UHRF家族蛋白在调控DNA甲基化的建立与维持中的作用及分子机制研究

DNA methylation is one of the most important epigenetic modifications in mammals and plays a critical role in embryonic development, differentiation and cell reprogramming. However, the mechanisms for establishment and maintenance of DNA methylation patterns in mammalian cells as well as the mechanisms for DNA methylation reprogramming during cell fate reprogramming remain to be well characterized. We and others have demonstrated that UHRF1 is essential for targeting DNMT1 to DNA replication forks for DNA maintenance methylation and that UHRF1 mediates the stimulatory effect of H3K9me2/3 on DNA methylation in part through its H3K9me2/3 binding activity. Furthermore, we uncovered recently that UHRF1 and its closely related UHRF2 negatively regulate DNA de novo methylation by inducing DNMT3A degradation. In this proposal, we will study: 1) The mechanism(s) by which UHRF1 targets DNMT1 to DNA replication forks in S phase of cell cycle; 2) The roles of negative regulation of DNMT3A by UHRF1 and UHRF2 in establishing and maintenance of DNA methylation patterns；3) The role of underlying mechanisms for the contribution of inhibiting DNA maintenance methylation in DNA demethylation during somatic cell reprogramming. This project will shed light on the regulatory mechanisms of DNA maintenance and de novo methylation and provide new insight into the DNA demethylation processes in somatic cell reprogramming.

DNA甲基化在哺乳动物胚胎发育、细胞分化及重编程过程中起重要作用，但DNA甲基化谱式的建立、维持及重编程的机制还有待深入研究。UHRF1是一个将DNMT1招募至DNA复制叉从而实现DNA维持性甲基化的关键蛋白。我们最近的工作表明UHRF1能通过其特异的甲基化H3K9结合能力介导了组蛋白H3K9甲基化对DNA甲基化的正调控；还发现UHRF1和UHRF2能通过降解DNMT3A负调控起始性DNA甲基化。在这个项目中我们拟：1) 进一步解析UHRF1在DNA复制期特异招募DNMT1实现维持性DNA甲基化的分子机制; 2) 解析UHRF1和UHRF2对DNMT3A的负调控在DNA甲基化谱式的建立与维持中的作用及意义；3）研究在体细胞重编程过程中抑制DNA维持性甲基化在DNA去甲基化中的作用及分子机制。该项目的顺利实施有望增强对DNA 甲基化修饰调控的分子机制及其在体细胞重编程中的作用的理解。

本项目旨在深入研究UHRF家族蛋白在调控DNA甲基化的建立与维持中的作用及分子机制。我们在本项目拟研究的三方面内容都取得了重要进展。在第一方面，我们深入解析了UHRF1的H3K9甲基化结合能力在介导DNA维持性甲基化方面的作用，发现该结合能力并不是介导DNMT1的维持性甲基化所必须，但起着重要辅助性作用。在第二方面，我们发现UHRF1和UHRF2对DNMT3A具有负调控作用，其分子机制是通过泛素化修饰DNMT3A从而导致DNMT3A的降解。我们认为这个调控途径可能促进了肿瘤细胞中整体DNA甲基化水平的下降。在第三方面我们的工作表明Oct4特别是Nanog能够抑制UHRF1与半甲基化DNA的结合。这方面的工作表明胚胎干细胞核心转录因子确实可能通过抑制维持性甲基化来重编程细胞表观修饰谱式。