Dominant role of DNA methylation over H3K9me3 in ERV silencing during embryonic endoderm development [RNA-Seq]

Silencing of endogenous retroviruses (ERVs) is largely mediated by repressive chromatin modifications, such as H3K9me3 and DNA methylation. Their impact on ERV silencing differs in various cell types and, no systematic analyses on the interdependence between H3K9me3 and DNA methylation have been performed in differentiated cells. Here we show that deletion of the H3K9me3 HMTase Setdb1 in mouse embryonic endoderm results in ERV de-repression in only a subset of endoderm cells. We found that de-repression is restricted to visceral endoderm descendants and does not occur in definitive endoderm cells. Deletion of Setdb1 in visceral endoderm progenitors resulted in loss of H3K9me3 and reduced DNA methylation of IAPEz, consistent with up-regulation of this ERV family. In definitive endoderm cells, loss of Setdb1 did not affect H3K9me3 nor DNA methylation, suggesting Setdb1-independent pathways for maintaining these modifications. Importantly, Dnmt1 ko resulted in ERV de-repression in both visceral and definitive endoderm cells, while H3K9me3 was unaltered. Thus, our data suggest a dominant role of DNA methylation over H3K9me3 in ERV silencing in endoderm cells. Our findings suggest, that H3K9me3 is not sufficient for ERV silencing, but rather critical for maintaining high DNA methylation. RNA-seq of Embryo/ES/XEN/DE cells内源性逆转录病毒（endogenous retroviruses, ERVs）的沉默主要由抑制性染色质修饰介导，例如组蛋白H3赖氨酸9三甲基化（H3K9me3）与DNA甲基化。二者对ERV沉默的影响因细胞类型而异，目前尚未在分化细胞中开展针对H3K9me3与DNA甲基化之间相互依赖关系的系统性分析。本研究结果显示，在小鼠胚胎内胚层中敲除组蛋白甲基转移酶（histone methyltransferase, HMTase）Setdb1后，仅在内胚层的部分亚群中出现ERV去抑制现象。我们发现，去抑制现象仅局限于脏壁内胚层的子代细胞，并未在定型内胚层细胞中发生。在脏壁内胚层祖细胞中敲除Setdb1会导致H3K9me3缺失，并降低IAPEz的DNA甲基化水平，这与该ERV家族的表达上调相一致。而在定型内胚层细胞中，Setdb1的缺失既不影响H3K9me3水平，也不改变DNA甲基化状态，这提示存在不依赖Setdb1的途径来维持这两种修饰。尤为重要的是，Dnmt1基因敲除（Dnmt1 ko）会导致脏壁内胚层与定型内胚层细胞均出现ERV去抑制，而H3K9me3水平未发生改变。综上，我们的数据表明，在内胚层细胞的ERV沉默过程中，DNA甲基化相较于H3K9me3发挥主导作用。我们的研究结果提示，H3K9me3并不足以独立完成ERV沉默，而是对维持高水平的DNA甲基化至关重要。本研究包含胚胎/胚胎干细胞/胚性内胚层细胞/定型内胚层细胞的RNA测序（RNA-seq）数据。