转录因子RFX2调控小鼠精子细胞发育基因转录及表观遗传状态的机理

Diverse morphologically different cell types appear during mammalian spermatogenesis; after meiosis, the morphological change of spermatids is especially dramatic, accompanied by the high degree condensation of chromatins and the silencing of transcription of genes. It has been known that many so-called "master switch" transcription factors play important roles in the differentiation of cells. Previously, through bioinformatics analysis, we found that transcription factor RFX2 may regulate the transcription of multiple genes; the spermatogenesis of its gene knockout mice stopped at the step 12 spermatids. In this application, we propose to carry out in-depth study of how RFX2 regulates gene transcription at the genome-wide scale, focusing on understanding how it contributes to the establishment and maintenance of the epigenetic status of the spermatids. We will first compare the transcriptomes of the spermatids of the wild-type and RFX2 gene knockout mice, including protein coding and non-coding RNA genes; then investigate how the methylation of DNA and histones changes after gene knockout, paying more close attention to the 5-hydroxymethylation of cytosines in the genome; finally identify direct target genes of RFX2, particularly other transcription factors, aiming to the construction of a gene regulatory network. The accomplishment of these researches will strengthen our understanding of how transcription factors are involved in the establishment and maintenance of epigenetic modifications as well as will help us to identify key nodes in the gene regulatory network of mammalian spermatogenesis.

哺乳动物精子发生过程中会出现多种形态各异的细胞；减数分裂完成后，随着染色质的高度凝缩和基因转录沉默，精子细胞的形态变化尤为剧烈。我们前期通过生物信息学分析，发现转录因子RFX2可能调控大量生精细胞高度表达和特异表达基因的转录；基因敲除小鼠表现为精子发生阻滞在球形精子阶段。本申请计划深入研究RFX2在整个基因组范围对基因转录的调控，重点研究其如何影响精子细胞的表观遗传状态。我们将1）比较野生型和RFX2敲除小鼠蛋白编码基因和非编码RNA基因的表达变化；2）研究DNA和组蛋白甲基化的变化，特别关注DNA羟甲基的变化；3）进而鉴定RFX2的直接靶基因，特别注重受其调控的转录因子；4）利用精原干细胞转基因和移植进一步研究受RFX2调控的二级转录因子是否能部分挽救敲除小鼠的缺陷。这些工作的完成，能够揭示转录因子对于表观遗传状态建立和维护的作用，还有助于发现哺乳动物精子发生中基因调控网络中的关键节点。

哺乳动物精子发生过程是一个复杂且高度有序的过程，包括精原细胞的增殖分化、减数分裂以及精子形变等事件。每一步的正常进行都依赖于大量基因的精确表达调控。根据本实验室对不同类型生精细胞进行RNA-seq分析表明，与其他组织相比，睾丸中有大量睾丸特异表达基因，且大部分都在减数分裂后期的圆形精子中高表达。因此，找到这些基因中的关键转录因子并对其进行深入研究可以更好地阐明精子发生的分子机制。经过生物信息学分析，我们发现Rfx2和Sox30是两个睾丸特异表达的转录因子，而且它们都被同一个基因——Mybl1调控。通过分别构建这两个基因的敲除小鼠我们发现，任意一个基因的缺失都会导致雄性小鼠不育，且精子发生都阻滞在圆形精子时期。高通量测序表明我们Rfx2调控小鼠精子鞭毛组装相关基因和某些睾丸特异表达基因，而Sox30可能通过参与染色中心形成调控大量蛋白编码基因和lncRNA。这说明Rfx2和Sox30是两个小鼠精子发生中的关键转录因子。