DNMT1通过与RNA聚合酶polII相互作用而导致抑癌基因启动子区域异常甲基化的机制研究

项目名称： DNMT1通过与RNA聚合酶polII相互作用而导致抑癌基因启动子区域异常甲基化的机制研究

项目编号： No.81472637

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 李志广

作者单位： 大连医科大学

项目金额： 72万元

中文摘要： 抑癌基因的失活与肿瘤的发生发展密切相关。启动子区域异常甲基化是多种肿瘤抑癌基因失活的重要方式，但这种异常甲基化的形成机制一直不清楚。DNMT1是重要的DNA甲基化酶。我们前期的研究表明该酶与RNA聚合酶polII存在直接相互作用，且两者在染色体上的结合位点有很大程度的重叠。polII的主要功能之一是识别并结合基因的启动子。据此本项目提出假说，认为DNMT1通过与polII结合作用于抑癌基因启动子区域，从而导致其异常甲基化。本项目拟确定polII与DNMT1直接结合的蛋白结构域；将该结构域定点突变后，观察在致癌条件下抑癌基因启动子区域甲基化的动态变化；运用深度测序技术，对该变化在全基因组内进行分析，以验证我们的假说。最后我们还尝试筛选能够通过特异阻断DNMT1与polII结合而抑制抑癌基因启动子异常甲基化的药物。本项目能够揭示抑癌基因失活的表观调控机制，为抗肿瘤药物的开发提供新的理论依据。

中文关键词： 肿瘤发生；DNA甲基化；基因组学；生物信息学；二代测序技术

英文摘要： The inactivation of tumor suppressor gene (TSG) is closely associated with tumor incidence and development. Abbarrent methylation at promoter regions is one of important ways for TSG inactivation in many types of tumor. However, the underlying mechanism remains elusive. Our previous studies indicate there exist direct interaction between DNMT1 and RNA polymerase polII and these two proteins are co-precipitated in immuno-precipitation experiments. Moreover, the binding regions on chromosomes of these two proteins are mostly overlapped. Based on these observations, we propose that DNMT1 is brought to gene promoters by interacting with polII and lead to the aberrant promoter methylation of TSG with the assistance of other carcinogenic factors. Here, we will first determine the protein domain necessary for their binding, and mutate such domain in polII. Then, we will transfect mutated and wild type polII　genes into cells repectively and investigate promoter methylation alterations under carcinogenic conditions. Finally, BS-seq and ChIP-seq will be employed to reveal the changes of DNA methylation, as well as DNMT1 and polII binding, within the whole genome. We will also try to screen drugs that can specifically suppress abbarrent methylation at TSG promoters via blocking the interaction between DNMT1 and polII. This project will illucidate the epigenetic mechanims leading to TSG inactivation, and guide the development of new anti-tumor drugs.

英文关键词： Tumorigenesis;DNA Methylation;Genomics;Bioinformatics;Next-Generation Sequencing Technology