Distinct roles of KAP1, HP1 and G9a/GLP in silencing of the two-cell-specific retrotransposon MERVL in mouse ES cells
© Maksakova et al.; licensee BioMed Central Ltd. 2013
Received: 14 December 2012
Accepted: 8 May 2013
Published: 4 June 2013
In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenous retroviruses (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 dispensable for this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but the molecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed.
Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10C and IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively low levels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1 influences 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-dependent H3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressed in HP1α and HP1β KO ESCs. However, like KAP1, HP1α and HP1β are only modestly enriched at MERVL relative to IAP LTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed in the 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes is a common feature in each of these KO lines.
Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, are recruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalytic activity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1β KO ESCs may occur predominantly as a consequence of indirect effects, in association with activation of a subset of 2C-specific genes.
Endogenous retrovirus-like sequences (ERVs) are fossils of ancient retroviral integrations into the mammalian germline. Multiple independent colonization events have led to the accumulation of over 400 different ERV families with defined transcriptional patterns, often limited to specific developmental stages and cell types. Based on the similarity of their reverse transcriptase genes, ERVs are grouped into three classes: I, II and III, most closely related to exogenous gammaretroviruses, betaretroviruses and spumaretroviruses, respectively . Most ERVs in each class are no longer capable of transcription and/or retrotransposition due to the accumulation of mutations and/or efficient targeting by host silencing mechanisms that act at various stages of the viral life cycle . Nevertheless, many ERVs possess functional regulatory sequences that direct transcription at specific developmental stages and/or in specific tissues. A number of ERV subfamilies are particularly active early in embryogenesis , likely due to selection for expression and in turn retrotransposition at those stages in development that maximize the likelihood of germline transmission.
The class III MT subfamily of MaLR retrotransposons, for example, which comprise less than 5% of the mouse genome , account for 13% of all transcripts in the fully grown oocyte . 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 and 37,172 solitary long terminal repeats (LTRs) in the C57BL/6 genome (based on Repeatmasker analysis), are among the first sequences to be transcribed in the early two-cell (2C) embryo and account for nearly 4% of the mouse transcriptome at the 2C stage [3, 5–7]. Class II intracisternal A-type particle (IAP) ERVs on the other hand, of which there are well over 600 full-length copies in the mouse genome, only account for 0.6% of the 2C transcriptome . Intriguingly, MERVL expression may be essential for development beyond the four-cell stage , perhaps due to the exaptation of ERV LTRs as promoters for essential genes [8, 9]. Indeed, MERVL-driven genic transcripts are abundant at the 2C stage [3, 10] and in mouse embryonic stem cells (mESCs) that show 2C-like features [11, 12]. Given that such transcripts are not detectable at later developmental stages, it is likely that the LTR promoters of such ‘chimeric’ genes are regulated by the same epigenetic mechanisms that govern the ERVs from which they are derived.
To minimize the generally deleterious effects associated with retrotransposition, a number of pathways have evolved to inhibit transcription of ERVs. DNA methylation, mediated by the de novo methyltransferases DNMT3A and DNMT3B and the maintenance methyltransferase DNMT1, plays a critical role in proviral silencing in somatic tissues [13–15] including fibroblasts [16, 17], as well as in late germline development [18, 19]. Surprisingly however, while class I and II ERVs show broad DNA demethylation in G9a and GLP knockout (KO) mESCs , neither of these lysine methyltransferases (KMTases), which dimethylate lysine 9 of histone H3 (H3K9), are required for silencing of these ERVs. Furthermore, while IAP transcript levels are elevated in DNMT1-deficient relative to wild-type (wt) mESCs [21, 22], this difference increases dramatically in mESCs cultured in the absence of leukemia inhibitory factor (LIF) , indicating that a DNA methylation-independent mechanism may also operate in undifferentiated ESCs to silence such ERVs.
Indeed, we recently reported that in mESCs, numerous class I and II ERVs, including MMERVK10C, MusD and ETn elements, are de-depressed in the absence of the H3K9 KMTase SETDB1/ESET, while IAP elements show the highest level of activation in the absence of both DNMT1 and SETDB1 [23, 24]. Furthermore, robust silencing of each of these ERVs is dependent upon H3K9me3 deposited by SETDB1 [24, 25] and the corepressor KAP1/TRIM28/TIF1-β, which directly interacts with the KAP1 interaction domain (KID) of SETDB1 . As KAP1 can directly interact with any one of several hundred Krüppel-associated box zinc finger proteins (KRAB-ZFPs), we and others have proposed that the KAP1/SETDB1 complex may generally be recruited to ERVs by KRAB-ZFPs that recognize specific ERV sequences [23, 24, 27–30]. Curiously however, while class III MERVL elements are also upregulated in KAP1 KO mESCs , we observed only modest upregulation of these ERVs (approximately 2-fold) in SETDB1 KO mESCs .
Heterochromatin protein 1 (HP1) proteins, which encode chromo- and chromoshadow domains, function in both structural and gene regulatory pathways in eukaryotes [31–33]. These H3K9me ‘readers’ modulate gene expression in part through binding to H3K9me2/3 via the chromodomain [34–39]. In addition, HP1 proteins directly interact with the PxVxL motif of KAP1 via their chromoshadow domain independent of H3K9 methylation state [29, 40–42]. Intriguingly, this interaction is required for transcriptional silencing of reporter genes [34, 43] as well as of the nonimprinted Mest allele in embryonal carcinoma cells . Surprisingly however, we recently showed that depletion of HP1α (encoded by Cbx5), HP1β (encoded by Cbx1) and/or HP1γ (encoded by Cbx3), alone or in combination, does not lead to derepression of class I or class II ERVs in mESCs , raising the question: do HP1 proteins play a role in repression of class III ERVs?
Here, using genetic knockouts and/or RNAi, we analyzed the roles of KAP1, HP1α, HP1β as well as G9a and GLP, in transcriptional silencing of MERVL elements in mESCs. Our results indicate that MERVL expression is induced as a consequence of both direct and indirect effects, the former due to loss of H3K9me2 and the latter in association with derepression of genes normally expressed at the 2C stage.
Class II ERVs are upregulated in KAP1- and SETDB1-deficient cells, while MERVL ERVs are upregulated exclusively in KAP1-deficient cells
MERVL elements and chimeric transcripts are upregulated in G9a and GLP KO mESCs
LTRs that are derepressed in mESCs deficient in SETDB1 or the H3K4me1/2 demethylase LSD1/KDM1A can function as alternative promoters for downstream genes [11, 23], and the specific ERV families upregulated in these KOs contribute to the majority of such aberrantly expressed chimeric transcripts. To determine whether naturally occurring chimeric transcripts that initiate in MERVL/MT2 LTRs and splice to downstream genic exons are also upregulated in G9a- or GLP-depleted mESCs, we first identified all protein-coding genes represented in both RefSeq and ENSEMBL databases with an MT2 LTR as the annotated exon 1 (Additional file 2). Among the 43 genes on this list, 10 and 6 were upregulated >4-fold in KAP1 and G9a KO mESCs, respectively, relative to their wt parent lines, as determined by meta-analysis of previously published RNA-seq data [12, 28]. Strikingly, of the six upregulated genes in the G9a KO line, five are also upregulated in the KAP1 KO line. The MT2B LTR-driven gene Zfp352, which is upregulated dramatically in both of these KO lines, is induced approximately 80-fold in Glp KD mESCs, as determined by qRT-PCR analysis (Additional file 1: Figure S1C), confirming that at least a subset of MERVL LTR-driven genic transcripts are silenced by G9a and GLP. Together, these data demonstrate that the G9a/GLP complex is not only required for silencing of intact MERVL elements but also plays a critical role in silencing of a subset of the annotated genes that initiate in MERVL LTRs.
MERVL elements are direct targets of the G9a/GLP H3K9 KMTase complex
MERVL and MERVL-promoted chimeric genes are upregulated in HP1α and HP1β KO mESCs
To determine whether genes that initiate in MERVL LTRs are also derepressed in HP1-depleted ESCs, we calculated the expression levels of each of the 43 annotated MT2-initiated genic transcripts. Four and six of these genes were upregulated >4-fold in the HP1α and HP1β KO lines, respectively, relative to the wt parent line HM1 (Additional file 2). Strikingly, four of the genes in each case were also among the genes upregulated in the KAP1 and G9a KO lines. Zfp352 for example, showed an increase in expression of 14- and 28-fold in the HP1α and HP1β KO lines, respectively. To determine whether additional unannotated genic transcripts (that is transcripts not present in the RefSeq or ENSEMBL databases) initiate in MERVL/MT2 LTRs in HP1 mutant cells, we identified all transcripts in our paired-end RNA-seq data in which one of the mate pairs aligns to an LTR element and the other to an annotated genic exon and scored all genes having >5 unique support reads for each in HM1 (wt), HP1α and HP1β KO mESCs. While ERVs of all three classes are represented among constitutively expressed chimeric transcripts in wt mESCs (Additional file 1: Figure S2C), the number of MERVL LTR-driven chimeric genes is higher in HP1 KO mESCs, with MT2 transcripts contributing 19% and 28% of all chimeric transcripts in the HP1α (15 of 78 total) and HP1β KO (28 of 102 total) lines, respectively, compared to 12% (5 out of 43) in wt cells (Additional file 1: Figure S2C). Together, these data demonstrate that HP1α and HP1β are required for silencing of MERVL elements as well as genic transcripts that initiate in MERVL LTRs.
HP1α and HP1β are only modestly enriched at the 5′LTR of MERVL
To determine whether HP1 proteins are enriched specifically at MERVL LTRs, HP1α and HP1β binding was analyzed via ChIP-qPCR using chromatin extracts isolated from HM1 wt and HP1 KO mESCs. An approximately 8-fold higher level of enrichment of HP1α was detected in the 5′LTR region of IAPEz elements relative to the immunoglobulin G (IgG) control immunoprecipitation (IP), consistent with our previous observations  (Figure 5C). This enrichment was lost in HP1α KO mESCs, confirming the specificity of the HP1α antibody. Importantly, HP1α binding was also reduced at IAPEz elements and at the promoter region of the single-copy imprinted gene Mest following KD of Kap1 (Additional file 1: Figure S2D-E), confirming that HP1α binding to these loci is KAP1-dependent . In contrast, the level of HP1α enrichment observed in the 5′LTR region of MERVL elements was only approximately 2-fold higher than the IgG control IP (Figure 5D), despite comparable numbers of genomic copies recognized by the MERVL LTR (519 elements) and IAP LTR (638 elements) primer pairs, which were also used in the analysis of H3K9 methylation state described above. A similar pattern was observed for HP1β, which is enriched approximately 7-fold and 2-fold at IAPEz and MERVL LTRs, respectively (Figure 5E-F). Thus, while MERVL elements are upregulated in HP1α and HP1β KO mESCs, 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 and HP1β, KO mESCs
Consistent with the observation that a subset of 2C genes are upregulated in these KO lines, genes shown previously to be expressed exclusively in early embryogenesis (confirmed to be 2C-specific in the analysis described above), including Zfp352, Zscan4d and Tdpoz3-4 (Figure 6D), are upregulated in HP1α and HP1β KO mESCs as well as KAP1 and G9a KO mESCs (Figure 6E). Moreover, the top five genes showing the greatest difference in expression between the 2C stage and the oocyte and 8C stages: Zfp352, Gm5039, Gm8994, B020004J07Rik and Dub1a, also showed dramatic upregulation in HP1α and HP1β KO mESCs (Additional file 1: Figure S3A-B). Taken together, these observations reveal that, like KAP1 and G9a KO ESCs, HP1 KO mESCs show a significant increase in expression of a specific subset of 2C-specific genes, many of which are regulated by MERVL LTR promoters.
Unlike class I and II ERVs, MERVL elements are neither bound by SETDB1 nor marked by H3K9me3, and deletion of SETDB1 does not dramatically induce expression of these class III ERVs. Furthermore, while deletion of KAP1, HP1α and HP1β promotes upregulation of MERVL elements, we did not detect significant enrichment of these factors at MERVL elements. While we cannot rule out the possibility that our ChIP assay is not sufficiently sensitive to detect binding of these chromatin factors to MERVL elements, we did detect significant enrichment of KAP1, HP1α and HP1β at IAP elements in the same experiments. A compelling explanation for the apparently distinct mechanisms underlying repression of these ERVs may be that MERVL elements are not bound by any of the over 300 KRAB-ZFPs encoded in the mouse genome [62, 63]. While the striking diversity in KRAB-ZFPs was likely driven by selection for KRAB-ZFPs that recognize specific motifs within ERVs , MERVL-specific KRAB-ZFPs may simply not have evolved since the two bursts of MERVL retrotransposition in the mouse genome, which occurred 2 and 4 million years ago .
While this manuscript was under preparation, Macfarlan et al. reported that MERVL elements are upregulated in G9a KO mESCs , although the molecular basis of aberrant MERVL expression was not addressed. Here, we confirm this observation, and extend it to include mESCs deficient in the related KMTase GLP. Furthermore, we show that MERVL elements are bound by G9a and that maintenance of MERVL repression is dependent upon the catalytic activity of G9a but not GLP. As these elements are bound by G9a and marked by H3K9me2 in a G9a-dependent manner, we propose that the G9a/GLP complex acts directly at MERVL ERVs to maintain these elements in a silent state (Figure 7A). In contrast to IAP ERVs, MERVL ERVs show significantly higher levels of H3K9me2 than H3K9me3 enrichment. Taken together with the observation that HP1α and HP1β are detected at IAP but not MERVL ERVs, our results indicate that these H3K9 methylation ‘readers’ bind preferentially to H3K9me3 marked regions in vivo, though they are apparently not required for SETDB1-dependent silencing .
Although the molecular basis of H3K9me2-mediated transcriptional repression of MERVL elements remains to be determined, we propose that this pathway is particularly important in early embryogenesis. Consistent with this model, G9a and Glp mRNA levels are relatively low in the 2C embryo (Figure S3C) and H3K9me2 is depleted on the paternal genome at this stage [65–67], perhaps explaining why MERVL elements and zygotic genes driven by the MERVL promoters are expressed at the 2C stage and silenced shortly thereafter [3, 10].
Intriguingly, depletion of several chromatin factors in mESCs including HP1α and HP1β (this study), G9a/GLP (this study and ), LSD1 , KAP1 , RYBP  and ZFP42 (REX1) , leads to upregulation of MERVL elements. Depletion of a subset of these factors, including KAP1, G9a, LSD1 and RYBP, also leads to derepression of genes highly expressed in preimplantation embryos, such as Zfp352 and Zscan4[12, 68]. Here, we show that the same is true for HP1α and HP1β KO mESCs. Derepression of MERVL elements in mESCs deficient in each of these chromatin factors may be due at least in part to an increase in the number of cells expressing 2C-specific genes, which may in turn stimulate MERVL expression (Figure 7B).
Complicating interpretation of these observations is the fact that a significant number of genes expressed specifically at the 2C stage initiate transcription within MERVL LTRs, such as Zfp352 and Tdpoz4, or are located within a few kb of a MERVL LTR [11, 12]. Thus, it is not clear whether induction of a subset of the 2C-specific genes in the above mentioned KO mESCs induce MERVL expression as a ‘secondary effect’, or whether aberrant genic transcription of 2C genes in these KO lines results predominantly as a direct consequence of perturbation of MERVL silencing per se. These alternative explanations are not necessarily mutually exclusive.
In summary, our results indicate that G9a and GLP play a direct role in silencing of MERVL ERVs and genes driven by MERVL LTR promoters via G9a-mediated deposition of H3K9me2, while the KAP1-interacting H3K9 KMTase SETDB1 is neither recruited to MERVL elements nor required for their repression. Conversely, the results presented here are consistent with the model that induction of MERVL expression following deletion of KAP1, HP1α and HP1β occurs primarily via indirect effects. Given that expression of MERVL elements, unlike other ERVs, is normally restricted to the 2C embryo, any perturbation of mESCs that induces a nuclear milieu permissive for expression of 2C-specific genes may indirectly induce expression of these ERVs as well as genes driven by MERVL promoters.
Cell culture, RNA isolation, qRT-PCR
Mouse ESC lines, including HP1α and HP1β KOs and their corresponding wt line HM1  and G9a and GLP KOs and their corresponding wt line TT2  were cultured in DMEM supplemented with 15% FBS (Thermo Scientific HyClone, 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-inhibitory factor and 2 mM L-glutamine on gelatinized plates. RNA was isolated using GenElute™ mRNA miniprep kit (Sigma-Aldrich, St Louis, MO, USA) and reverse transcribed using SuperScript III (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. Quantitative RT-PCR was carried out using SsoFAST™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on StepOne™ Software v2.1 (Applied Biosystems, Foster City, CA, USA). Data are presented as mean +/- standard deviations of three technical replicates. Primer efficiencies were 95 to 105%. Dissociation curve analysis was performed after the end of the PCR to confirm the presence of a single and specific product. Corresponding ERV primers detect 519 MERVL elements, 202 MMERVK10C elements and 638 IAPEz elements. Primer sequences are listed in Additional file 3.
For RNA collection, 7,000 mESCs per well of a 96-well plate were plated into antibiotic-free ES medium the day before transfection. Transfection was performed according to the manufacturer’s protocol, using 100 nM of each siRNA (Dharmacon siGENOME SMARTpool) and 0.4 μl DharmaFECT 1 reagent per well (Thermo Scientific Dharmacon, Lafayette, CO, USA). The first day after transfection, a fraction of cells was transferred into a 12-well plate into antibiotic-free ES medium, and the transfection was repeated on the third day. The following day (approximately 30 h) after the second KD, most of the cells were collected for RNA for confirmation of KD efficiency (day 1 after the second KD), and the rest were plated onto 3.5-cm dishes for expansion and collection of RNA to monitor ERV derepression on day 4 after the second KD. For ChIP on day 4 after the second KD, the same steps were performed accounting for the increased volumes, with the cells plated onto a 12-well plate for the first transfection, transferred onto two 6 cm dishes for the second transfection and onto 4 to 6 × 10 cm dishes for collection on day 4 post second transfection, with approximately 2 × 105 cells saved at day 1 (30 h) for the RNA analysis of KD efficiency.
Native ChIP (N-ChIP) and cross-linked ChIP
For N-ChIP, 1 × 107 ES cells for each cell line were resuspended in douncing buffer (10 mM Tris–HCl pH 7.5, 4 mM MgCl2, 1 mM CaCl2) and homogenized through a 25 G5/8 needle syringe for 20 repetitions. Following 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 incubated on ice for 5 min. 1 ml of hypotonic buffer (0.2 mM EDTA pH 8.0, 0.1 mM benzamidine, 0.1 mM PMSF, 1.5 mM DTT) was added and the sample incubated on ice for 1 h. Cellular debris was pelleted and the supernatant was recovered. Protein A (Millipore, 16–156) and G Sepharose (Millipore, 16–266) (Millipore, Billerica, MA, USA) beads were blocked with single-stranded salmon sperm DNA and BSA, washed and resuspended in IP buffer (10 mM Tris–HCl (pH 8.0), 1% Triton X-100, 0.10% deoxycholate, 0.10% SDS, 90 mM NaCl, 2 mM EDTA). Blocked protein A/G beads were added to the digested chromatin fractions and rotated at 4°C for 2 h to preclear chromatin. An aliquot of a 100 μl of precleared chromatin was purified by phenol-chloroform extraction, and DNA fragment sizes were analyzed and confirmed to correspond to 1 to 3 nucleosome fragments. Chromatin was subdivided into aliquots for 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, Carlsbad, CA, USA), and control IgG (1 μl, Sigma-Aldrich I8140) were added to each tube and rotated at 4°C for 1 hour. The antibody-protein-DNA complex was precipitated by adding 20 μl of the blocked protein A/G beads and rotated at 4°C overnight (O/N). The complex was washed and eluted; IP’d material was purified using the QIAquick PCR Purification Kit (Qiagen, Venlo, Netherlands).
Cross-link ChIP for HP1 proteins was performed as described by Metivier et al. . Briefly, 2 × 107 ES cells were harvested. Chromatin was cross-linked in 10 ml of PBS + 1% formaldehyde for 10 min at room temperature (RT). Reaction was quenched with 0.125 M glycine and incubated at RT for 5 min. Cells were washed two times with PBS and resuspended in 1 ml of collection buffer. The solution was incubated on ice for 10 min, then for 10 min at 30°C. Cells were lysed by vortexing pellets sequentially in 1 ml buffer A, 1 ml buffer B, followed by incubation of cell pellets at RT in 1 ml of lysis buffer for 10 min. Chromatin was sonicated using the Diagenode Bioruptor (Diagenode, Liege, Belgium) at high setting for 18 min at intervals of 30 sec ‘on’ and 30 sec ‘off’ to achieve fragment lengths of 250 to 500 bp. For each IP, 2 × 106 cell-equivalents of precleared chromatin were used and diluted 2.5 times in IP buffer. Antibodies specific for HP1α (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 and rotated at 4°C O/N. To each tube, 40 ul of precleared beads were added, and tubes were rotated at 4°C for 2 h. The complex was washed and eluted off the beads. The cross-links were reversed O/N at 65°C, and the DNA was treated with Proteinase K and RNase A. DNA was purified using the Qiaquick PCR Purification Kit (Qiagen) and analyzed by qPCR with respect to input using SsoFAST™ EvaGreen Supermix (Bio-Rad). Primer sequences are listed in Additional file 3.
Cross-linked ChIP for G9a and KAP1 were performed similarly with several modifications. Approximately 5 × 106 TT2 or G9a KO mESCs were harvested cross-linked in 0.75% formaldehyde in PBS for 10 min, quenched with glycine, and lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA) (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS). Chromatin was sonicated 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 incubated O/N at 4°C with 4 μg of purified mouse IgG (Sigma-Aldrich), mouse monoclonal anti-G9a (R&D Systems 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 buffer and eluted by shaking in 0.1 M NaHCO3, 1% SDS, 20 mM DTT for 15 min. Cross-links were reversed by heating samples at 95°C for 5 min in the presence of 300 mM NaCl. DNA was RNase A-treated, purified and qPCR was performed as described above. Primers used or ChIP-qPCR analyses detect: 638 IAP ERVs, 519 MERV-L ERVs, 202 MMERVK10C ERVs, 637 MERV-L pol internal regions, as determined by in silico PCR on the UCSC genome browser.
RNA-seq, data normalization and Z-score calculation
where RPKMA and RPKMB are RPKMs in the region of interest of A and B samples respectively, and rAB = NA/NB, where Nx is the total number of aligned reads used for normalization.
KAP1 ChIP-seq data analysis
In order to compare the coverage of KAP1 and SETDB1 among all families of ERVs and generate the average density of KAP1 in the genomic regions flanking ERVs, we mined the published KAP1 ChIP-seq data set . Sequence reads for KAP1 were remapped to mm9 (NCBI 37) using BWA v0.5.9  and default parameters. Reads having identical coordinates were collapsed into a single read, and reads with mapQ > =10 passed to FindPeaks 3.1  (with a fixed directional read extension of 300 bp) for generation of an unthresholded coverage WIG file to be visualized in the UCSC genome browser . This coverage file was used to calculate KAP1 RPKM values for various regions of interest. Subsequently, we identified all enriched regions with a peak-height ≥10 and generated a thresholded coverage WIG file for KAP1, using FindPeaks. This WIG file was used to generate the profiles at the genomic regions flanking ERVs.
The KAP1 profile was generated at the genomic flanks of intact elements (flanked by two identical LTRs), which satisfied the length criteria, for three ERV families: MMERVK10C, IAPEz and MERVL. The mean density of KAP1 for each family was calculated for 50 bp bins within 6 kb distance at 5′ and 3′ flanks of elements by agglomerating the coverage inside the bins for all elements of one family and dividing this number by the number of elements and total number of aligned reads in the KAP1 IP.
Detection of chimeric transcripts
Chimeric transcripts containing ERV and genic sequences were identified by exploiting the genomic locations of paired-end reads. Mate-pair reads separated by more than one standard deviation from the mean fragment size were identified, and those mate-pairs containing one read in an ERV located upstream of the first exon of a gene, and the other read in an annotated genic exon of that gene, were enumerated. The number of chimeric mate-pairs was calculated for each chimeric transcript and the transcripts with the number of chimeric mate-pairs >5 were scored as valid transcripts.
Immunoprecipitation and Western blotting
Whole-cell extracts were prepared by lysing cells in modified RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 0.1% SDS). Nuclear extracts were prepared as previously described  except nuclei were extracted for 30 min in 20 mM HEPES pH 7.9, 500 mM KCl, 1.5 mM MgCl2, 25% glycerol. Protein concentrations were determined by Bradford assay (Bio-Rad) using BSA as a standard. For immunoprecipitation, 200 μg of nuclear extract was diluted to approximately 170 mM KCl incubated with 1 μg of mouse monoclonal anti-KAP1 (Abcam ab22553), mouse monoclonal anti-G9a (R&D Systems PP-A8620-00), or mouse IgG (Sigma-Aldrich) and samples were rotated overnight at 4°C. Complexes were precipitated with protein G-Dynabeads (Invitrogen), washed three times with ice-cold wash buffer (20 mM HEPES pH 7.9, 200 mM KCl, 1% NP-40, 10% glycerol) and eluted by boiling beads in SDS-PAGE sample buffer containing DTT. For Western blot analysis, protein extracts or immunoprecipitated samples were separated on 7.5% or 10% SDS-PAGE gels, transferred to nitrocellulose membranes, blocked with 4% skim milk in Tris-buffered saline (TBS: 20 mM Tris–HCl pH 7.5, 100 mM NaCl) and probed overnight at 4°C with primary antibodies diluted in TBS containing 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 rabbit anti-HP1β (Cell Signaling 2613) (Cell Signaling Technology, Danvers, MA, USA) and 1:500 rabbit anti-Pol II large subunit (Santa Cruz Biotechnology sc-899) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were subsequently washed in TBS-T, incubated in IRDye-conjugated secondary antibodies diluted 1:20,000 in 2% milk in TBS-T washed again in TBS-T and scanned on the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, ME, USA).
Availability of supporting data
The data sets supporting the results of this article are available in NCBI's Gene Expression Omnibus repository , GEO Series accession number GSE47370 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47370).
We thank Yoichi Shinkai for the G9a and GLP KO mESCs, Stephen Smale for the HP1α antibody, and Dixie Mager and Julie Brind’Amour for helpful discussions. We also thank Misha Bilenky for technical support. This work was supported by Canadian Institutes of Health Research (CIHR) grants 77805 and 92090 to ML. ML is a CIHR New Investigator. SJ is a Scholar of the Michael Smith Foundation for Health Research (MSFHR) and MK was supported by a MSFHR postdoctoral fellowship.
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