Pervasive transcription and endosiRNAs restrain transposons during global demethylation in embryonic stem cells [ChIP-Seq 2]. Mus musculus

DNA methylation and other repressive epigenetic marks are erased in the mammalian germline and transposable elements (TEs) acquire the potential to be transcribed. This is a critical phase for genome defense and complementary TE silencing pathways are required to limit their activity. We find overlapping sense/antisense transcription in TEs in mouse embryonic stem cells, with an increase of sense transcription induced by acute deletion of Dnmt1, leading to increased abundance of small RNAs. These small RNAs are loaded into ARGONAUTE2 (AGO2) suggesting an endosiRNA based silencing mechanism. Reduction of Dicer and Ago2 levels reveals that small RNAs are involved in an immediate response to transposon activation by demethylation, while the deposition of repressive histone marks represents an ensuing chronic response. Dicer dependent endosiRNAs which map to TEs are also found in vivo during primordial germ cell development. Our results suggest that TE antisense transcription acts as a trap that restrains acute transposon activity through small RNAs during epigenetic reprogramming in the germline. Overall design: ChIP-Seq (2 biological replicates of 6 samples with technical replicates, 1 biological replicate of 6 samples)

DNA甲基化（DNA methylation）与其他抑制性表观遗传标记在哺乳动物生殖系中被清除，转座因子（transposable elements，TEs）由此获得转录潜能。这是基因组防御的关键阶段，需要互补的转座因子沉默通路来限制其活性。我们在小鼠胚胎干细胞的转座因子中发现存在有义/反义转录的重叠现象：急性敲除Dnmt1可诱导有义转录上调，进而提升小RNA的丰度。此类小RNA可被装载至ARGONAUTE2（AGO2）中，提示存在基于内生小干扰RNA（endosiRNA）的沉默机制。通过降低Dicer与Ago2的表达水平，我们发现小RNA参与了去甲基化引发的转座子激活的即时应答，而抑制性组蛋白标记的沉积则代表后续的慢性应答。在体内原始生殖细胞（primordial germ cell）发育过程中，同样可检测到靶向转座因子的Dicer依赖型内生小干扰RNA。本研究结果表明，在生殖系表观遗传重编程阶段，转座因子的反义转录可作为"陷阱"，通过小RNA通路抑制转座子的急性激活。
整体实验设计：染色质免疫沉淀测序（ChIP-Seq），包含6个样本的2次生物学重复并附带技术重复，以及6个样本的1次生物学重复。