小鼠单倍体胚胎干细胞二倍体化的表观遗传调控机制

Mouse haploid embryonic stem cells (haESCs) tend to readily self-diploidize over time during culture. Failure in maintenance of the haploid status largely limits the application of haESCs in basic scientific research and the clinical applications. However, the molecular mechanisms underlying diploidization of haESCs remains enigmatic. Our preliminary results show that haESCs have a significantly higher efficiency of diploidization at early stage than at late stage, and genome-wide DNA methylation levels are correlated with the diploidization efficiency. On the basis of these findings, this project attempts to reveal the molecular mechanisms of haESCs diploidization in the perspective of epigenetic regulation. We apply fluorescence-activated cell sorting to collect haploid and diploid haESCs at early and late stage, respectively. These four types of haESCs are used in this study. We generate their transcriptome, genome-wide DNA methylation, maps of nucleosome positions, and key histone modifications. Through comparisons between haploid and diploid haESCs, between haESCs with low and high diploidization efficiency with assistance of bioinformatics tools, we can discover changes in gene expression and epigenetic landscape along the diploidization of haESCs, identify the key genes critical to the diploidization of haESCs, validate their functions and reveal their roles in the diploidization of haESCs. These cells can be injected into mature oocytes to produce viable and fertile progenies because of their germline competence. The results of this study will greatly enhance the application of haESCs in genetic screening and assisted reproduction. Our accumulative expertise in previous work on epigenetic regulatory mechanisms for mouse somatic reprogramming provides a solid technique support to this study.

小鼠单倍体胚胎干细胞（haESCs）培养过程中趋向于二倍体化，极大阻碍其在基础研究与临床上的应用。但其二倍体化的机制并不清楚。我们预实验发现haESCs二倍体化效率早期显著高于晚期，而且与DNA甲基化有关系。基于此，本项目从表观遗传调控方向解析haESCs二倍体化的分子机制。通过分选早期与晚期haESCs的单倍体与二倍体状态的细胞，共得到四类haESCs。制备它们的转录组、DNA甲基化、核小体定位、与组蛋白修饰图谱。分别比较单倍体与二倍体状态haESCs，及不同转化效率haESCs，分析haESCs二倍体化过程的基因表达与表观修饰变化，整合构建haESCs二倍体化过程的表观遗传调控网络，验证haESCs二倍体化中起重要作用的基因的功能，揭示其作用机制。本项目结果将促进haESCs在遗传学研究与临床辅助生殖中的应用。我们在细胞命运转化的表观遗传调控机制的前期工作基础可为本项目提供技术支撑。

由于单倍体中每个基因只有一个拷贝而不存在对基因功能改变或缺失的补偿作用，在遗传学研究中容易操作，尤其是在隐性性状的筛查研究中具有巨大价值，因此近年来单倍体胚胎干细胞（haESCs）诱导与全能性研究成为热点。大量研究发现，haESCs在培养过程中会自发二倍体化。这极大阻碍其在基础研究与临床上的应用。为揭示haESCs自发二倍体化的机制，本项目通过分选早期与晚期haESCs的单倍体与二倍体状态的细胞。制备转录组、DNA甲基化、核小体定位、与组蛋白修饰数据。分析haESCs二倍体化过程的基因表达与表观修饰变化，构建haESCs二倍体化过程的调控网络。结果发现，haESCs的全局核小体定位模式、不同转录因子靶点与核小体之间的特定拓扑空间关系、启动子与增强上的组蛋白修饰、及基因表达谱，均与ESCs高度相似，而与小鼠成纤维细胞、圆形精子显著不同。值得指出的是，haESC中部分与细胞周期调控相关的增强子呈现关闭状态，也可能与自发二倍化有关。深入分析发现，haESCs自发二倍化过程中，显著上调的差异表达基因极光激酶Aurora家族、CDK和Polo样激酶为中心构成的调控网络，主要调控细胞核分裂、细胞器分裂、染色体分离、姐妹染色单体分离。这表明haESCs的自发二倍化可能是由于细胞分裂发生异常造成的，而且主要发生在G2到M期，包括的关键基因有Aurka、Aurkb、Cdk1、Plk1、Mps1与Bub3等。而晚期haESCs自发二倍化率下降与细胞有丝分裂无关，主要由于与p53表达下降导致的细胞凋亡通路活性下降密切相关。细胞凋亡通路活性降低导致更多单倍体状态haESCs存活，在晚期haESCs中单倍体状态haESCs比例升高，从而自发二倍化率变低。进一步分析发现，启动子H3K4me3信号上升，上调有丝分裂检查点复合物基因集及Aurkb的转录，从而促进haESCs自发二倍化。而细胞凋亡通路活性下降与基因启动子组蛋白修饰没有紧密关系。本项目系统阐述了haESC的基因表达与染色质状态特征，揭示了在haESCs自发二倍化与传代过程中二倍化率下降中起重要作用的关键信号通路与基因，及其调控机制。这为解决haESCs的自发二倍化问题和拓展haESCs的应用提供理论依据。