以纤毛虫原生动物－嗜热四膜虫为模式：DNA复制延伸相关的表观遗传学研究

项目名称： 以纤毛虫原生动物－嗜热四膜虫为模式：DNA复制延伸相关的表观遗传学研究

项目编号： No.31470064

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 高珊

作者单位： 中国海洋大学

项目金额： 30万元

中文摘要： 大量研究显示，DNA的复制延伸与组蛋白嵌入及核小体组装过程高度相关。但迄今对表观遗传因子与DNA复制相互作用机理的了解高度匮乏，尤其对组蛋白转录后修饰以及变体如何参与该复制的过程欠缺了解，这也构成了当今表观遗传学研究的热点问题之一。申报人最近以四膜虫为模式动物，刻画了H3K27me1特异性敲除对DNA复制延伸的影响等并取得了富有意义的发现。本项目拟在此基础上开展如下研究：1）以四膜虫单核小体为内参，借助染色质免疫共沉淀-高通量测序等方法，构建多种表观遗传因子的全基因组分布图谱；2）在高通量测序信息基础上，比对已知DNA复制起点图谱，筛选参与该复制过程的关键因子；3）利用基因敲除和标记技术，探讨和阐明参与复制延伸过程的关键因子；4）在上述工作基础上，揭示表观遗传信息影响DNA复制上下游通路的调控机制。本研究将为解答"表观遗传信息影响DNA复制的作用机制"这一科学命题提供重要的佐证和新的认知。

中文关键词： 复制；TXR1；H3K27me1；核小体；四膜虫

英文摘要： Previous studies have shown that DNA replication is highly cooridinated with histone deposition. However, the epigenetic mechanism regulating DNA replication is still not clear because we know little about which epigenetic marks, including histone PTMs (post-translational modifications) and histone variants, are involved in DNA replication, not to mention their distribution along chromosomes. Recently, the applicants and colleagues revealed that DNA replication elongation is severely impaired by deficiency of H3K27me1, which is the first to show that perturbation of epigenetic information can directly affect replication elongation. In this proposal, we will use the ciliated protozoan - Tetrahymena thermophila - as a model organism, to investigate the involvement of additional epigenetic factors in this pathway that couples propagation of both genetic and epigenetic information. In brief, mono-nucleosomes will be purified by MNase (micrococcal nuclease) digestion and sucrose gradient centrifugation. Using mono-nucleosome as input, chromatin immunoprecipitation (ChIP) will be performed with specific histone modification antibodies or affinity tag antibodies, the purified samples from which will be analyzed by deep sequencing. This will allow us to provide the first high-resolution genome-wide distribution maps for

英文关键词： Replication；TXR1；H3K27me1；nucleosome；Tetrahymena