piRNA pathway targets active LINE1 elements to establish repressive H3K9me3 mark in germ cells. Mus musculus

Transposable elements (TEs) occupy large fraction of metazoan genomes and pose constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small non-coding piwi interacting (pi)RNAs recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments. Overall design: Total of 34 libraries were analyzed. In case of ChIP libraries, every 'input' sample was used for normalization of the respective ChIP (H3K9me3 or H3) sample; the input libraries preceed ChIP libraries in the list below. There are four replicates (input-ChIP pairs) for H3K9me3 ChIP on liver cells, two for ChIP on testicular somatic cells for each genotype (Miwi2 Het and KO); and four for ChIP on male germ cells for each genotype (one from FACS-sorted germ cells, and three from MACS-sorted germ cells). Each replicate has a Het/KO pair, and the respective libraries were cloned from the material isolated from littermates. One H3 ChIP for each genotype is included, with respective inputs.

转座因子（Transposable elements, TEs）占据了后生动物基因组的较大比例，并持续对基因组完整性构成威胁。这种威胁在生殖细胞中尤为关键，因为转座因子诱导的基因组改变会传递给子代。小非编码Piwi互作RNA（piwi interacting RNAs, piRNAs）可识别并沉默生殖细胞中的多种转座因子。在小鼠中，piRNA介导的转座子沉默与其序列上CpG DNA甲基化的建立相关，但piRNA沉默的具体机制及其基因组靶标范围仍未明确。本研究证实，除DNA甲基化外，piRNA通路还可维持生殖细胞中长散在核元件（long interspersed nuclear elements, LINEs）上高水平的抑制性组蛋白修饰H3K9me3。piRNA依赖的染色质抑制仅靶向具有转座活性的LINE家族的全长元件，这展现了piRNA通路可在海量基因组转座子片段中精准识别活性转座元件的卓越能力。

实验设计概述：共分析34个测序文库。对于染色质免疫共沉淀（ChIP）文库，每个Input样本均用于对应ChIP（H3K9me3或组蛋白H3）样本的标准化校正，且下方列表中Input文库均位于对应ChIP文库之前。肝细胞H3K9me3 ChIP实验设有4组重复（Input-ChIP配对样本）；各基因型（Miwi2杂合子Het与敲除子KO）的睾丸体细胞ChIP实验各设2组重复；各基因型的雄性生殖细胞ChIP实验各设4组重复（1组来自荧光活化细胞分选FACS纯化的生殖细胞，3组来自磁活化细胞分选MACS纯化的生殖细胞）。每组重复均包含Het/KO配对样本，对应文库均从同窝小鼠的分离材料中构建。此外，各基因型均设置1组组蛋白H3 ChIP样本及对应Input文库。