Genome stability relies on epigenetic mechanisms that enforce repression of endogenous retroviruses (ERVs). Current evidence suggests that distinct chromatin-based mechanisms repress ERVs in cells of embryonic origin (histone methylation dominant) vs. more differentiated cells (DNA methylation dominant). However, the latter aspect of this model has not been tested. Remarkably, and in contrast to the prevailing model, we find that repressive histone methylation catalyzed by the enzyme SETDB1 is critical for suppression of specific ERV families and exogenous retroviruses in committed B-lineage cells from adult mice. The profile of ERV activation in SETDB1-deficient B cells is distinct from that observed in corresponding embryonic tissues, despite the loss of repressive chromatin modifications at all ERVs. We provide evidence that, on loss of SETDB1, ERVs are activated in a lineage-specific manner depending on the set of transcription factors available to target proviral regulatory elements. These findings have important implications for genome stability in somatic cells, as well as the interface between epigenetic repression and viral latency.epigenetics | histone methylation | repression | transposable elements | retroviruses R epressive chromatin is a biochemical platform for many cellular processes (1), including gene silencing during development and the suppression of potentially mutagenic repetitive elements, such as endogenous retroviruses (ERVs). The formation and maintenance of repressive chromatin rely on epigenetic modification of its DNA and nucleosome components, including hypermethylation of CpG dinucleotides by DNA methyltransferases (DNMTs). DNA methylation patterns are conserved during replication, and this modification is thought to provide the most stable form of repression (1). An important modification for initiating repressive chromatin formation is trimethylation of histone H3 at the lysine 9 position (H3K9me3) by two major histone methyltransferases (HMTs). Suppressor Of Variegation 3-9 Homolog (SUV39H)1/2 complexes maintain this modification at constitutive heterochromatin and most long interspersed nuclear elements (LINEs), which together encompass the vast majority of H3K9me3 in chromatin (2, 3). In contrast, the HMT called SET Domain, Bifurcated 1 (SETDB1 a.k.a. ESET) is targeted primarily to ERVs, which must be silenced to maintain genome and transcriptome integrity (4).Long terminal repeat (LTR)-containing ERVs represent ∼10% of human and mouse genomes (5). Although most are defunct genomic artifacts, many species contain a substantial number of active ERVs (6). In mice, ERV expression is tissue and strain specific. The intracisternal A particle (IAP) and early transposon/ Mus musculus type D (ETn/MusD) families of ERVs are expressed predominantly in early embyronic tissues. One consequence of this de-repression is the acquisition of spontaneous germ-line mutations, with IAP and ETn retrotransposition accounting for nearly 15% of these genomic alterations in mice (5). Likewise, B lymph...