H3K9me3-binding proteins are dispensable for SETDB1/H3K9me3-dependent retroviral silencing

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.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.Endogenous retroviral sequences (ERVs) are relics of ancient retroviral integration into the germline. These parasitic elements are abundant in mammals, occupying approximately 8% of the mouse genome and 10% of the human genome [1,2]. ERVs are subdivided into three diverse classes based on the similarity of their reverse transcriptase genes or their relationship to different genera of exogenous retroviruses. In the mouse, class I ERVs, similar to gammaretroviruses, include active families such as murine leukaemia viruses (MLVs) and murine retroviruses that use tRNAGln (GLN). Class II ERVs are similar to alpha- and betaretroviruses and include Mus musculus ERV using tRNALys type 10C (MMERVK10C), the highly retrotranspositionally active intracisternal A-type particles (IAPEz) and early transposon/Mus musculus type D retrovirus (ETn/MusD) families. Class III ERVs, the oldest and most ab