Piwi蛋白和piRNA调控DNA去甲基化在生殖细胞的发育和分化中的功能研究

During germ cell development and differentiation, Piwi proteins and Piwi-interacting RNAs (piRNAs) are highly expressed in two distinct phases: the gonocyte stage and the pachytene spermatocyte to round spermatid stage. Meanwhile, the open chromatins structures adapt to a low levels of repressive epigenetic marks at these stages. At the gonocyte stage, piRNAs and Piwi proteins Mili and Miwi2 are primarily involved in de novo DNA methylation, and function in transposon silencing, thereby protecting the genome integrity of prospermatogonia or gonocytes at 12.5 to 18.5 dpc. However, involvement of Miwi, Mili and pachytene piRNA in postnatal testes remains unknown. Indeed, we observe an increase of LINE1 and IAP both in Mili and Miwi deletion testis, and mutant germ cells show increased level of 5hmC, with Tet2 and Tet3 are up-regulated. We suggest that Mili or Miwi with piRNA pathyway suppress transposons by protecting against conversion of 5mC to 5hmC in germ cell development and differentiation. To gain insight into the function of Miwi, Mili and pachytene piRNA in adult spermatogenesis, we will perform 5hmC-Seq using mouse testis of wildtype and mutant of Miwi, Mili, and Milic-ko. Our results will reveal a role for Piwi/piRNAs in maintaining DNA methylation by inhibition of TET proteins activity and provide a new transcriptional regulation of 5mC and 5hmC modification in germ cell development and differentiation.

在生殖细胞发育分化过程中，在性原细胞和粗线期精母细胞到圆形精子这两个阶段，细胞染色体结构处于开放的状态，表现出低水平的抑制性表观遗传修饰，小鼠的Piwi蛋白和piRNA在这些阶段大量表达。在性原细胞阶段（12.5～18.5 dpc），Mili和Miwi2/piRNA通路沉默转座子并参与对转座子建立DNA重新甲基化。Piwi/piRNA在精母细胞到圆形精子阶段的功能还不明确。我们发现小鼠敲除Mili或Miwi都导致成年睾丸中LINE1和IAP表达上调，睾丸的5hmC水平上升，Tet2和Tet3的表达升高；因此推测Mili或Miwi/piRNA通路抑制了piRNA靶序列的5mC去甲基化，维持转录沉默状态。本项目以Piwi/piRNA通路为出发点，利用Miwi、Mili、Tet2和Tet3敲除小鼠，探索小鼠精子发生过程中，Mili或Miwi/piRNA调控piRNA靶序列的5mC去甲基化的机制。