染色质三维结构变异在成熟阻滞型无精症发生中的作用机制研究

Azoospermia due to maturation arrest (MA) is an important type of male infertility, patients are characterized by spermatogenesis arrested in a certain cell stage, but the specific mechanism is still unknown. Our preliminary work had verified that spermatogenesis was closely related to three-dimensional chromatin structure. We found that the higher order chromatin organization was drastically reorganized, and pachytene spermatocytes (pacSC) was especially special, topologically associated domains and chromatin loops almost completely disappeared, but still had transcriptional activity. Therefore, we propose a hypothesis that the variation of three-dimensional chromatin structure of pacSC has a certain relationship with the pathogenesis of MA. In order to verify this hypothesis, this application will isolate and purify the pacSC of patients with MA and obstructive azoospermia, to construct the chromatin interaction map of pacSC by Hi-C technology with a small number of cells, and obtain sample subtraction matrix, then screen the variation area of the interaction map by combining with ChIP-Seq, FISH and other techniques. Moreover, we will explore the open-chromatin state and expression levels of target genes by utilizing ATAC-Seq, RNA-Seq and other methods. Taken together, we will analyze the role and mechanism on the variation of three-dimensional chromatin structure from the epigenetics-gene regulatory networks in the pathogenesis of MA, and this application will provide novel clues for the cause of MA.

成熟阻滞型（MA）无精症是男性不育中的一种重要类型，患者表现为精子发生停滞在某一特定的细胞阶段，但其具体机制不明。我们前期研究已证实精子发生与染色质三维结构密切相关，我们发现小鼠精子发生中的高阶染色质结构呈现大幅重塑现象，其中粗线期精母细胞（pacSC）尤其特别，拓扑相关结构域及染色质环基本完全消失，但仍具有转录活性。由此我们提出假说：pacSC的染色质三维结构变异与MA发生具有一定关系。为验证此假说，本项目拟分离纯化MA患者及梗阻性无精症患者的pacSC，通过少量细胞Hi-C技术构建染色质互作图谱，获得样本差减矩阵，结合ChIP-Seq、FISH等技术筛选染色质互作图谱的差异区域，再结合ATAC-Seq、RNA-Seq等方法探索差异区域的染色质开放状态及基因表达水平，从表观遗传-基因调控双层网络解析染色质三维结构变异在MA发生中的作用及机制，为探讨MA的病因提供新线索。

染色质构象异常已被证实与多种疾病的发生有关，但其在男性不育中的具体机制不明。本项目结合Hi-C、ATAC-seq、RNA-seq、scATAC-seq、scRNA-seq及ChIP-seq等多组学研究技术，以人类睾丸组织、小鼠睾丸组织及生殖相关细胞系为研究对象，从表观遗传-基因调控网络，对染色质构象在哺乳动物精子发生及不育中的调控机制进行了一系列的研究。.我们构建了小鼠精子发生中的三维基因组互作图谱，探索了三维结构在精子发生中的基因调控机制；构建了人类精子发生中的染色质可接近性位点图谱，揭示了细胞特异性的转录调控机制；研究了溴蛋白抑制剂JQ1引发雄性不育的作用机制，发现JQ1在小鼠精子发生中能通过改变染色质构象来调控基因转录；研究了PIWI相互作用RNA piR-36249在男性不育及睾丸癌发生中的作用机制，发现piR-36249可通过靶向OAS2 mRNA，和/或结合于DHX36蛋白，来调控睾丸癌细胞的生物学功能。以上研究结果，对精子发生中的基因调控机制进行了补充，为探讨男性不育的病因提供了新的线索，为男性不育的个性化诊治提供了新的理论基础。本项目已发表SCI论文4篇，分别发表在Genes & diseases、iScience、Human molecular genetics和Non-coding RNA Research期刊上。