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Distinct roles of KAP1, HP1 and G9a/GLP in silencing of the two-cell-specific retrotransposon MERVL in… Maksakova, Irina A; Thompson, Peter J; Goyal, Preeti; Jones, Steven J; Singh, Prim B; Karimi, Mohammad M; Lorincz, Matthew C Jun 4, 2013

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RESEARCH Open AccessDistinct roles of KAP1, HP1 and G9a/GLP insilencing of the two-cell-specific retrotransposonMERVL in mouse ES cellsIrina A Maksakova1, Peter J Thompson1, Preeti Goyal1, Steven JM Jones2, Prim B Singh3,Mohammad M Karimi1,2 and Matthew C Lorincz1*AbstractBackground: In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenousretroviruses (ERVs), including IAP, ETn and MMERVK10C, is dependent upon the H3K9 methyltransferase (KMTase) SETDB1/ESET and its binding partner KAP1/TRIM28. In contrast, the H3K9 KMTases G9a and GLP and HP1 proteins are dispensablefor this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but themolecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed.Results: Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10Cand IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively lowlevels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1influences MERVL expression independent of SETDB1. In contrast to class I and class II ERVs, MERVL and MERVL LTR-driven genic transcripts are also upregulated following depletion of G9a or GLP, and G9a binds directly to these ERVs.Consistent with a direct role for H3K9me2 in MERVL repression, these elements are highly enriched for G9a-dependentH3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressedin HP1α and HP1β KO ESCs. However, like KAP1, HP1α and HP1β are only modestly enriched at MERVL relative to IAPLTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed inthe 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes isa common feature in each of these KO lines.Conclusions: Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, arerecruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalyticactivity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1β KO ESCs may occur predominantly as aconsequence of indirect effects, in association with activation of a subset of 2C-specific genes.Keywords: G9a, GLP, HP1, KAP1, MERVL, ERV, Chimeric transcripts, Preimplantation, H3K9me2, mESCBackgroundEndogenous retrovirus-like sequences (ERVs) are fossils ofancient retroviral integrations into the mammaliangermline. Multiple independent colonization events haveled to the accumulation of over 400 different ERV familieswith defined transcriptional patterns, often limited to spe-cific developmental stages and cell types. Based on thesimilarity of their reverse transcriptase genes, ERVs aregrouped into three classes: I, II and III, most closely re-lated to exogenous gammaretroviruses, betaretrovirusesand spumaretroviruses, respectively [1]. Most ERVs ineach class are no longer capable of transcription and/orretrotransposition due to the accumulation of mutationsand/or efficient targeting by host silencing mechanismsthat act at various stages of the viral life cycle [2]. Never-theless, many ERVs possess functional regulatory se-quences that direct transcription at specific developmentalstages and/or in specific tissues. A number of ERV* Correspondence: mlorincz@mail.ubc.ca1Department of Medical Genetics, Life Sciences Institute, 2350 HealthSciences Mall, University of British Columbia, Vancouver, British Columbia V6T1Z3, CanadaFull list of author information is available at the end of the article© 2013 Maksakova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.Maksakova et al. Epigenetics & Chromatin 2013, 6:15http://www.epigeneticsandchromatin.com/content/6/1/15subfamilies are particularly active early in embryogenesis[3], likely due to selection for expression and in turnretrotransposition at those stages in development thatmaximize the likelihood of germline transmission.The class III MT subfamily of MaLR retrotransposons,for example, which comprise less than 5% of the mousegenome [4], account for 13% of all transcripts in the fullygrown oocyte [3]. The related class III ERV MERVL/MuERV-L (mouse ERV with a leucine tRNA primer-binding site), of which there are 656 full-length copies and37,172 solitary long terminal repeats (LTRs) in the C57BL/6 genome (based on Repeatmasker analysis), are among thefirst sequences to be transcribed in the early two-cell (2C)embryo and account for nearly 4% of the mouse transcrip-tome at the 2C stage [3,5-7]. Class II intracisternal A-typeparticle (IAP) ERVs on the other hand, of which there arewell over 600 full-length copies in the mouse genome, onlyaccount for 0.6% of the 2C transcriptome [3]. Intriguingly,MERVL expression may be essential for development be-yond the four-cell stage [5], perhaps due to the exaptationof ERV LTRs as promoters for essential genes [8,9]. Indeed,MERVL-driven genic transcripts are abundant at the 2Cstage [3,10] and in mouse embryonic stem cells (mESCs)that show 2C-like features [11,12]. Given that such tran-scripts are not detectable at later developmental stages, it islikely that the LTR promoters of such ‘chimeric’ genes areregulated by the same epigenetic mechanisms that governthe ERVs from which they are derived.To minimize the generally deleterious effects associatedwith retrotransposition, a number of pathways have evolvedto inhibit transcription of ERVs. DNA methylation, mediatedby the de novo methyltransferases DNMT3A and DNMT3Band the maintenance methyltransferase DNMT1, plays acritical role in proviral silencing in somatic tissues [13-15]including fibroblasts [16,17], as well as in late germline de-velopment [18,19]. Surprisingly however, while class I and IIERVs show broad DNA demethylation in G9a and GLPknockout (KO) mESCs [20], neither of these lysinemethyltransferases (KMTases), which dimethylate lysine 9 ofhistone H3 (H3K9), are required for silencing of these ERVs.Furthermore, while IAP transcript levels are elevated inDNMT1-deficient relative to wild-type (wt) mESCs [21,22],this difference increases dramatically in mESCs cultured inthe absence of leukemia inhibitory factor (LIF) [21], indicat-ing that a DNA methylation-independent mechanism mayalso operate in undifferentiated ESCs to silence such ERVs.Indeed, we recently reported that in mESCs, numerousclass I and II ERVs, including MMERVK10C, MusD andETn elements, are de-depressed in the absence of theH3K9 KMTase SETDB1/ESET, while IAP elements showthe highest level of activation in the absence of bothDNMT1 and SETDB1 [23,24]. Furthermore, robust silen-cing of each of these ERVs is dependent upon H3K9me3deposited by SETDB1 [24,25] and the corepressor KAP1/TRIM28/TIF1-β, which directly interacts with the KAP1interaction domain (KID) of SETDB1 [26]. As KAP1 candirectly interact with any one of several hundred Krüppel-associated box zinc finger proteins (KRAB-ZFPs), we andothers have proposed that the KAP1/SETDB1 complexmay generally be recruited to ERVs by KRAB-ZFPs thatrecognize specific ERV sequences [23,24,27-30]. Curiouslyhowever, while class III MERVL elements are alsoupregulated in KAP1 KO mESCs [28], we observed onlymodest upregulation of these ERVs (approximately 2-fold)in SETDB1 KO mESCs [23].Heterochromatin protein 1 (HP1) proteins, which en-code chromo- and chromoshadow domains, function inboth structural and gene regulatory pathways in eukary-otes [31-33]. These H3K9me ‘readers’ modulate gene ex-pression in part through binding to H3K9me2/3 via thechromodomain [34-39]. In addition, HP1 proteins directlyinteract with the PxVxL motif of KAP1 via theirchromoshadow domain independent of H3K9 methylationstate [29,40-42]. Intriguingly, this interaction is requiredfor transcriptional silencing of reporter genes [34,43] aswell as of the nonimprinted Mest allele in embryonal car-cinoma cells [42]. Surprisingly however, we recentlyshowed that depletion of HP1α (encoded by Cbx5), HP1β(encoded by Cbx1) and/or HP1γ (encoded by Cbx3), aloneor in combination, does not lead to derepression of class Ior class II ERVs in mESCs [25], raising the question: doHP1 proteins play a role in repression of class III ERVs?Here, using genetic knockouts and/or RNAi, we analyzedthe roles of KAP1, HP1α, HP1β as well as G9a and GLP, intranscriptional silencing of MERVL elements in mESCs.Our results indicate that MERVL expression is induced as aconsequence of both direct and indirect effects, the formerdue to loss of H3K9me2 and the latter in association withderepression of genes normally expressed at the 2C stage.ResultsClass II ERVs are upregulated in KAP1- and SETDB1-deficient cells, while MERVL ERVs are upregulatedexclusively in KAP1-deficient cellsWe recently showed that silencing of many class I and IIERV families is maintained in mESCs by SETDB1-mediated deposition of H3K9me3 [23,24]. Consistent witha previous report [28], we also found that the SETDB1-associated corepressor KAP1 is required for repression ofmany of the same ERVs [24], supporting an essential rolefor the KAP1-SETDB1 complex [44] in proviral repression.Curiously however, while MERVL elements were alsoreported to be dramatically upregulated in KAP1 KOmESCs [11,28], our genome-wide analysis revealed min-imal derepression of this class III ERV family in SETDB1KO mESCs [23]. Reanalysis of RNA-sequencing (RNA-seq)data from SETDB1 KO [23] and KAP1 KO mESCs [28],confirmed that many class I and II families are derepressedMaksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 2 of 16http://www.epigeneticsandchromatin.com/content/6/1/15in both cell lines (Figure 1A-B). IAPEz and MMERVK10C(a close relative of IAP [45]) ERVs, for example, areupregulated 14- and 100-fold, respectively, in the SETDB1KO, versus 24- and 26-fold, respectively, in the KAP1 KO.In contrast, while MERVL elements, composed of threeannotations in the UCSC genome browser: MT2_Mm(LTR), MERVL-int (internal region) and ORR1A3-int(Figure 1A), are upregulated 27-fold in the KAP1 KO, theyare upregulated less than 3-fold in the SETDB1 KO(Figure 1B). Knockdown (KD) of Setdb1 or Kap1 by RNAifollowed by qRT-PCR yielded similar results (Figure 1C),ruling out the possibility that the distinct phenotypesobserved in SETDB1 versus KAP1 KO mESCs are due todifferences in genetic background. Thus, while silencing ofclass III MERVL elements is indeed dependent uponKAP1, depletion of SETDB1 has a relatively modest effecton MERVL expression, revealing that KAP1 plays a role insilencing of this ERV subfamily independent of SETDB1.To determine whether MERVL elements are bound byKAP1 and/or SETDB1, we performed meta-analysis ofpublished KAP1 and SETDB1 chromatin immunopre-cipitation sequencing (ChIP-seq) datasets [46,47]. Nu-merous class I and class II ERVs, including IAPsubfamilies, show significant enrichment of both factorsand a strong positive correlation between the two(Figure 1D), consistent with our previous observationsthat these ERV families are marked by H3K9me3 in aSETDB1-dependent manner [23]. In contrast, MERVL isone of a small group of ERVs showing no detectable en-richment of SETDB1 and low levels of cumulative KAP1binding relative to most SETDB1-bound class I and IIERVs (Figure 1D). While class III MaLR ERVs ORR1AFigure 1 MERVL ERVs are derepressed upon KAP1 but not SETDB1 depletion, while MMERVK10C and IAP ERVs are upregulatedfollowing depletion of both. (A) Repbase annotations of the LTR and internal regions of full-length MERVL, IAPEz and MMERVK10C elements areshown. Black bars indicate qPCR amplicons for LTRs of each family of element and the internal pol gene region for MERVL. (B) Deregulation oftransposable element families in KAP1 and SETDB1 KO mESCs. RNA-seq data for SETDB1 [23] and KAP1 [28] KO lines and the corresponding wtcell lines were used to calculate Z-score values for all annotated retroelements and plotted as shown. (C) KAP1 and SETDB1 were depleted byRNAi in wt TT2 mESCs, and reactivation of MERVL and MMERVK10C elements was determined by qRT-PCR. Mean expression (+/−SD) of each ERV(normalized to β-actin) relative to a scrambled siRNA pool (Scram) is shown for three technical replicates. (D) MERVL is among a small group oftransposable elements bound by KAP1 but not SETDB1. RPKM (*10) values generated from published ChIP-seq data for KAP1 [46] and SETDB1[47] were plotted for all retroelements. Numerous class I and class II ERVs, including IAP subfamilies are enriched for both proteins, which aregenerally strongly correlated. MERVL elements are modestly enriched for KAP1, but show relatively low levels of SETDB1 coverage. ChIP-seq,chromatin immunoprecipitation sequencing; RPKM, reads per kilobase per million mapped reads.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 3 of 16http://www.epigeneticsandchromatin.com/content/6/1/15and MTA are enriched for KAP1 at levels similar toMERVL, only MERVL is upregulated in KAP1 KOmESCs. To determine whether KAP1 binding at MERVLelements is associated with enrichment of KAP1 in theunique regions flanking these proviruses, we analyzedKAP1 enrichment in the nonrepetitive sequencesflanking all 656 full-length MERVL, 298 MMERVK10Cand 599 IAPEz elements. As shown previously forH3K9me3 [25,48], KAP1 binding is clearly higher in theimmediate flanks of IAP elements when analyzed in ag-gregate, decreasing to background levels with increasingdistance to the provirus (Figure 2A). MMERVK10CFigure 2 Unique regions flanking IAPEz but not MERVL elements are highly enriched in KAP1. (A) Profiling of KAP1 in the flankingsequence of all full-length IAPEz, MERVL and MMERVK10C ERVs. KAP1 ChIP-seq reads [46] from wt mESCs were aligned to the mouse genome(mm9), and the density profile of unique reads mapping to the 6 kb regions flanking all annotated intact MERVL (656), MMERVK10C (298) andIAPEz (599) elements, was plotted as shown. (B-C) Heat maps of KAP1 enrichment in the genomic regions flanking 599 IAPEz and 656 MERVLelements in wt mESCs. KAP1 ChIP-seq reads [46] were aligned to the mouse genome (mm9), and the density of uniquely aligned reads, mappingto the 6 kb regions flanking all intact ERVs of the specified families, was plotted. Reads extending into the ERV are due to in silico extension ofaligned reads by 300 bp. (D) ChIP and qPCR analysis of KAP1 in TT2 wt mESCs at the LTRs of IAPEz, MMERVK10C and MERVL, as well as theMERVL pol internal region. IgG, negative control IP. Data are mean enrichment from three technical replicates as a percentage of the inputchromatin and error bars represent SD. IgG, immunoglobulin G; IP, immunoprecipitation; SD, standard deviation.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 4 of 16http://www.epigeneticsandchromatin.com/content/6/1/15elements also show enrichment in proximal versus distalflanking regions, although to a lesser extent. This patternis common to multiple individual IAP elements (Figure 2B);consistent with the hypothesis that KAP1 binds directly tothis class II ERV and spreads into neighboring genomic re-gions. In contrast, enrichment of KAP1 is not detected atthe flanks of MERVL elements when analyzed in aggre-gate (Figure 2A) and is sparsely detected at the flanks ofonly a small fraction of MERVL elements when analyzedindividually (Figure 2C). Furthermore, in contrast to thesequences flanking individual IAP elements, enrichment isevenly distributed across the 6 kb regions flanking the fewindividual MERVL elements that show KAP1 binding intheir flanks, indicating that the low level of KAP1 enrich-ment observed within these elements may reflect thechromatin state of the locus/integration site, rather thandirect recruitment of KAP1 to these elements. Alterna-tively, in the absence of SETDB1-mediated H3K9me3deposition, spreading of KAP1 into the regions flankingMERVL elements may not occur, leading to a relativelylow level of focal KAP1 enrichment within the regulatoryregion of these ERVs. As an alternative approach to quan-tify KAP1 enrichment specifically within MERVL elements,we conducted ChIP using a KAP1-specific antibody.Consistent with the ChIP-seq data, KAP1 enrichment wasdetected at IAP and to a lesser extent at MMERVK10CLTRs (Figure 2D). In contrast, no enrichment wasdetected at LTR or pol internal regions of MERVL, usingprimers that detect 519 and 637 elements, respectively,as determined by in silico PCR. While we cannot ruleout the possibility that relatively weak, localized bindingof KAP1 to MERVL LTRs plays a direct, SETDB1-independent role in silencing of these elements,SETDB1/H3K9me3 is routinely observed at repressednative loci and transgenes bound by KAP1 [23,26,29].Furthermore, KAP1 mutants that cannot interact withSETDB1 are defective in silencing of KAP1-boundtransgenes [26]. Thus, our observations are also consis-tent with the hypothesis that derepression of MERVLelements in KAP1-deficient mESCs occurs as a conse-quence of indirect effects.MERVL elements and chimeric transcripts are upregulatedin G9a and GLP KO mESCsWhile we have shown previously that silencing of class Iand II ERVs is not dependent upon G9a [20], we did not ad-dress whether this H3K9me1/2 KMTase plays a role in re-pression of class III ERVs. As MERVL elements wererecently shown to be marked by H3K9me2 [11], we nexttested whether G9a and the closely related KMTase GLP,Figure 3 MERVL ERVs are derepressed in G9a- and GLP-deficient mESCs and MERVL silencing is dependent on G9a catalytic activity.(A) Upregulation of MERVL in G9a and GLP KO mESCs. Expression of MERVL, MMERVK10C and IAPEz ERVs in TT2 wt, G9a and GLP KO mESCs wasanalyzed by qRT-PCR. Mean (+/−SD) expression levels relative to the wt line for three technical replicates (normalized to β-actin) are shown. (B)Catalytic activity of G9a but not GLP is required for MERVL silencing. G9a or GLP KO mESCs stably expressing wt or catalytic mutant G9a (C1168A)or GLP (C1201A) [50] transgenes, respectively, were assessed for MERVL expression by qRT-PCR, as described above. (C) MERVL but notMMERVK10C or IAPEz ERVs are upregulated upon KD of Glp. Relative expression of ERVs was determined by qRT-PCR, as described above. (D)Efficiency of each KD was determined by qRT-PCR with primers specific for Kap1 and Glp, as described above. SD, standard deviation.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 5 of 16http://www.epigeneticsandchromatin.com/content/6/1/15which form a heterodimer [49], are required for silencing ofMERVL ERVs. Quantitative RT-PCR analysis of G9a KOand GLP KO mESCs revealed that MERVL elements areupregulated approximately 8-fold and approximately 13-foldrespectively, relative to their parent line TT2 (Figure 3A). Incontrast, MMERVK10C, similar to IAP ERVs (Figure 3Aand [20]), showed no change in expression in either the G9aor GLP KO lines. Analysis of G9a or GLP KO mESCs stablyexpressing wt or catalytic mutants of G9a (G4, C1168A) orGLP (L4, C1201A), respectively [50], revealed that MERVLsilencing is dependent only upon catalytically active G9a(Figure 3B). This result may be explained by the fact thatwhile G4 and L4 catalytic mutants form heteromeric com-plexes with wt GLP and G9a, respectively, H3K9me2 levelsare restored only in the GLP KO line rescued with the L4catalytic mutant [50], implicating G9a as the critical H3K9KMTase in the context of the G9a/GLP heterodimer. Tran-sient depletion of GLP also disrupts MERVL silencing(siRNAs directed against G9a did not yield efficient deple-tion of G9a mRNA), with a 14-fold increase in MERVLexpression observed 4 days post siRNA transfection(Figure 3C-D). In contrast, as expected, no increase inMMERVK10C or IAPEz expression was observed follow-ing KD of Glp. While MERVL expression was induced ap-proximately 16-fold following KAP1 KD, MMERVK10Cwas induced only approximately 5-fold in this experimentand IAPEz – only 1.5-fold, likely due to DNA methylation-mediated repression [23] and/or insufficient depletion ofthe protein. Importantly, KAP1 and LSD1 protein levelsare not reduced in GLP or G9a KO mESCs (Additionalfile 1: Figure S1A), indicating that derepression of MERVLelements in these cells is not due to destabilization ofthese proteins, which were previously implicated inMERVL silencing [11,28]. Furthermore, while interactionsbetween KAP1 and HP1β and G9a and HP1β were clearlydetected by co-immunoprecipitation (co-IP), as reportedpreviously [40,41,51], no interaction between KAP1 andG9a was detected under the same conditions (Additionalfile 1: Figure S1B), indicating that G9a is unlikely to regu-late MERVL elements via direct interaction with KAP1.LTRs that are derepressed in mESCs deficient in SETDB1or the H3K4me1/2 demethylase LSD1/KDM1A can func-tion as alternative promoters for downstream genes [11,23],and the specific ERV families upregulated in these KOscontribute to the majority of such aberrantly expressedchimeric transcripts. To determine whether naturally oc-curring chimeric transcripts that initiate in MERVL/MT2LTRs and splice to downstream genic exons are alsoupregulated in G9a- or GLP-depleted mESCs, we first iden-tified all protein-coding genes represented in both RefSeqand ENSEMBL databases with an MT2 LTR as the anno-tated exon 1 (Additional file 2). Among the 43 genes on thislist, 10 and 6 were upregulated >4-fold in KAP1 and G9aKO mESCs, respectively, relative to their wt parent lines, asdetermined by meta-analysis of previously published RNA-seq data [12,28]. Strikingly, of the six upregulated genes inthe G9a KO line, five are also upregulated in the KAP1 KOline. The MT2B LTR-driven gene Zfp352, which isupregulated dramatically in both of these KO lines, is in-duced approximately 80-fold in Glp KD mESCs, as deter-mined by qRT-PCR analysis (Additional file 1: Figure S1C),confirming that at least a subset of MERVL LTR-drivengenic transcripts are silenced by G9a and GLP. Together,these data demonstrate that the G9a/GLP complex is notonly required for silencing of intact MERVL elements butalso plays a critical role in silencing of a subset of the anno-tated genes that initiate in MERVL LTRs.MERVL elements are direct targets of the G9a/GLP H3K9KMTase complexTo determine whether G9a is directly bound at MERVL el-ements, we performed cross-linked ChIP in wt and G9aKO mESCs using a G9a-specific antibody. In contrast toKAP1, G9a was specifically enriched in the LTR and polgene regions of MERVL (Figure 4A) and was also detectedat IAPEz and MMERVK10C LTRs (Figure 4B). Import-antly, G9a was depleted at these regions in the G9a KOline, confirming the specificity of this antibody. To deter-mine whether MERVL elements are marked by H3K9me2in a G9a-dependent manner, we analyzed all three states ofH3K9 methylation in wt and G9a KO mESCs by nativeChIP (N-ChIP). H3K9me2 is highly enriched at theMERVL 5′LTR/promoter region in wt mESCs and reducedto near background levels in G9a KO mESCs (Figure 4C).In contrast, the 5′LTR/promoter regions of MMERVK10Cand IAPEz ERVs show relatively low but clearly detectablelevels of H3K9me2 in wt mESCs (Figure 4D-E), as shownpreviously [20]. The converse is true for H3K9me3, con-sistent with our previous observations that class II ERVs,including IAPEz and MMERVK10C, are directly regulatedby SETDB1 [23,24] and the results presented above, whichreveal that MERVL elements are directly regulated by G9aand GLP. Taken together, these results indicate that theG9a/GLP heterodimer plays a direct role in silencing ofMERVL elements.MERVL and MERVL-promoted chimeric genes areupregulated in HP1α and HP1β KO mESCsHP1 proteins are thought to play an important role in tran-scriptional silencing via binding to methylated H3K9, leadingto chromatin compaction and heterochromatin spreading[29,35,37]. While these corepressors have highest affinity forH3K9me3, they can also bind H3K9me2 in vitro and havebeen reported to directly interact with both KAP1 and theG9a/GLP complex [35,40,41,51]. To determine whetherHP1 proteins are required for silencing of MERVL elements,we generated RNA-seq data for mESCs deficient in HP1α orHP1β and the wt parent line HM1 (described in [25]).Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 6 of 16http://www.epigeneticsandchromatin.com/content/6/1/15Intriguingly, MERVL elements are among the most highlyupregulated retrotransposons in both HP1α and HP1βKO lines (Figure 5A), showing increases in expression of6-fold and 11-fold, respectively, relative to the parentHM1 mESC line. These elements are also among the fewERVs upregulated in both HP1 and KAP1 KO mESCs(Additional file 1: Figure S2A). Consistent with the RNA-seq data, qRT-PCR analysis revealed that MERVL ele-ments are upregulated approximately 4-fold and approxi-mately 6-fold in HP1α and HP1β KO mESCs, respectively(Figure 5B). Importantly, analysis of RNA-seq reads thatuniquely align to specific full-length MERVL elements, ofwhich there are 656 genomic copies, reveals that the sameelements are de-repressed in the HP1α KO and HP1β KOlines (Additional file 1: Figure S2B).To determine whether genes that initiate in MERVLLTRs are also derepressed in HP1-depleted ESCs, wecalculated the expression levels of each of the 43 anno-tated MT2-initiated genic transcripts. Four and six ofthese genes were upregulated >4-fold in the HP1α andHP1β KO lines, respectively, relative to the wt parentline HM1 (Additional file 2). Strikingly, four of thegenes in each case were also among the genesupregulated in the KAP1 and G9a KO lines. Zfp352 forexample, showed an increase in expression of 14- and28-fold in the HP1α and HP1β KO lines, respectively.To determine whether additional unannotated genictranscripts (that is transcripts not present in the RefSeqor ENSEMBL databases) initiate in MERVL/MT2 LTRsin HP1 mutant cells, we identified all transcripts in ourpaired-end RNA-seq data in which one of the matepairs aligns to an LTR element and the other to an an-notated genic exon and scored all genes having >5unique support reads for each in HM1 (wt), HP1α andHP1β KO mESCs. While ERVs of all three classes arerepresented among constitutively expressed chimerictranscripts in wt mESCs (Additional file 1: Figure S2C),the number of MERVL LTR-driven chimeric genes isFigure 4 G9a is bound at MERVL and H3K9me2 enrichment at MERVL elements is dependent on G9a. (A-B) ChIP and qPCR of G9a in TT2wt and G9a KO mESCs at LTR of MERVL and MERVL internal region and LTRs of IAPEz and MMERVK10C. IgG, negative control IP. Data are meanenrichment from three technical replicates as a percentage of the input chromatin and error bars represent SD. (C-E) N-ChIP and qPCR wasperformed for H3K9me1 (me1), H3K9me2 (me2), H3K9me3 (me3) and IgG as a negative control in TT2 wt and G9a KO mESCs at the LTRs ofMERVL, IAPEz and MMERVK10C. Data are mean enrichment (+/−SD) for three technical replicates normalized to input. IgG, immunoglobulin G; IP,immunoprecipitation; SD, standard deviation.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 7 of 16http://www.epigeneticsandchromatin.com/content/6/1/15higher in HP1 KO mESCs, with MT2 transcripts con-tributing 19% and 28% of all chimeric transcripts in theHP1α (15 of 78 total) and HP1β KO (28 of 102 total)lines, respectively, compared to 12% (5 out of 43) in wtcells (Additional file 1: Figure S2C). Together, thesedata demonstrate that HP1α and HP1β are required forsilencing of MERVL elements as well as genic tran-scripts that initiate in MERVL LTRs.HP1α and HP1β are only modestly enriched at the 5′LTRof MERVLTo determine whether HP1 proteins are enriched specific-ally at MERVL LTRs, HP1α and HP1β binding was ana-lyzed via ChIP-qPCR using chromatin extracts isolatedfrom HM1 wt and HP1 KO mESCs. An approximately8-fold higher level of enrichment of HP1α was detected inthe 5′LTR region of IAPEz elements relative to the im-munoglobulin G (IgG) control immunoprecipitation (IP),consistent with our previous observations [24] (Figure 5C).This enrichment was lost in HP1α KO mESCs, confirmingthe specificity of the HP1α antibody. Importantly, HP1αbinding was also reduced at IAPEz elements and at thepromoter region of the single-copy imprinted gene Mestfollowing KD of Kap1 (Additional file 1: Figure S2D-E),confirming that HP1α binding to these loci is KAP1-dependent [42]. In contrast, the level of HP1α enrichmentobserved in the 5′LTR region of MERVL elements wasonly approximately 2-fold higher than the IgG control IP(Figure 5D), despite comparable numbers of genomic cop-ies recognized by the MERVL LTR (519 elements) andIAP LTR (638 elements) primer pairs, which were alsoused in the analysis of H3K9 methylation state describedabove. A similar pattern was observed for HP1β, which isenriched approximately 7-fold and 2-fold at IAPEz andMERVL LTRs, respectively (Figure 5E-F). Thus, whileMERVL elements are upregulated in HP1α and HP1β KOmESCs, these KAP1-interacting factors, like KAP1 itself,are enriched at relatively low levels at MERVL LTRs.2C-specific genes are induced in KAP1, G9a, HP1α andHP1β, KO mESCsIntriguingly, an increase in the percentage of cells permis-sive for MERVL expression and upregulation of numeroustranscripts normally expressed at the 2C stage was recentlyreported for KAP1-, LSD1- and G9a-deficient mESCs [12].These observations raise the possibility that MERVLFigure 5 MERVL ERVs are derepressed in HP1α and HP1β KO mESCs but HP1α and HP1β show relatively low enrichment at theseERVs. (A) RNA-seq analysis of retroelement expression in HP1α and HP1β KO mESCs. RNA-seq data for HP1α and HP1β KO mESCs and the HM1parent line was generated, and Z-scores were calculated for all retroelements and plotted. Note that MERVL elements show relatively high levelsof derepression in both KO lines. (B) MERVL elements are upregulated in HP1α and HP1β KO mESCs. Expression of MERVL, IAP and MMERVK10CERVs was analyzed by qRT-PCR in HM1 wt, HP1α and HP1β KO lines. Mean (+/−SD) expression levels (normalized to β-actin) are shown, relativeto the wt line for three technical replicates. (C-F) IAPEz elements show significantly higher levels of HP1α and HP1β enrichment than do MERVLelements. Cross-linked ChIP was performed with antibodies specific for HP1α or HP1β in HM1 and the corresponding KO cell line. IgG was usedas a negative control. The level of enrichment for each IP was determined by qPCR, and the mean and standard deviation of three technicalreplicates are shown for each experiment. IgG, immunoglobulin G; IP, immunoprecipitation; SD, standard deviation.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 8 of 16http://www.epigeneticsandchromatin.com/content/6/1/15elements are derepressed in these KO lines at least in partas a result of indirect effects of establishment of a cellularfate permissive for MERVL transcription. To determinewhether 2C-specific genes are also upregulated in HP1αand/or HP1β KO mESCs, we first generated a list of genesexpressed specifically at this embryonic stage, based onpublished RNA-seq data from single blastomeres [52]. Weidentified genes that are expressed at levels at least 4-foldhigher at the 2C stage than the oocyte or eight-cell stages(oocyte <2C >8C). Of 264 such 2C-‘specific’ genes, 11%and 6% were upregulated relative to their wt parent lines inKAP1 and G9a KO mESCs, respectively (Figure 6A). Strik-ingly, the transcription start sites (TSSs) of nine of the six-teen 2C-specific genes upregulated in the G9a KO lineoverlap with or are within 5 kb of a MERVL/MT2 LTR.Similarly, analysis of the HP1α and HP1β KO RNA-seqdatasets presented above revealed that 7% and 15% of 2C-specific genes are upregulated in these lines respectively,relative to the parent HM1 line. Taken together, 16% (42/264) of 2C genes are upregulated in one or both of theHP1 KO lines (Figure 6B). 2C-specific genes are signifi-cantly overrepresented among the genes upregulated inthese KO mESCs, as only 1% of all genes in the blastomereRNA-seq data (of which there are 26,155) were identifiedas 2C-specific. Furthermore, of the nine 2C-specific genesupregulated in all four KO lines (Figure 6C), seven initiatetranscription either within an MT2 LTR or within 5 kb ofan MT2, MT2A or MT2C LTR, whereas only 4.5% (1387/30321) of all RefSeq genes are within 5 kb of an MT2,MT2A or MT2C annotated LTR.Consistent with the observation that a subset of 2Cgenes are upregulated in these KO lines, genes shown pre-viously to be expressed exclusively in early embryogenesis(confirmed to be 2C-specific in the analysis describedabove), including Zfp352 [53], Zscan4d [54] and Tdpoz3-4[55] (Figure 6D), are upregulated in HP1α and HP1β KOmESCs as well as KAP1 and G9a KO mESCs (Figure 6E).Moreover, the top five genes showing the greatest differ-ence in expression between the 2C stage and the oocyteand 8C stages: Zfp352, Gm5039, Gm8994, B020004J07Rikand Dub1a, also showed dramatic upregulation in HP1αand HP1β KO mESCs (Additional file 1: Figure S3A-B).Taken together, these observations reveal that, like KAP1and G9a KO ESCs, HP1 KO mESCs show a significant in-crease in expression of a specific subset of 2C-specificgenes, many of which are regulated by MERVL LTRpromoters.DiscussionMERVL elements are present in all placental mammals,suggesting that a common mammalian ancestor was colo-nized at least 70 million years ago. Several bursts of amplifi-cation have subsequently occurred in a number of lineages,including the mouse [56], which now harbors 600 to 700full-length copies and approximately 37,000 solitary LTRsin the C57BL/6 genome. Intriguingly, a subset of MERVLLTRs may have been domesticated to serve as gene pro-moters specifically at the two-cell stage, when MERVL LTRpromoters are highly transcribed [3,10]. While a small sub-set of sequences derived from ERVs may play a positive roleby providing regulatory signals or encoding exapted pro-teins, proviral integration events are more likely to com-promise host fitness. To counteract this threat, multiplemechanisms directed at various stages of the viral life cyclehave evolved, including at the transcriptional level [57-60].Our results reveal that distinct H3K9 methylation-basedmechanisms of transcriptional silencing are used againstdifferent ERV families. At numerous class I and II ERVs,SETDB1 is recruited by KAP1 [44,61], which in turn inter-acts with one of multiple KRAB-zinc finger proteins thatpresumably recognize specific sequences within these ERVsto promote H3K9me3-mediated transcriptional silencing[44,61] (Figure 7A). Indeed, these parasitic elements aredramatically upregulated in KAP1- and SETDB1-deficientmESCs [24,28]. The relatively high levels of H3K9me3[23,24], KAP1 and HP1 detected in the LTR and flankinggenomic regions of class I and II ERVs, particularly IAP ele-ments, is most consistent with a ‘spreading’ model, wherebydeposition of H3K9me3 induces binding of HP1 proteins[35,36] and in turn KAP1 and SETDB1, which promotesdeposition of H3K9me3 at neighboring nucleosomes in aprocess that occurs reiteratively. Curiously however, class Iand II ERVs are not derepressed in mESCs deficient in HP1proteins [25], leaving the role of HP1 proteins at class I andII ERVs in question.Unlike class I and II ERVs, MERVL elements are neitherbound by SETDB1 nor marked by H3K9me3, and deletionof SETDB1 does not dramatically induce expression ofthese class III ERVs. Furthermore, while deletion of KAP1,HP1α and HP1β promotes upregulation of MERVL ele-ments, we did not detect significant enrichment of thesefactors at MERVL elements. While we cannot rule out thepossibility that our ChIP assay is not sufficiently sensitiveto detect binding of these chromatin factors to MERVL el-ements, we did detect significant enrichment of KAP1,HP1α and HP1β at IAP elements in the same experiments.A compelling explanation for the apparently distinctmechanisms underlying repression of these ERVs may bethat MERVL elements are not bound by any of the over300 KRAB-ZFPs encoded in the mouse genome [62,63].While the striking diversity in KRAB-ZFPs was likelydriven by selection for KRAB-ZFPs that recognize specificmotifs within ERVs [64], MERVL-specific KRAB-ZFPsmay simply not have evolved since the two bursts ofMERVL retrotransposition in the mouse genome, whichoccurred 2 and 4 million years ago [56].While this manuscript was under preparation, Macfarlanet al. reported that MERVL elements are upregulated inMaksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 9 of 16http://www.epigeneticsandchromatin.com/content/6/1/15G9a KO mESCs [12], although the molecular basis of aber-rant MERVL expression was not addressed. Here, we con-firm this observation, and extend it to include mESCsdeficient in the related KMTase GLP. Furthermore, weshow that MERVL elements are bound by G9a and thatmaintenance of MERVL repression is dependent upon theFigure 6 Two-cell embryo-specific genes are induced in HP1α, HP1β, KAP1 and G9a KO mESCs. A list of two-cell (2C) specific genes wasproduced from single blastomere expression data [52] by identifying genes expressed at levels 4-fold higher at the 2C stage than the oocyte or8C stages (Oo < 2C > 8C). (A-B) Venn diagrams illustrating the overlap between this gene list and the list of genes upregulated at least 4-fold inKAP1, G9a [12,28], HP1α or HP1β KO mESCs are shown. The percentage of genes upregulated in the KO that are also 2C-specific is displayedabove, while the percentage of 2C-specific genes that are also upregulated in the KO are presented below. (C) The nine genes upregulated in allfour KO lines are listed, along with the distance of the nearest MERVL LTR (MT2) to the transcription start site (TSS). (D) Confirmation of theexpression pattern of previously identified 2C-specific genes. RPKM values, derived by division of reads per million (RPM) values from RNA-seqdata generated from pooled single blastomeres [52] by transcript length, are presented for Zfp352, Zscan4d, Zscan4c, Tdpoz3 and Tdpoz4 genes.(E) Expression levels of these 2C-specific genes was determined in G9a, KAP1, HP1α and HP1β KO mESCs as well as their wt parent lines usingour RNA-seq data (HP1α and HP1β) or previously published RNA-seq data (G9a and KAP1 [12,28]), and Z-score values (see Materials and Methods)for each are presented.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 10 of 16http://www.epigeneticsandchromatin.com/content/6/1/15catalytic activity of G9a but not GLP. As these elements arebound by G9a and marked by H3K9me2 in a G9a-dependent manner, we propose that the G9a/GLP complexacts directly at MERVL ERVs to maintain these elements ina silent state (Figure 7A). In contrast to IAP ERVs, MERVLERVs show significantly higher levels of H3K9me2 thanH3K9me3 enrichment. Taken together with the observationthat HP1α and HP1β are detected at IAP but not MERVLERVs, our results indicate that these H3K9 methylation‘readers’ bind preferentially to H3K9me3 marked regionsin vivo, though they are apparently not required forSETDB1-dependent silencing [25].Although the molecular basis of H3K9me2-mediatedtranscriptional repression of MERVL elements remainsto be determined, we propose that this pathway is par-ticularly important in early embryogenesis. Consistentwith this model, G9a and Glp mRNA levels are relativelylow in the 2C embryo (Figure S3C) and H3K9me2 is de-pleted on the paternal genome at this stage [65-67], per-haps explaining why MERVL elements and zygotic genesFigure 7 Overview of transcriptional silencing mechanisms acting at different ERV families and the effect of chromatin factor depletionon mESC fate. (A) The mechanism of transcriptional silencing of class I and II ERVs is distinct from that acting on the class III ERV MERVL. KAP1 isrecruited to numerous class I and II ERVs, including IAP and MMERVK10C elements, via an interaction between the RBCC domain of KAP1 and theKRAB box of KRAB-ZFPs, which presumably bind directly to specific sequences within these ERVs. The SUMOylated bromodomain of KAP1 recruitsSETDB1, which deposits the repressive H3K9me3 mark. MERVL elements in contrast, are bound by G9a and marked by H3K9me2 in a G9a-dependent manner. As these ERVs are upregulated in the absence of G9a or GLP, we propose that the G9a/GLP complex directly regulatesMERVL expression via deposition of H3K9me2. (B) Depletion of specific chromatin factors, including KAP1 and HP1 proteins, may promote MERVLexpression via indirect effects. MERVL transcripts and MERVL-driven genic transcripts are abundant at the 2C embryonic stage, but are rapidlydepleted at subsequent stages, including the blastocyst, from which mESCs are derived. While a small fraction of wt mESCs continually enter atransient state associated with expression of multiple 2C-specific genes, the percentage of these cells increases dramatically in mESCs depleted ofKAP1, G9a/GLP and LSD1 [11,12]. One or more of the 2C-specific genes commonly induced in these cells as well as in HP1α and HP1β KO mESCsmay indirectly promote transcription of MERVL elements and MERVL LTR-driven genes.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 11 of 16http://www.epigeneticsandchromatin.com/content/6/1/15driven by the MERVL promoters are expressed at the2C stage and silenced shortly thereafter [3,10].Intriguingly, depletion of several chromatin factors inmESCs including HP1α and HP1β (this study), G9a/GLP(this study and [12]), LSD1 [11], KAP1 [28], RYBP [68] andZFP42 (REX1) [69], leads to upregulation of MERVL ele-ments. Depletion of a subset of these factors, includingKAP1, G9a, LSD1 and RYBP, also leads to derepression ofgenes highly expressed in preimplantation embryos, suchas Zfp352 and Zscan4 [12,68]. Here, we show that thesame is true for HP1α and HP1β KO mESCs. Derepressionof MERVL elements in mESCs deficient in each of thesechromatin factors may be due at least in part to an increasein the number of cells expressing 2C-specific genes, whichmay in turn stimulate MERVL expression (Figure 7B).Complicating interpretation of these observations is thefact that a significant number of genes expressed specific-ally at the 2C stage initiate transcription within MERVLLTRs, such as Zfp352 [53] and Tdpoz4 [55], or are locatedwithin a few kb of a MERVL LTR [11,12]. Thus, it is notclear whether induction of a subset of the 2C-specificgenes in the above mentioned KO mESCs induce MERVLexpression as a ‘secondary effect’, or whether aberrant genictranscription of 2C genes in these KO lines results pre-dominantly as a direct consequence of perturbation ofMERVL silencing per se. These alternative explanations arenot necessarily mutually exclusive.ConclusionsIn summary, our results indicate that G9a and GLP play adirect role in silencing of MERVL ERVs and genes drivenby MERVL LTR promoters via G9a-mediated depositionof H3K9me2, while the KAP1-interacting H3K9 KMTaseSETDB1 is neither recruited to MERVL elements nor re-quired for their repression. Conversely, the resultspresented here are consistent with the model that induc-tion of MERVL expression following deletion of KAP1,HP1α and HP1β occurs primarily via indirect effects.Given that expression of MERVL elements, unlike otherERVs, is normally restricted to the 2C embryo, any per-turbation of mESCs that induces a nuclear milieu permis-sive for expression of 2C-specific genes may indirectlyinduce expression of these ERVs as well as genes driven byMERVL promoters.MethodsCell culture, RNA isolation, qRT-PCRMouse ESC lines, including HP1α and HP1β KOs and theircorresponding wt line HM1 [25] and G9a and GLP KOsand their corresponding wt line TT2 [70] were cultured inDMEM supplemented with 15% FBS (Thermo ScientificHyClone, Logan, UT, USA), 20 mM HEPES, 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.05 mM streptomycin, leukemia-inhibitoryfactor and 2 mM L-glutamine on gelatinized plates. RNAwas isolated using GenElute™ mRNA miniprep kit (Sigma-Aldrich, St Louis, MO, USA) and reverse transcribed usingSuperScript III (Invitrogen, Carlsbad, CA, USA) as per themanufacturer’s instructions. Quantitative RT-PCR was car-ried out using SsoFAST™ EvaGreen Supermix (Bio-Rad,Hercules, CA, USA) on StepOne™ Software v2.1 (AppliedBiosystems, Foster City, CA, USA). Data are presented asmean +/− standard deviations of three technical replicates.Primer efficiencies were 95 to 105%. Dissociation curveanalysis was performed after the end of the PCR to confirmthe presence of a single and specific product. Correspond-ing ERV primers detect 519 MERVL elements, 202MMERVK10C elements and 638 IAPEz elements. Primersequences are listed in Additional file 3.RNAiFor RNA collection, 7,000 mESCs per well of a 96-wellplate were plated into antibiotic-free ES medium the daybefore transfection. Transfection was performed accordingto the manufacturer’s protocol, using 100 nM of eachsiRNA (Dharmacon siGENOME SMARTpool) and 0.4 μlDharmaFECT 1 reagent per well (Thermo ScientificDharmacon, Lafayette, CO, USA). The first day after trans-fection, a fraction of cells was transferred into a 12-wellplate into antibiotic-free ES medium, and the transfectionwas repeated on the third day. The following day (approxi-mately 30 h) after the second KD, most of the cells werecollected for RNA for confirmation of KD efficiency (day 1after the second KD), and the rest were plated onto 3.5-cmdishes for expansion and collection of RNA to monitorERV derepression on day 4 after the second KD. For ChIPon day 4 after the second KD, the same steps wereperformed accounting for the increased volumes, with thecells plated onto a 12-well plate for the first transfection,transferred onto two 6 cm dishes for the second transfec-tion and onto 4 to 6 × 10 cm dishes for collection on day 4post second transfection, with approximately 2 × 105 cellssaved at day 1 (30 h) for the RNA analysis of KD efficiency.Native ChIP (N-ChIP) and cross-linked ChIPFor N-ChIP, 1 × 107 ES cells for each cell line wereresuspended in douncing buffer (10 mM Tris–HClpH 7.5, 4 mM MgCl2, 1 mM CaCl2) and homogenizedthrough a 25 G5/8 needle syringe for 20 repetitions. Fol-lowing addition of 1.25 μl (50 U/μl) of MNase(Worthington Biochemicals Corp., Lakewood Township,NJ, USA), the sample was incubated at 37°C for 7 min.The reaction was quenched with 0.5 M EDTA and incu-bated on ice for 5 min. 1 ml of hypotonic buffer(0.2 mM EDTA pH 8.0, 0.1 mM benzamidine, 0.1 mMPMSF, 1.5 mM DTT) was added and the sample incu-bated on ice for 1 h. Cellular debris was pelleted and thesupernatant was recovered. Protein A (Millipore, 16–Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 12 of 16http://www.epigeneticsandchromatin.com/content/6/1/15156) and G Sepharose (Millipore, 16–266) (Millipore,Billerica, MA, USA) beads were blocked with single-stranded salmon sperm DNA and BSA, washed andresuspended in IP buffer (10 mM Tris–HCl (pH 8.0), 1%Triton X-100, 0.10% deoxycholate, 0.10% SDS, 90 mMNaCl, 2 mM EDTA). Blocked protein A/G beads wereadded to the digested chromatin fractions and rotated at4°C for 2 h to preclear chromatin. An aliquot of a 100 μlof precleared chromatin was purified by phenol-chloroform extraction, and DNA fragment sizes wereanalyzed and confirmed to correspond to 1 to 3 nucleo-some fragments. Chromatin was subdivided into aliquotsfor each IP. Antibodies specific for unmodified H3 (5 μl,Sigma-Aldrich H9289), H3K9me1 (5 ul, Abcam ab8896;),H3K9me2 (5 μl, Abcam ab1220) (Abcam, Cambridge, UK),H3K9me3 (5 μl, Active Motif 39161) (Active Motif, Carls-bad, CA, USA), and control IgG (1 μl, Sigma-AldrichI8140) were added to each tube and rotated at 4°C for1 hour. The antibody-protein-DNA complex was precipi-tated by adding 20 μl of the blocked protein A/G beads androtated at 4°C overnight (O/N). The complex was washedand eluted; IP’d material was purified using the QIAquickPCR Purification Kit (Qiagen, Venlo, Netherlands).Cross-link ChIP for HP1 proteins was performed asdescribed by Metivier et al. [71]. Briefly, 2 × 107 ES cellswere harvested. Chromatin was cross-linked in 10 ml ofPBS + 1% formaldehyde for 10 min at room temperature(RT). Reaction was quenched with 0.125 M glycine andincubated at RT for 5 min. Cells were washed two timeswith PBS and resuspended in 1 ml of collection buffer.The solution was incubated on ice for 10 min, then for10 min at 30°C. Cells were lysed by vortexing pellets se-quentially in 1 ml buffer A, 1 ml buffer B, followed byincubation of cell pellets at RT in 1 ml of lysis buffer for10 min. Chromatin was sonicated using the DiagenodeBioruptor (Diagenode, Liege, Belgium) at high setting for18 min at intervals of 30 sec ‘on’ and 30 sec ‘off ’ toachieve fragment lengths of 250 to 500 bp. For each IP, 2× 106 cell-equivalents of precleared chromatin were usedand diluted 2.5 times in IP buffer. Antibodies specific forHP1α (10 μl, academic, S. Smale), HP1β (5 μl, NEB,HP1beta (D2F2) XP™ rabbit mAb 8676) and control IgG(1 μl, Sigma-Aldrich I8140) were added into each IP androtated at 4°C O/N. To each tube, 40 ul of preclearedbeads were added, and tubes were rotated at 4°C for 2 h.The complex was washed and eluted off the beads. Thecross-links were reversed O/N at 65°C, and the DNAwas treated with Proteinase K and RNase A. DNA waspurified using the Qiaquick PCR Purification Kit(Qiagen) and analyzed by qPCR with respect to inputusing SsoFAST™ EvaGreen Supermix (Bio-Rad). Primersequences are listed in Additional file 3.Cross-linked ChIP for G9a and KAP1 were performedsimilarly with several modifications. Approximately 5 × 106TT2 or G9a KO mESCs were harvested cross-linked in0.75% formaldehyde in PBS for 10 min, quenched with gly-cine, and lysed in ice-cold radioimmunoprecipitation assaybuffer (RIPA) (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS). Chromatin wassonicated with the Bioruptor on 30 sec ‘on’ and 30 sec ‘off ’to achieve 200 to 600 bp fragment size for each sample.The samples were divided into three equal aliquots and in-cubated O/N at 4°C with 4 μg of purified mouse IgG(Sigma-Aldrich), mouse monoclonal anti-G9a (R&D Sys-tems PP-A8620-00) (R&D Systems, Minneapolis, MN,USA), or mouse monoclonal anti-KAP1 (Abcam Ab22553).Samples were precipitated with 30 μl protein-G Dynabeads(Invitrogen), washed three times with ice-cold RIPA bufferand eluted by shaking in 0.1 M NaHCO3, 1% SDS, 20 mMDTT for 15 min. Cross-links were reversed by heating sam-ples at 95°C for 5 min in the presence of 300 mM NaCl.DNA was RNase A-treated, purified and qPCR wasperformed as described above. Primers used or ChIP-qPCRanalyses detect: 638 IAP ERVs, 519 MERV-L ERVs, 202MMERVK10C ERVs, 637 MERV-L pol internal regions, asdetermined by in silico PCR on the UCSC genome browser.RNA-seq, data normalization and Z-score calculationRNA-seq libraries were constructed from mRNA as de-scribed in Morin et al. [72] from 10 μg of DNaseI-treated total RNA, and 75 bp paired-end sequencing wasperformed on an Illumina Genome Analyzer followingthe recommended protocol (Illumina Inc., Hayward, CA,USA). Sequence reads were aligned to the mouse refer-ence genome (mm9) using BWA v0.5.9 [73] with Smith-Waterman alignment disabled and annotated exon-exonjunctions compiled from Ensembl [74], RefSeq [75] andUCSC [76] (downloaded from http://genome.ucsc.eduon 17 August 2011). To quantify expression levels andthe strength of KAP1 and SETDB1 marks, we calculatedreads per kilobase per million mapped reads (RPKM)[77,78] for genomic regions of interest. For pair-wisesample comparisons, an empirical Z-score was calcu-lated assuming the distribution of RPKMs for each sam-ple followed a Poisson model:Z−score ¼ RPKMA−RPKMBð Þ=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiRPKMA−rABRPKMBð Þpwhere RPKMA and RPKMB are RPKMs in the region ofinterest of A and B samples respectively, and rAB = NA/NB, where Nx is the total number of aligned reads usedfor normalization.KAP1 ChIP-seq data analysisIn order to compare the coverage of KAP1 and SETDB1among all families of ERVs and generate the average densityof KAP1 in the genomic regions flanking ERVs, we minedthe published KAP1 ChIP-seq data set [46]. Sequence readsMaksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 13 of 16http://www.epigeneticsandchromatin.com/content/6/1/15for KAP1 were remapped to mm9 (NCBI 37) using BWAv0.5.9 [79] and default parameters. Reads having identicalcoordinates were collapsed into a single read, and readswith mapQ> =10 passed to FindPeaks 3.1 [80] (with a fixeddirectional read extension of 300 bp) for generation of anunthresholded coverage WIG file to be visualized in theUCSC genome browser [81]. This coverage file was used tocalculate KAP1 RPKM values for various regions of inter-est. Subsequently, we identified all enriched regions with apeak-height ≥10 and generated a thresholded coverageWIG file for KAP1, using FindPeaks. This WIG file wasused to generate the profiles at the genomic regionsflanking ERVs.The KAP1 profile was generated at the genomic flanksof intact elements (flanked by two identical LTRs), whichsatisfied the length criteria, for three ERV families:MMERVK10C, IAPEz and MERVL. The mean density ofKAP1 for each family was calculated for 50 bp bins within6 kb distance at 5′ and 3′ flanks of elements by agglomer-ating the coverage inside the bins for all elements of onefamily and dividing this number by the number of ele-ments and total number of aligned reads in the KAP1 IP.Detection of chimeric transcriptsChimeric transcripts containing ERV and genic sequenceswere identified by exploiting the genomic locations ofpaired-end reads. Mate-pair reads separated by more thanone standard deviation from the mean fragment size wereidentified, and those mate-pairs containing one read in anERV located upstream of the first exon of a gene, and theother read in an annotated genic exon of that gene, wereenumerated. The number of chimeric mate-pairs was cal-culated for each chimeric transcript and the transcriptswith the number of chimeric mate-pairs >5 were scored asvalid transcripts.Immunoprecipitation and Western blottingWhole-cell extracts were prepared by lysing cells in modi-fied RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1%NP-40, 0.25% deoxycholate, 0.1% SDS). Nuclear extractswere prepared as previously described [82] except nucleiwere extracted for 30 min in 20 mM HEPES pH 7.9,500 mM KCl, 1.5 mM MgCl2, 25% glycerol. Protein con-centrations were determined by Bradford assay (Bio-Rad)using BSA as a standard. For immunoprecipitation, 200 μgof nuclear extract was diluted to approximately 170 mMKCl incubated with 1 μg of mouse monoclonal anti-KAP1(Abcam ab22553), mouse monoclonal anti-G9a (R&D Sys-tems PP-A8620-00), or mouse IgG (Sigma-Aldrich) andsamples were rotated overnight at 4°C. Complexes wereprecipitated with protein G-Dynabeads (Invitrogen),washed three times with ice-cold wash buffer (20 mMHEPES pH 7.9, 200 mM KCl, 1% NP-40, 10% glycerol) andeluted by boiling beads in SDS-PAGE sample buffercontaining DTT. For Western blot analysis, protein extractsor immunoprecipitated samples were separated on 7.5% or10% SDS-PAGE gels, transferred to nitrocellulose mem-branes, blocked with 4% skim milk in Tris-buffered saline(TBS: 20 mM Tris–HCl pH 7.5, 100 mM NaCl) and probedovernight at 4°C with primary antibodies diluted in TBScontaining 0.1% Tween-20 (TBS-T): 1:5000 mouse anti-KAP1 (Abcam), 1:1000 mouse anti-G9a (R&D Systems),1:1000 mouse anti-GLP (R&D Systems PP-B0422-00),1:2000 rabbit anti-LSD1 (Abcam ab17721), 1:500 rabbitanti-HP1β (Cell Signaling 2613) (Cell Signaling Technology,Danvers, MA, USA) and 1:500 rabbit anti-Pol II large sub-unit (Santa Cruz Biotechnology sc-899) (Santa Cruz Bio-technology, Santa Cruz, CA, USA). Blots were subsequentlywashed in TBS-T, incubated in IRDye-conjugated secondaryantibodies diluted 1:20,000 in 2% milk in TBS-T washedagain in TBS-T and scanned on the Odyssey infrared im-aging system (LI-COR Biosciences, Lincoln, ME, USA).Availability of supporting dataThe data sets supporting the results of this article are avail-able in NCBI's Gene Expression Omnibus repository [83],GEO Series accession number GSE47370 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47370).Additional filesAdditional file 1: Figures S1-S3 and supporting figure legends.Additional file 2: Table of annotated genic transcripts initiating in aMERV-L LTR. All protein-coding genes that initiate in an annotatedMERV-L/MT2 LTR in both RefSeq and ENSEMBL databases were identifiedand cumulative RPKM values at annotated exons for each gene werequantified for KAP1 [28] and G9a [12] KO ESCs, as well as their wt parentlines using previously published RNA-seq datasets. Z-score and fold-change values were calculated based on these RPKM values. RPKM, Z-score and fold-change values were also generated from RNA-seq datagenerated from HP1α and HP1β KO lines and their wt parent line HM1.Additional file 3: Table of primer sequences used in this study.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsIM performed most experiments, PT performed qRT-PCR of Zfp352, ChIP-qPCR of G9a and KAP1 and Western blotting, PG performed ChIP-qPCR ofHP1s, MK performed bioinformatics analyses, with support from SJ, PScontributed the HP1 KO lines, IM and ML designed the study and wrote themanuscript. All authors read and approved the final manuscript.AcknowledgementsWe thank Yoichi Shinkai for the G9a and GLP KO mESCs, Stephen Smale forthe HP1α antibody, and Dixie Mager and Julie Brind’Amour for helpfuldiscussions. We also thank Misha Bilenky for technical support. This work wassupported by Canadian Institutes of Health Research (CIHR) grants 77805 and92090 to ML. ML is a CIHR New Investigator. SJ is a Scholar of the MichaelSmith Foundation for Health Research (MSFHR) and MK was supported by aMSFHR postdoctoral fellowship.Author details1Department of Medical Genetics, Life Sciences Institute, 2350 HealthSciences Mall, University of British Columbia, Vancouver, British Columbia V6TMaksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 14 of 16http://www.epigeneticsandchromatin.com/content/6/1/151Z3, Canada. 2British Columbia Cancer Agency, Genome Sciences Centre, 675West 10th Avenue, Vancouver, British Columbia V5Z 4S6, Canada.3Fächereverbund Anatomie, Institut für Zell and Neurobiologie, Charite -Universitätsmedizin, Charitéplatz 1, 10117, Berlin, Germany.Received: 14 December 2012 Accepted: 8 May 2013Published: 4 June 2013References1. 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Nucleic Acids Res 2002,30:207–10.doi:10.1186/1756-8935-6-15Cite this article as: Maksakova et al.: Distinct roles of KAP1, HP1 andG9a/GLP in silencing of the two-cell-specific retrotransposon MERVL inmouse ES cells. Epigenetics & Chromatin 2013 6:15.Maksakova et al. Epigenetics & Chromatin 2013, 6:15 Page 16 of 16http://www.epigeneticsandchromatin.com/content/6/1/15


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