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. 2011 Jul 20;4(1):12.
doi: 10.1186/1756-8935-4-12.

H3K9me3-binding Proteins Are Dispensable for SETDB1/H3K9me3-dependent Retroviral Silencing

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Free PMC article

H3K9me3-binding Proteins Are Dispensable for SETDB1/H3K9me3-dependent Retroviral Silencing

Irina A Maksakova et al. Epigenetics Chromatin. .
Free PMC article

Abstract

Background: Endogenous retroviruses (ERVs) are parasitic sequences whose derepression is associated with cancer and genomic instability. Many ERV families are silenced in mouse embryonic stem cells (mESCs) via SETDB1-deposited trimethylated lysine 9 of histone 3 (H3K9me3), but the mechanism of H3K9me3-dependent repression remains unknown. Multiple proteins, including members of the heterochromatin protein 1 (HP1) family, bind H3K9me2/3 and are involved in transcriptional silencing in model organisms. In this work, we address the role of such H3K9me2/3 "readers" in the silencing of ERVs in mESCs.

Results: We demonstrate that despite the reported function of HP1 proteins in H3K9me-dependent gene repression and the critical role of H3K9me3 in transcriptional silencing of class I and class II ERVs, the depletion of HP1α, HP1β and HP1γ, alone or in combination, is not sufficient for derepression of these elements in mESCs. While loss of HP1α or HP1β leads to modest defects in DNA methylation of ERVs or spreading of H4K20me3 into flanking genomic sequence, respectively, neither protein affects H3K9me3 or H4K20me3 in ERV bodies. Furthermore, using novel ERV reporter constructs targeted to a specific genomic site, we demonstrate that, relative to Setdb1, knockdown of the remaining known H3K9me3 readers expressed in mESCs, including Cdyl, Cdyl2, Cbx2, Cbx7, Mpp8, Uhrf1 and Jarid1a-c, leads to only modest proviral reactivation.

Conclusion: Taken together, these results reveal that each of the known H3K9me3-binding proteins is dispensable for SETDB1-mediated ERV silencing. We speculate that H3K9me3 might maintain ERVs in a silent state in mESCs by directly inhibiting deposition of active covalent histone marks.

Figures

Figure 1
Figure 1
Catalytically active SETDB1 is required for endogenous retrovirus silencing. (A) Profiling of trimethylated lysine 9 of histone 3 (H3K9me3) along the length of IAPEz endogenous retroviruses (ERVs) in the TT2 wild type (TT2 wt) and Setdb1 knockout (Setdb1 KO) mouse embryonic stem cells (mESCs) (see Figure S3 in Additional file 1 for profiles of murine leukaemia virus (MLV), MusD, MMERVK10C and GLN ERVs). The profile was generated by aligning chromatin immunoprecipitation assay sequencing (ChIP-seq) reads from TT2 wt and Setdb1 KO mESCs [19] to the consensus sequence of IAPEz. H3K9me3 enrichment levels are presented as reads per kilobase per million mapped reads values (RPKM). (B) Profiling of H3K9me3 and H4K20me3 in the genomic regions flanking 599 IAPEz elements in TT2 wt and Setdb1 KO mESCs (see Figure S4 in Additional file 1 for MusD and MLV profiles). H3K9me3 ChIP-seq reads from TT2 wt (C57BL/6 ± CBA) and Setdb1 KO mESCs [19] were used, along with H4K20me3 ChIP-seq from the wt V6.5 mESCs (129SvJae ± C57BL/6) [18]. Reads were aligned to the mouse genome (mm9), and the density of reads mapping to the 7-kb regions flanking intact IAPEz ERV families was plotted for H3K9me3 in TT2 wt and Setdb1 KO mESCs and for H4K20me3 in V6.5 wt mESCs. Vertical lines indicate the 5' and 3' boundaries of the ERV. The average mappability for 50-bp reads was confirmed to be, on average, uniform in the assayed 7 kb region (data not shown), ruling out the possibility of mapping bias. (C) Setdb1 deletion was induced with 4-hydroxytamoxifen (4-OHT) in mESCs containing no transgene (KO), a wt transgene (KO TG+) or a transgene with a mutation rendering SETDB1 catalytically inactive (KO C1243A) [20]. Expression is normalized to β-actin relative to wt. Data are presented as means ± standard deviations (SD) for three technical replicates. (D) To establish the expression levels of Setdb1 in the KO and transgenic lines, quantitative RT-PCR (qRT-PCR) was performed with Setdb1-specific primers, and expression was normalized to β-actin relative to wt. Data are presented as means ± SD for three technical replicates.
Figure 2
Figure 2
Expression of heterochromatin protein 1 genes and ERVs in the Cbx5-/- and Cbx1-/- mESCs. (A) qRT-PCR with primers specific for Cbx5 (encoding HP1α) Cbx1 (encoding HP1β), Cbx3 (encoding HP1γ) and the pluripotency factor Nanog in the Cbx5-/- and Cbx1-/- mESC lines confirms the KOs and reveals compensatory upregulation of the genes encoding the remaining HP1 proteins in the Cbx5-/- line. Expression levels were normalized to β-actin relative to wt, and the data are presented as means ± SD for three technical replicates. (B) Western blot analysis of whole-cell lysates confirms the lack of expression of HP1α and HP1β in the Cbx5-/- and Cbx1-/- mESC lines, respectively. (C) Expression of representative ERV families in the HM1 (wt), Cbx5-/- and Cbx1-/- mESCs was determined by qRT-PCR. Expression levels were normalized to β-actin relative to wt. Data are presented as means ± SD of four independent experiments, each of which was performed in triplicate. (D) Northern blot analysis of RNA isolated from the parental HM1,Cbx5-/- and Cbx1-/- mESC lines using probes specific for ETnII, MusD and IAP ERVs are shown. RNA from Dnmt1-/- mESCs, in which IAP elements are upregulated approximately fourfold [20,86] and MusD elements are upregulated approximately 1.5-fold [86], was used as a control.
Figure 3
Figure 3
DNA methylation and chromatin marks at ERVs in Cbx5-/- and Cbx1-/- mESCs. (A) An approximately 600-bp fragment of the LTR and downstream region of ETnII/MusD ERVs was analyzed by bisulphite sequencing using primers that detect 105 ETnII/MusD elements, according to in silico PCR analysis (UCSC Genome Browser, http://genome.ucsc.edu/). (B) An approximately 400-bp fragment of the LTR and downstream region of IAP ERVs was analyzed by bisulphite sequencing using primers that detect 1,461 IAP elements. (C) Bisulphite-sequenced molecules were binned into four categories, depending on the number of methylated CpG sites detected, and the data are presented as the percentage of all clones for each cell line in each bin. While the ETnII/MusD family shows no difference in DNA methylation, several IAP molecules in the Cbx5-/- cell line exhibit partial demethylation. (D) Native ChIP (N-ChIP) with antibodies against H3K9me3, H4K20me3 and pan-H3 was followed by qPCR using primers specific for major satellite repeats and IAP, MLV and MusD ERVs, and the data are presented as means ± SD for three technical replicates. The level of H4K20me3 was reduced by more than 50% at major satellite repeats in the Cbx5-/- mESCs but remained at the same level at ERVs.
Figure 4
Figure 4
HP1β plays a role in H4K20me3 but not H3K9me3 spreading from ERVs into flanking genomic DNA. N-ChIP was performed with H3K9me3-, H4K20me3- and pan-H3-specific antibodies using chromatin isolated from HM1, Cbx5-/- and Cbx1-/- mESCs. The level of enrichment of these modifications at the flanks of two full-length IAP elements on chromosomes 2 and 5 as well as at positions approximately 1 kb, 2 kb and 3.5 kb distal to these flanking regions, was determined by qPCR. Data are presented as means ± SD of three technical replicates, and pairs of control and experimental samples with *P < 0.05 and **P < 0.01 (two-tailed Student's t-test) are shown. H3K9me3 enrichment levels across these genomic regions as determined using our previously published ChIP-seq data sets [19] are also shown for wt and Setdb1 KO mESCs.
Figure 5
Figure 5
Silencing kinetics and reactivation of ERV reporters integrated in a specific genomic site. (A) Scheme for targeting of ERV reporters into a specific genomic site in mESCs via recombinase-mediated cassette exchange (RMCE). The mESC line HA36 contains a hygromycin B and herpes simplex virus thymidine kinase (HyTK) cassette between inverted Lox sites (L1 and 1L). MFG-green fluorescent protein (GFP), MusD-GFP and IAP-GFP proviral reporter cassettes, which contain the Moloney murine leukaemia virus, MusD (approximately +130 bp of downstream sequence) and IAP (approximately +450 bp of downstream sequence) LTRs, respectively, flanked by L1 and 1L sites, were cotransfected into the HA36 line with a Cre recombinase expression vector. Negative selection with ganciclovir eliminated cells with the original HyTK cassette, yielding pools of cells harbouring the proviral reporter cassettes predominantly integrated at the same site. (B) The kinetics of silencing of the MFG, MusD and IAP cassettes after reactivation of the RMCE pool with siRNA against Setdb1 are shown. (C) GFP-negative cells were sorted at day 12 postinduction with Setdb1 siRNA. Robust reactivation of GFP } expression from each of these pools of cells was observed upon secondary Setdb1 knockdown (KD). Flow cytometry data are presented as contour plots and histograms of 10,000 viable (propidium iodide (PI)-negative) cells.
Figure 6
Figure 6
Reactivation of ERV reporters and ERVs upon siRNA-mediated KD of H3K9me3-binding proteins. (A) The percentage of enhanced green fluorescent protein-positive mESCs with reactivated ERV reporters was determined by flow cytometry (upper panel) on day 5 after the second transfection with siRNA against specified H3K9me3 readers. At least 10,000 cells were collected for each sample. Data are presented as means ± SD of three biological replicates. KD efficiency was determined by qRT-PCR at 30 hours after the second siRNA transfection (lower panel). Data are presented as means ± SD of three technical replicates. (B) Relative expression of ERVs at day 5 after the second KD with the indicated siRNA pool (upper panel), along with the efficiency of each KD, as determined by qRT-PCR at 30 hours after the second siRNA transfection (lower panel) is presented as means ± SD of three technical replicates. For each amplicon, expression was normalized to β-actin relative to scramble siRNA KD. (C) Western blot analysis of HP1 proteins in single- and triple-KD cells at day 5 after the second siRNA transfection is shown. H3 was used as a loading control.

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