EWSR1 regulates PRDM9-dependent histone 3 methylation and links recombinant hotspots to the chromosomal axis

Meiotic recombination in mammals requires recombination hotspot activation through the action of the histone 3 lysine-4 and lysine-36 methyltransferase PRDM9 to ensure successful double-strand break initiation and repair. Here using a Ewsr1 male germ cell conditional knockout mouse, we show that EWSR1 increases the efficiency of PRDM9-dependent H3K4/K36 trimethylation at the adjacent nucleosomes in vivo, and directs the hotspot choices for double-strand breaks formation and their subsequent repair into sufficient number of crossovers properly positioned along the centromere-telomere axis. We performed ChIP-seq with antibodies against H3K4me3 and H3K36me3 using chromatin extracted from control, Prdm9 PR/SET domain mutant and Ewsr1 conditional knockout mouse spermatocytes, and compared it to previously generated ChIP-seq data for H3K4me3 and H3K36me3 in the same cell type. We also performed ChIP-seq with antibody against single strand DNA binding protein DMC1 in spermatocytes isolated from control, Prdm9 PR/SET domain mutant, Prdm9 heterozygous and Ewsr1 conditional knockout mouse to analyze meiotic DNA double strand formation level

哺乳动物减数分裂重组需通过组蛋白3赖氨酸4与赖氨酸36甲基转移酶PRDM9的作用激活重组热点，以保障双链断裂的有效启动与修复。本研究利用Ewsr1雄性生殖细胞条件性敲除小鼠，证实EWSR1可在体内提升PRDM9依赖的H3K4/K36三甲基化在邻近核小体上的修饰效率，并调控双链断裂形成的热点选择，使得后续修复产生的交换事件数量充足且沿着丝粒-端粒轴精准排布。本研究针对对照、Prdm9 PR/SET结构域突变及Ewsr1条件性敲除小鼠的精母细胞提取染色质，利用靶向H3K4me3与H3K36me3的抗体开展染色质免疫共沉淀测序（ChIP-seq），并将所得数据与先前已发布的同类型细胞中H3K4me3和H3K36me3的ChIP-seq数据集进行比对。此外，本研究还针对对照、Prdm9 PR/SET结构域突变、Prdm9杂合突变及Ewsr1条件性敲除小鼠的分离精母细胞，利用靶向单链DNA结合蛋白DMC1（single strand DNA binding protein DMC1）的抗体开展ChIP-seq实验，以分析减数分裂DNA双链的形成水平。