piRNA/PIWI在哺乳动物精子发育过程的表观遗传调控与功能机制

A unique feature in spermiogenesis, the postmeiotic developmental stage of male germ cells, is the remarkable compaction of sperm chromatin. The drastic changes of chromatin structure from early haploid spermatids to mature sperm suggests that epigenetic regulation should play critical roles in this specific stage of male germ cell development; however, whether and how epigenetic regulation functions in this process have remained largely unexplored. piRNAs are a novel class of animal germ cell-specific small noncoding RNAs that interact to PIWI family proteins. Recent studies indicate that piRNAs, in complex with PIWI proteins, prevent the activity of mobile genetic elements from destabilizing DNA in animal germ cells at the epigenetic or/and post-transcriptional levels. Thus, piRNA pathway has been implicated as a unique innate immune system in animal germ cells to antagonize transposon and retrotransposon for protecting the integrity of germ cell genome, which is essential to gametogenesis in various organisms. Our preliminary results showed that murine PIWI (MIWI) directly interacts to histone acetyltransferase CBP/p300 and represses CBP/p300-mediated acetylation of H3K14. More importantly, we found that H3K14Ac is present in spermatocytes, but completely absent in hapliod spermatids in wildtype mice. By contrast, we found that H3K14Ac is still detectable in haploid spermatids, strongly implicating that MIWI is involved in regulation of H3K14Ac in haploid spermatids. In this research plan, we will study the physiological role and mechanism of MIWI/piRNA-mediated epigenetic regulation in spermatid development and sperm maturation. We will investigate the molecular mechanisms of PIWI/piRNA in regulation of histone acetylation and identifiy the downstream target genes that are controlled by PIWI/piRNAs epigenetically during spermiogenesis. Additionally, we will examine whether and how PIWI/piRNA-regulated histone modifications in spermatids are involved in heterochromatinization during sperm maturation. The obtained results may bring new insights into molecular information of abnormal development of spermatids and dysregulation of sperm maturation, and also may provide novel biomarkers for diagnosis and therapeutics of male infertility.

染色质结构在精子形成期剧烈变化，表明表观遗传调控在此发育阶段具有重要作用，但目前对此发育阶段的表观遗传调控功能机制尚知之甚少。piRNA是新近在动物生殖细胞中发现的一类小非编码RNA。一些证据表明，piRNA与PIWI形成复合物，在表观遗传和转录后水平沉默转座遗传元件、维持生殖细胞基因组稳定性和完整性。我们的前期研究发现，小鼠PIWI（MIWI）与组蛋白乙酰基转移酶CBP/p300相互作用、抑制CBP/p300介导的H3K14Ac，并且发现MIWI与H3K14Ac在单倍体精子细胞中的去除密切相关。本项目将研究小鼠精子发育过程piRNA/PIWI介导的表观遗传调控与功能机制，调查其介导组蛋白修饰的分子机制及调控的相关靶基因，探索相关组蛋白修饰与异染色质化等事件之间的关系。项目研究获得结果将阐析精子发育过程piRNA/PIWI介导表观遗传调控的功能机制，为相关男性不育症的诊治等研究提供新靶标。

已有的证据表明，piRNA及其结合蛋白PIWI特异性地在动物生殖细胞中表达，可能在表观遗传及转录后水平控制生殖细胞基因组中的转座元件或/和编码基因，对动物的生殖细胞发育分化至关重要，但相关的作用机制还不完全清除。在该项目支持下，我们调查了小鼠单倍体精子细胞中表达的粗线期piRNA及其结合蛋白MIWI（小鼠PIWI）在精子形成过程的功能机制，主要获得了以下研究结果，包括：1）发现MIWI可能通过与一个组蛋白乙酰基转移酶（HAT）相互作用并抑制该HAT介导的一个H3位点乙酰化修饰，可能与该位点乙酰化修饰在单倍体精子细胞中的缺失相关；2）发现粗线期piRNA与其结合蛋白MIWI和脱腺苷酶CAF1形成pi-RISC复合物，依赖于piRNA百万级不同的序列，以类似于miRNA的作用机制，通过促进mRNA的脱腺苷化，介导精子细胞中大量mRNA的降解；3）发现piRNA作为MIWI蛋白的配体，诱导MIWI蛋白构象改变，促进其结合到APC/C复合物的底物结合亚基上，进而诱导其泛素化修饰；4）发现HIWI（人PIWI）蛋白泛素化修饰拮抗突变导致精子细胞发育发生大量缺陷、男性不育。..共发表了项目资助标注论文5篇（其中3篇IF>10.0），包括Dev Cell (2013, 24:13-25; IF:14.03)、Cell Res (2014, 24:254-7; IF:12.98)、Cell Res (2014, 24:680-700; IF:12.98)和Cancer Res (2014, 74:4720-30; IF:9.28)等4篇研究论文及1篇应邀综述论文WIREs RNA (2014, 5:733-45; IF:6.15），目前还有2篇重要研究论文在投稿准备中。此外，在此项目执行期间，已培养3名博士研究生毕业取得博士学位、3名硕士研究生毕业取得硕士学位。