Setdb1 is required for persistence of H3K9me3 and repression of endogenous retroviruses in mouse primordial germ cells

Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at ~E15.5 in prospermatogonia. Earlier in germline development however, the genome, including most retrotransposons, is progressively demethylated, with young ERVK and ERV1 elements retaining intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low input ChIP-seq method. Although these repressive histone modifications are predominantly found on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated LTRs and LINE1 elements. Germline-specific conditional knock-out (KO) of the H3K9 methyltransferase SETDB1 yields a decrease of both histone marks and DNA methylation at H3K9me3 enriched retrotransposon families. Strikingly, Setdb1-KO E13.5 PGCs show concomitant de-repression of many marked ERVs, including IAP, ETn and ERVK10C elements and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline. H3K9me3, H3K27me3 and expression profiles in Setdb1 WT, Het and KO male and female E13.5 PGCs.配子发生过程中，从头DNA甲基化会抑制内源性逆转录病毒（endogenous retroviruses, ERVs）的转录；该过程在卵母细胞中于出生后启动，在精原祖细胞中则约于胚胎期15.5天（E15.5）启动。然而在生殖系发育的更早阶段，包括大多数逆转录转座子在内的基因组会逐步发生去甲基化，年轻的ERVK与ERV1元件仍维持中等甲基化水平。当雌雄个体胚胎期13.5天（E13.5）的原始生殖细胞（primordial germ cells, PGCs）中DNA甲基化水平降至低点时，我们借助新近开发的低起始量染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）技术，探究了逆转录转座子是否存在组蛋白H3第9位赖氨酸三甲基化（H3K9me3）与组蛋白H3第27位赖氨酸三甲基化（H3K27me3）修饰标记。尽管这类抑制性组蛋白修饰在E13.5 PGCs中主要分布于不同的基因组区域，但它们同时也标记了部分甲基化的长末端重复序列（long terminal repeat, LTR）与长散在核元件1（long interspersed nuclear element 1, LINE1）。对组蛋白H3赖氨酸9甲基转移酶SETDB1进行生殖系特异性条件性敲除（conditional knock-out, KO）后，富集H3K9me3的逆转录转座子家族上的两类组蛋白修饰与DNA甲基化水平均出现下降。值得注意的是，SETDB1敲除的E13.5 PGCs中，诸多被标记的ERVs（包括IAP、ETn与ERVK10C元件）以及ERV邻近基因均出现了同步的去抑制现象，其中部分基因的表达变化呈现性别依赖性。此外，SETDB1缺陷会导致雄性PGCs数量减少，且雌雄个体均出现产后性腺功能减退症。综上，上述结果表明，在生殖系从头DNA甲基化启动之前，SETDB1是抵御前病毒表达的关键保护因子。本数据集涵盖SETDB1野生型（WT）、杂合型（Het）与敲除型（KO）雌雄E13.5 PGCs的H3K9me3、H3K27me3修饰谱与基因表达谱数据。