中华绒螯蟹顶体反应精子规避DNA损伤的表观遗传学机制

Proteins associated to chromatin structure (PACSs) and their binding site composition feature (including DNA methylation) are the key factors to determine the chromatin structure and the stability of DNA. We recently found that the acrosome-reacting sperm of Eriocheir sinensis with non-condensed nucleus presented both a large number of PACSs and large-scale DNA methylation. However, how both to maintain the stability of DNA in the acrosome-reacting sperm is coming to be determined. We suppose that there may be massive specific binding sites, which bind the corresponding PACSs in the acrosome-reacting sperm of this species to build a loose but relatively stable specific architecture of chromatin for maintaining the stability of DNA. Accordingly, we should identify, localize and quantify the PACSs and their modification types, analyze the composition feature of those sites binded with the PACSs, and explore both the chromatin composition and the relationships of those factors above with the stability of DNA in the AR sperm, using acrosome-reacting sperm in E. sinensis as materials, utilizing the technique of tandem mass spectrometry (MS-MS), immunoprecipitation (IP), immunocytochemistry (ICC), high-throughput DNA sequencing, electrophoresis, etc. and bioinformatics database resources, to illustrate the epigenetic mechanism to avoid damage of DNA in the acrosome-reacting sperm of E. sinensis based on the PACSs and their binding site composition feature (including DNA methylation). The results may provide a theoretical basis for protection and utilization of germplasm resources and elucidating the fertilization mechanisms of this species.

染色质结构相关蛋白(PACS)及其结合位点组成特征（包括DNA甲基化，下同）是决定染色质结构和稳定DNA的关键因素。我们发现非浓缩核的中华绒螯蟹顶体反应（AR）精子内存在大量PACS及大量DNA甲基化现象，但这些直接与DNA稳定相关的因素如何维系AR精子DNA的稳定这一科学问题尚待解决。我们推测，该物种精子染色质和DNA上存在大量特异位点并与相应的多种PACS结合，构成了松散但相对稳定的特定染色质结构以维系DNA的稳定。本项目拟以该物种AR精子为材料，通过串联质谱、免疫沉淀、细胞免疫荧光、高通量测序等技术及生物信息学数据库资源，分析AR精子PACS的种类、含量、分布、修饰类型及其结合位点特征，探讨AR精子染色质的组成及这些因素与DNA稳定的关系，以期在此基础上阐明中华绒螯蟹AR精子规避DNA损伤的表观遗传学机制。研究结果可为该物种种质资源的保护与利用及受精机理的探明提供理论依据。

染色质结构相关蛋白及基因组甲基化是决定染色质结构和稳定DNA的关键因素，但它们保护AR精子DNA的机制还不清楚。因此本项目通过串联质谱、免疫荧光定位等技术及生信方法，探讨了AR精子染色质结合蛋白组成、基因组甲基化及染色质时空结构的特征，分析了精子基因表达调控方式及miRNA参与骨架蛋白基因表达的调控网络，进而阐释了AR精子规避DNA损伤的表观遗传学机制。结果显示，AR精子中有370个差异表达的精子染色质结合蛋白（DESP）（与未AR精子相比，下同），且多上调表达、分布于核，属于细胞骨架蛋白或碱性蛋白。其功能包括构建染色质转运通道、遗传物质主动运输、DNA甲基化及组蛋白甲基化和去乙酰化、构建核骨架和染色质高级结构、参与小GTPase介导的信号通路、参与AR过程。AR和非AR精子基因组上的C总甲基化率都高达9%以上，且AR精子基因组上的mC含量略多，但不同染色体上C的甲基化密度差异较大。CpG岛上mC的含量超过36%。两样本间共有3050个差异甲基化基因，其中97%低表达或不表达。高密度的甲基化和不活跃的基因都有利于稳定精子基因组。精子中基因表达的调控方式是多样的，可将精子基因分为7类。大量miRNA参与细胞骨架蛋白的靶向调控。AR过程中，进一步的甲基化增强了DNA的稳定性，少量残存组蛋白的甲基化和去乙酰化及组蛋白变体增加了染色质一级结构的稳定性，碱性蛋白和EF手性域蛋白松散结合稳定了染色质高级结构，后者再以核骨架为依附来稳定遗传物质，DNA甲基化、miRNA和lncRNA精准调控基因表达减少遗传物质参与的活动而降低DNA损伤的概率。同时，以DESP参与的辐射臂收缩、顶体囊外翻和顶体管前伸为动力，以染色质高级结构及遗传物质依附的核骨架和染色质转运通道为转运单元，以主动运输提高转运效率，以小GTPase介导的信号通路调控转运过程，进一步规避DNA的损伤。