调控合子DNA 甲基化及合子转录激活的母源因子的鉴定及其作用机理

During animal embryogenesis, the zygotic genome is inactivated for transcription upon fertilization and its transcription will occur after a certain period of development. The transcriptional activation of the zygotic genome is closely related to DNA demethylation of the genome, a process controlled by maternal factors. However, it remains unclear which maternal factors are involved in DNA demethylation of the zygotic genome following fertilization and how they function and regulate embryonic development. This project aims to identify maternal factors that regulating DNA demethylation of the zygotic genome and to investigate their roles in zygotic gene activation and embryonic development. To achieve these goals, ENU mutagenesis will be performed in zebrafish; mutants with the disability of DNA demethylation during the cleavage period will be identified by immunostaining with anti-5mC antibody; and the mutant genes will be mapped and cloned. The mutant embryos will be investigated for any defects in zygotic gene activation, germ layer formation and patterning, and organogenesis. The mechanisms of DNA demethylation of the identified factors will also be investigated through biochemical and genetic approaches. The accomplishment of this study would allow the discovery of new demethylation factors and new demethylation pathways, ultimately deepening our understanding of zygotic gene activation and embryonic development in vertebrates.

在动物胚胎发育过程中，受精后合子基因组处于转录抑制状态，到一定阶段合子基因才转录激活。合子基因的转录激活与基因组DNA 去甲基化密切相关，合子基因组DNA的去甲基化由母源因子调控。目前对于参与脊椎动物受精卵合子基因组去甲基化的母源因子和作用机理所知甚少，亟待利用好的实验系统去探究。本项目拟以斑马鱼为对象，通过ENU 大规模诱变，甲基化胞嘧啶（5mC）抗体免疫染色筛选，鉴定基因组DNA 甲基化水平明显上升的突变体，从而发现具有DNA 去甲基化功能的母源因子，并研究它们的作用机理和对合子基因激活、胚层形成与分化、组织器官发育的影响。本项目可望发现调控合子基因组甲基化水平的新的去甲基化因子和信号通路，加深对胚胎早期发育调控的分子机理的认识。

在动物胚胎发育过程中，受精后合子基因组处于转录抑制状态，到一定阶段合子基因组才激活转录。本项目致力于鉴定调控脊椎动物早期胚胎基因组中的DNA和组蛋白修饰及合子基因组激活的母源因子，研究它们的作用机制和对胚胎发育重要过程的影响。通过在斑马鱼中开展ENU诱变，建立了10个DNA甲基化水平上升的突变家系，对部分家系的突变表型进行了进一步的分析。发现尿嘧啶DNA糖基化酶Ung参与了合子基因组DNA的去甲基化，该去甲基化提供了组蛋白修饰潜能，促进合子基因组的激活。发现亲本基因组增强子区域的H3K27ac修饰在受精后迅速擦除，到合子基因组激活时才在高甲基化区域重新建立，但启动子区域的H3K27ac修饰在4细胞期即开始重建且与DNA甲基化无关，组蛋白的乙酰基化修饰与母源表达的组蛋白乙酰基转移酶密切相关。发现复制前起始复合体成员cdc6突变导致胚胎细胞周期停滞在S期、染色体异常分离、胚胎致死。发现Gbf1蛋白通过Arf1-COPI介导的囊泡转运，以细胞自主性方式调控内皮细胞的存活，进而影响血管发育和稳态维持。建立了在未成熟卵母细胞中进行遗传操作的方法，发现在卵母细胞中敲降DNA甲基转移酶基因dnmt1导致早期胚胎中DNA甲基化水平的大幅下降，胚胎在原肠作用前即大量死亡，说明早期胚胎中DNA的重甲基化对于胚胎的发育非常重要。发现一个新的重要母源膜蛋白因子Huluwa，它通过促进Axin降解而促进beta-catenin信号，母源huluwa的突变导致胚胎不能形成组织中心和体轴，说明它是一个关键的组织中心决定因子。这些研究成果加深了我们对于脊椎动物早期发育的调控机制的理解。有关研究成果发表在Science、Molecular Cell、Human Molecular Genetics、Development、Journal of Molecular Cell Biology、Journal of Biological Chemistry等期刊上。