Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells

Transposable elements (TEs) comprise roughly forty per cent of mammalian genomes(1). TEs have played an active role in genetic variation, adaptation, and evolution through the duplication or deletion of genes or their regulatory elements(2-4) and TEs themselves can act as alternative promoters for nearby genes resulting in non-canonical regulation of transcription(5,6). However, TE activity can lead to detrimental genome instability(7), and hosts have evolved mechanisms to appropriately silence TE mobility(8,9). Recent studies have demonstrated that a subset of TEs, endogenous retroviral elements (ERVs) containing long terminal repeats (LTRs), are silenced through trimethylation of histone H3 on lysine 9 (H3K9me3) by ESET (also known as SETDB1, SET domain bifurcated 1, or KMT1E)(10) and a co-repressor complex containing KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28)(11) in mouse embryonic stem cells (ESCs). Here we show that the replacement histone variant H3.3 is enriched at class I and class II ERVs, notably early transposon (ETn)/MusD and intracisternal A-type particles (IAPs). Deposition at a subset of these elements is dependent upon the H3.3 chaperone complex containing ATRX (alpha thalesemia/mental retardation syndrome X)(12) and DAXX (Death-associated protein 6)(12-14). We demonstrate that recruitment of DAXX, H3.3, and KAP1 to ERVs are co-dependent and upstream of ESET, linking H3.3 to ERV-associated H3K9me3. Importantly, H3K9me3 is reduced at ERVs upon H3.3 deletion, resulting in derepression and dysregulation of adjacent, endogenous genes, along with increased retrotransposition of IAPs. Our study identifies a unique heterochromatin state marked by the presence of both H3.3 and H3K9me3 and establishes an important role for H3.3 in control of ERV retrotransposition in ESCs.