UHRF2 mediates resistance to DNA methylation reprogramming in primordial germ cells [RNAseq_PGCs]

In mammals, primordial germ cells (PGCs) undergo global erasure of DNA methylation with delayed demethylation of germline genes and selective retention of DNA methylation at young retrotransposons. However, the molecular mechanisms of persistent DNA methylation in PGCs remain unclear. Here we report that resistance to DNA methylation reprogramming in PGCs requires UHRF2, the paralog of the DNMT1 cofactor UHRF1. PGCs from Uhrf2 knock-out mice show loss of retrotransposon DNA methylation, while DNA methylation is unaffected in somatic cells of Uhrf2-/- mice. Furthermore, Uhrf2-deficient PGCs show precocious demethylation of germline genes and overexpress meiotic genes in females. To explore the dynamics of DNA methylation during epigenetic reprogramming in PGCs, we performed DNA methylation analysis by RRBS at all stages of PGC development in the mouse from E9.5 to E17.5. To study the role of Dnmt1 and Uhrf2 in PGCs, we performed DNA methylation analysis by RRBS in PGCs isolated from E13.5 embryos carrying a conditional knockout of Dnmt1 and PGCs isolated from Uhrf2-deficient and WT control E13.5 embryos. To expand our study of the role of Uhrf2, we also performed DNA methylation analysis by RRBS in Uhrf2-deficient and WT control E8.5 embryos, postnatal organs (brain, liver, heart) and spermatozoa. Finally, we studied the consequences of Uhrf2 deficiency on the transcriptome by performing RNA-seq in Uhrf2-deficient and WT control E8.5 embryos, and Uhrf2-deficient and WT control PGCs isolated from E13.5 embryos.在哺乳动物体内，原始生殖细胞（primordial germ cells, PGCs）会发生全基因组范围的DNA甲基化擦除，伴随生殖系基因的去甲基化进程延迟，且选择性保留年轻反转录转座子区域的DNA甲基化修饰。然而，PGCs中DNA甲基化持续存在的分子机制仍未明确。本研究发现，PGCs对DNA甲基化重编程的抵抗作用依赖于UHRF2——DNMT1辅助因子UHRF1的旁系同源蛋白。Uhrf2基因敲除小鼠的PGCs中反转录转座子的DNA甲基化水平出现丢失，而Uhrf2纯合敲除（Uhrf2-/-）小鼠的体细胞DNA甲基化则不受影响。此外，Uhrf2缺陷型PGCs会出现生殖系基因的早熟去甲基化，且在雌性个体中过度表达减数分裂相关基因。为探究PGCs表观遗传重编程过程中DNA甲基化的动态变化，我们对小鼠胚胎发育第9.5天（E9.5）至第17.5天（E17.5）的全部PGC发育阶段样本，采用简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）开展DNA甲基化分析。为研究Dnmt1与Uhrf2在PGCs中的功能，我们对两类样本开展RRBS甲基化分析：一类是携带Dnmt1条件性敲除的E13.5胚胎分离得到的PGCs，另一类是从Uhrf2缺陷型及野生型（wild type, WT）对照E13.5胚胎中分离的PGCs。为拓展Uhrf2功能的相关研究，我们还对Uhrf2缺陷型与WT对照的E8.5胚胎、出生后器官（脑、肝、心脏）以及精子样本开展了RRBS DNA甲基化分析。最后，我们通过RNA测序（RNA Sequencing, RNA-seq）对Uhrf2缺陷型与WT对照的E8.5胚胎，以及从E13.5胚胎中分离的Uhrf2缺陷型和WT对照PGCs进行检测，以探究Uhrf2缺陷对转录组的影响。