in which the single unpaired X chromosome in each pachytenenucleus is dramatically enriched for H3K9me2 relative to the auto-somes (Kelly et al. 2002). If H3K9me2 recruits HPL-2 to chromatin,we would expect to see an enrichment of HPL-2 on unpaired Xchromosomes as well. We observed that HPL-2 is not concentrated Figure2.  HPL-2associateswithchromatinindependentlyofH3K9me.( A )Normalizedmean Z  -scoresforHPL-2ChIP-chipsignaloverthelengthofeachchromosomeinwild-type(WT)embryosand met-2set-25 doublemutantembryos,whichlackH3K9me(SupplementalFig.S5).Thegenome-widePCCof HPL-2 ChIP-chip signal between WT and  met-2 set-25  is 0.79. Chromosome arms are marked with black bars (see Fig. 1). Supplemental Figure S6 showsdifferently normalized HPL-2 ChIP-chip signal. ( B  ) Immunofluorescence images of DNA, H3K9me2, and HPL-2 in WT and  met-2 set-25  hermaphroditegermline nuclei. WT and  met-2 set-25  samples were stained in parallel, and images were acquired using identical settings. Scale bar, 2  m m. ( C  ) Immu-nofluorescence images of DNA (blue), H3K9me2 (green), and HPL-2 (red) in two WT male (XO) germline nuclei. Arrows point to single unpaired Xchromosomes. Scale bar, 2  m m. ( D ) Percentage of hermaphrodites that were fertile (gray) or sterile (black) at 25 ° C in wild type (WT),  hpl-2  M+Z-,  hpl-2 M-Z-,  met-2 set-25  M+Z-, and  met-2 set-25  M-Z-. (M) Maternal supply of gene product, (Z) zygotic synthesis of gene product. Heterochromatin in  C. elegans  Genome Research 79 www.genome.org  Cold Spring Harbor Laboratory Presson April 25, 2018 - Published by genome.cshlp.orgDownloaded from   onand infactappearstobe absent from unpairedX chromosomes,despite their heavy enrichment for H3K9me2 (Fig. 2C). This ob-servation demonstrates that H3K9me2 is not sufficient to recruitHPL-2 to chromatin.Previous experiments demonstrated that  hpl-2  mutantsdisplay maternal-effect sterility at elevated temperature (25 ° C)(Coustham et al. 2006). Even though the distribution of HPL-2 in met-2 set-25 mutantslackingH3K9meappearssimilartowildtype,mutant worms may have impaired HPL-2 function in the absenceof H3K9me. To test this possibility, we compared the fertility of  met-2 set-25  M+Z- and M-Z- mutants at elevated temperature withthe fertility of   hpl-2  M+Z- and M-Z- mutants, where M representsa maternal supply and Z represents zygotic synthesis of the geneproduct (Fig. 2D). Similar to the absence of detectable H3K9me in met-2 set-25  embryos (Supplemental Fig. S5), immunostaining of  met-2 set-25  adult hermaphrodite germlines did not show any de-tectable H3K9me1 (data not shown), H3K9me2 (Fig. 2B), orH3K9me3(Hoetal.2014).Asexpected,0%of  hpl-2  M+Z-mutantsand 100% of   hpl-2  M-Z- mutants raised at 25 ° C were sterile. In-terestingly, 4% of   met-2 set-25 M+Z- mutants and only 32% of   met-2set-25  M-Z- mutants raised at 25 ° C were sterile, indicating that met-2 set-25  mutants display only weak maternal-effect sterility.Observing that  met-2 set-25  mutants showsignificantly lesssterilityin the M-Z- generation than  hpl-2  mutants suggests that HPL-2 canpromote fertility in the absence of H3K9me. Conversely, the pub-lished finding that  met-2 hpl-2  double mutants have more severedefects than either single mutant (Andersen and Horvitz 2007)suggests that H3K9me has HPL-2-independent roles and that re-duction of H3K9me and loss of HPL-2 lead to additive defects. HPL-2 and centromeric chromatin are generally anticorrelated Heterochromatin is typically concentrated in pericentric regions(Hennig 1999). In  Drosophila , heterochromatin boundaries havebeen shown to be hotspots for ectopic centromere formation(Olszaketal.2011),suggestingthatheterochromatinhelpstodefinecentromeres.  C. elegans  chromosomes are holocentric, with centro-meres distributed along their lengths (Albertson and Thomson1982), raising the interesting question of how the distribution of HPL-2 relates to dispersed centromeric regions. To explore this, wecompared the distributions of HPL-2 and the  C. elegans  CENP-Ahomolog HCP-3 (also known as CeCENP-A), a highly conservedcentromeric histone H3 variant (Buchwitz et al. 1999), in HPL-2-enriched arms and HPL-2-depleted centers. Using publishedCeCENP-AChIP-chipdataobtainedfromsimilarembryonicstages(Gassmann et al. 2012), we observed that, in arms, HPL-2 andCeCENP-A are anti-correlated: High HPL-2 signal is generally presentin regions with low CeCENP-A signal, and vice versa (Fig. 3A). Byexamining HPL-2 levels at the borders of CeCENP-A domains(Gassmannetal.2012),wefoundthat,inarms,CeCENP-AdomainsareflankedbyelevatedHPL-2levels(Fig.3B,C),creatingregionsthatresemble pericentric heterochromatin found in other organisms.However, in chromosome centers, CeCENP-A domains are notflanked by elevated HPL-2 (Fig. 3B,C), suggesting that HPL-2 is notcritical for centromere formation. HPL-2 peaks are enriched for repetitive sequences HP1proteinsinfissionyeast,flies,andmammalshavebeenshownto associate with regions containing or flanked by repetitive DNAelements (Partridge et al. 2000; Guenatri et al. 2004; de Wit et al.2005). Consistent with HPL-2 binding sites overlapping with re-petitive elements, we observed HPL-2-enriched arms to also beenriched for repetitive elements (Fig. 1A). Interestingly, aftercomparing HPL-2 peaks called using MA2C (Song et al. 2007) torepetitive regions, we found that HPL-2 peaks residing in chro-mosome arms as well as centers are enriched for repetitive ele-ments (genome in arms  =  18% repeats, HPL-2-bound regions  = 20% repeats; genome in centers  =  6% repeats, HPL-2-bound re-gions  =  15% repeats) (Fig. 4A). To determine if specific classes of repetitive elements are enriched for HPL-2 binding, we identifiedthe 10 species of repetitive elements with the highest numbers of individual repeats overlapping HPL-2 peaks (Fig. 4B). Strikingly,the repetitive elements CeRep5 and PALTTAA2 have 93% (1241/1339) and 62% (771/1236) of their individual repeats throughoutthegenomeboundbyHPL-2,respectively.Notably,mostofthetop10 species of HPL-2-bound repeats have higher fractions of in-dividualrepeatsboundbyHPL-2inarmscomparedtocenters(Fig.4B). These observations suggest that specific types of repetitiveelementsmay serve an importantrole in helpingto define regionswhereHPL-2binds.However,asmanyHPL-2peaksdonotoverlapwith any identified repetitive elements (data not shown), possiblerepeat-centric mechanisms of HPL-2 binding cannot account forall observed HPL-2 peaks. Interestingly, four of the top 10 speciesof HPL-2-bound repeats are transposons or possible transposons(PALTTAA2, PALTA5, HelitronY1A, and CELE1). Perhaps bindingof HPL-2 participates in preventing their expression. HPL-2 peaks in chromosome arms cover different genomicfeatures than HPL-2 peaks in chromosome centers To identify genic features on which HPL-2 may be enriched ordepletedrelativetotherestofthegenome,wedeterminedthetotalnumber of base pairs of all HPL-2 peaks that overlap defined ge-nomic categories: promoter, genic, exon, intron, and intergenic.Interestingly, we found that the prevalence of genomic featurescoveredbyHPL-2peaksresidinginarmsdiffersfromthoseresidingin chromosome centers. For example, HPL-2 peaks in arms areenrichedforintronregions(genomeinarms = 39%introns,HPL-2-boundregions = 49%introns),whileHPL-2peaksincentersarenot(genome in centers  =  30% introns, HPL-2-bound regions  =  19%introns) (Fig. 4A). Conversely, HPL-2 peaks in centers are enrichedfor promoter regions (genome in centers  =  10% promoter, HPL-2-boundregions = 28%promoter),whileHPL-2peaksinarmsarenot(genome in arms  =  9% promoter, HPL-2-bound regions  =  9% pro-moter) (Fig. 4A). Figure 4C shows individual genes with strikingintron enrichment or promoter enrichment of HPL-2. As HPL-2peaksareenrichedforgenicandpromoterregions,wehypothesizethat HPL-2 may serve direct roles in regulating at least some of thegenes to which it binds. HPL-2 preferentially associates with well-expressed genes Although HP1 is best known for its role in generating repressedchromatin, HP1 proteins have been observed to associate with ac-tivelyexpressedgenes.Forexample,in  Drosophila ,HP1Aisenrichedoverwell-expressedgenes(deWitetal.2007),andHP1Cisfoundinregions of active chromatin, where it promotes gene expression byinteracting with the transcriptional machinery (Kwon et al. 2010).TodeterminewhetherHPL-2preferentiallyassociateswithrepressedor active genes, we compared the levels of histone modificationsassociated with repressed (H3K9me2, H3K9me3, H3K27me3) oractive (H3K4me3, H3K36me3) transcriptional states, as well as RNApolymerase II (Pol II) and mRNA levels from previously publisheddata(Rechtsteineretal.2010;Liuetal.2011),onHPL-2-boundgenesandgenesnotboundbyHPL-2(Fig.5A).WefoundthatHPL-2-bound Garrigues et al. 80 Genome Research www.genome.org  Cold Spring Harbor Laboratory Presson April 25, 2018 - Published by genome.cshlp.orgDownloaded from 
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