Abstract
BackgroundEndogenous 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.ResultsWe 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.ConclusionTaken 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.
Highlights
Endogenous retroviruses (ERVs) are parasitic sequences whose derepression is associated with cancer and genomic instability
We have recently demonstrated by chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) that HP1a, HP1b and HP1g are enriched on IAPEz, MusD and murine leukaemia viruses (MLVs) ERV sequences in mouse embryonic stem cells (mESCs), albeit at modest levels, and that this binding is partially dependent on SETDB1-deposited H3K9me3 [20]
Catalytic activity of SETDB1 is largely required for ERV silencing We recently showed by ChIP-qPCR [20] and ChIP-seq [19] analyses that numerous class I and class II ERV families are marked by H3K9me3
Summary
Endogenous retroviruses (ERVs) are parasitic sequences whose derepression is associated with cancer and genomic instability. Class I ERVs, similar to gammaretroviruses, include active families such as murine leukaemia viruses (MLVs) and effort to counteract the potentially detrimental effects of ERVs, eukaryotic genomes have evolved multiple lines of defence against active exogenous and endogenous retroviruses [14], including DNA methylation and repressive histone modifications. IAP and ETn/ MusD retrotransposons, the two most active class II mouse ERV families and the source of numerous recent germline mutations [24], are among the families with the highest H3K9me enrichment levels These families are dramatically upregulated in SETDB1 knockout (SETDB1 KO) mESCs [19,20], confirming that they have a high potential for activation in the absence of H3K9me. The class III MERV-L and MaLR families, which are devoid of the H3K9me mark in mESCs, are repressed by the histone lysine-specific demethylase 1 (LSD1/KDM1A) [25], revealing that different ERV classes are regulated by distinct epigenetic modifications in these pluripotent cells
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