哺乳动物DNA甲基化表观遗传的核心机制与功能研究

In mammals, DNA methylation plays critical roles in various biological processes including embryonic development and differentiation, cell fate determination, genome stability and transcriptional regulation and pathological processes such as cancer. DNA methylation is a key component of epigenetic regulation, and the underlying mechanism for DNA methylation inheritance is DNMT1-mediated DNA maintenance methylation. Our preliminary data indicate that DNA maintenance methylation by DNMT1 is not only dependent on cofactor UHRF1, which recruits DNMT1 to replication forks during S phase of cell cycle, but also requires a negative regulator USP7. USP7 knockout results in progressive accumulation of DNA methylation, leading to cell proliferation defect, cell senescence and apoptosis. In this grant, we propose to: 1) determine the DNA methylation profiles in USP7 knockout cells; 2) investigate the molecular mechanisms by which USP7 negatively regulate DNMT1-mediated DNA methylation; 3) investigate if negative control by USP7 is a evolutionarily conserved mechanism for regulation of DNA methylation inheritance; 4) elucidate the biological significance of USP7-mediated negative regulation of DNA methylation; 5) investigate if USP7 plays a role in DNA methylation reprogramming occurred in early embryonic development and contributes to aberrant DNA methylation in cancers. Our study will provide novel insights into the mechanisms by which DNA methylation is established and maintained in mammals and the functional significance of appropriate regulation of DNA methylation.

哺乳动物DNA甲基化在多种生物学过程如胚胎发育分化、细胞命运决定、基因组稳定性、转录调控及病理过程如肿瘤发生发展均发挥重要作用。DNA甲基化是表观遗传调控的重要组成部分，其核心是由DNMT1介导的维持性甲基化。本实验室的前期研究结果发现DNMT1的维持性甲基化不仅需要UHRF1将DNMT1正确靶向DNA复制叉，而且需要USP7对DNMT1功能的负调控，敲除USP7导致DNA甲基化逐渐增高，细胞增殖的抑制、衰老和凋亡。在此基础上，本课题拟1）确定USP7敲除导致的DNA甲基化谱式的变化；2）阐明USP7负调控DNA甲基化的分子机制；3）阐明USP7负调控DNA甲基化的进化保守性；4）USP7负调控DNA甲基化的生物学功能；5）USP7负调控DNA甲基化与DNA甲基化重编程和肿瘤DNA甲基化异常的关系。该课题的研究将有助于进一步解析DNA甲基化建立与维持的分子机制及生物学功能。

哺乳动物DNA甲基化在多种生物学过程如胚胎发育分化、细胞命运决定、基因组稳定性、转录调控及病理过程如肿瘤发生发展均发挥重要作用。DNA甲基化是表观遗传调控的重要组成部分，其核心是由UHRF1特异识别DNA复制叉的半甲基化DNA并介导的组蛋白泛素化修饰和组蛋白泛素化修饰依赖的DNMT1招募。在本项目中，我们发现在多种细胞中敲除USP7都导致细胞DNA甲基化整体水平显著上升并产生大量的新的甲基化位点。分子机制研究发现USP7拮抗UHRF1催化的组蛋白H3和H2B的去泛素化，USP7通过其去泛素化酶活性及与DNMT1的相互作用负调控DNA复制叉上的组蛋白泛素化修饰从而避免组蛋白泛素化的过度积累和DNMT1的过度招募和DNA的过度甲基化。因此，我们的研究揭示USP7是DNA甲基化稳态遗传的一个重要调控因子。在本项目中我们还研究了DNMT1突变导致人类神经退行性疾病的分子机制。我们构建了多个模拟人类神经退行性疾病中发现的DNMT1突变的小鼠模型，发现纯合的小鼠在胚胎发育10.5天左右死亡，杂合子小鼠能正常成活但具有神经退行性表型如学习和记忆缺陷。分子机制的研究发现突变的DNMT1蛋白很不稳定，但不是通过蛋白酶体而是通过一个未知的蛋白酶进行降解。此外，我们的研究还表明突变DNMT1的杂合子小鼠具有神经细胞增殖与发育的异常。这一研究揭示了DNMT1杂合突变导致神经退行性疾病的分子机制。总的来说，我们的研究进一步解析了DNA甲基化建立与维持的分子机制及生物学功能。