介导人类基因组DNA甲基化修饰的非编码RNA的鉴定及其作用机理研究

DNA methlation modification have key regulatory roles in stem cell differentiation. In the process of cell differentiation, how the DNA methyltransferases recognize specific sites in genome for site-specidic DNA methylation is still an open question. Our previous work shows that in the Dnmt3b complexe there are non-coding RNAs (ncRNA) which may direct DNA methlation modification. To investigate the function of ncRNA on the DNA methlation modification during the process of human cell diffrentiation from embryonic stem cell toward neural stem cell, we will isolate RNA from RNA and Dnmt3 complexes and construct the RNA libraries. Then, we will obtain ncRNA sequence from Dnmt3a-ncRNA and Dnmt3b-ncRNA complexes by deep sequencing and identify ncRNAs which direct DNA methlation modification. Finally, we will study the role of members in Dnmt3a-ncRNA and Dnmt3b-ncRNA complexes on the DNA methlation modification and reveal valuable insight into the molecular mechanisms of RNA directing DNA methlation modification.The research of this project will not only identify a group of ncRNAs which direct DNA methlation modification, but also provide valuable method for the differentiation of embryonic stem cell toward neural stem cell by regulating ncRNAs which direct site-specific DNA methylation.

DNA甲基化修饰对干细胞的分化起着重要调控作用。但是在干细胞的分化过程中，DNA甲基化转移酶如何识别特定的DNA位点，催化其甲基化修饰？这个问题迄今没有被解析。我们在前期研究中发现，Dnmt3b复合物中存在非编码RNA,这些非编码RNA可能介导了DNA的甲基化修饰。本项目拟建立人ES细胞向神经细胞分化过程中，与DNMT3复合物结合的RNA 深度测序文库，获得与Dnmt3a和Dnmt3b复合物结合的非编码RNA的序列信息，鉴定出能够介导基因组DNA甲基化修饰的非编码RNA，研究参与DNA甲基化修饰的Dnmt3复合物的组成及各成员在DNA甲基化修饰中所起的作用，揭示非编码RNA介导DNA甲基化修饰的分子机制。本项目的实施不仅能够鉴定一批介导DNA甲基化修饰的非编码RNA，阐明细胞分化的表观遗传调控机制，而且能够建立通过非编码RNA调控DNA甲基化进而诱导ES细胞向神经细胞高效分化的新技术。

在干细胞的分化过程中，DNA甲基化修饰起着重要的调控作用。但是，DNA甲基转移酶识别基因组中的特定位点并对其进行甲基化修饰的分子机制目前尚不清楚。本项目以ES细胞向神经元分化过程为研究体系，采用RNA结合蛋白免疫沉淀技术（RNA Binding Protein Immunoprecipitation, RIP）分离Dnmt3a复合物和Dnmt3b复合物中的非编码RNA,分别建立这些RNA的深度测序文库。采用TopHat软件将reads映射到基因组上，采用Cufflinks软件对转录物进行组装，确定了非编码RNA在基因组上的分布情况，获得了与Dnmt3a和Dnmt3b复合物结合的非编码RNA的序列信息。我们发现了一批参与DNA甲基化修饰的非编码RNA，并对其中的部分非编码RNA进行了进详细分析。我们的研究揭示了非编码RNA 介导DNA 甲基化修饰的分子机制，为采用非编码RNA 调控DNA 甲基化进而诱导ES 细胞向神经细胞高效分化的技术体系的建立和应用奠定了基础。