CRISPR-Cas9遗传学DNA片段编辑技术研究原钙粘蛋白家簇的3D基因组拓扑结构及功能

The applicant has had outstanding track-records working on genomic organization, expression and functional research and discovered three closely-linked mammalian protocadherin gene clusters containing about 60 genes arrayed in tandem. The protocadherin proteins are expressed at synapses in a combinatorial manner and provide enormous neuronal cell-surface diversity for synaptic connectivity in the brain. Protocadherin clusters contain a variable region with more than a dozen highly-similar exons followed by a single set of downstream constant exons, similar to that of the drug-metabolizing UGT, immunoglobulin, and T cell receptor gene clusters. My laboratory recently developed a DNA-fragment editing (including DNA fragment inversion, duplication, deletion, and modification) method using CRISPR-Cas9 system with paired sgRNAs. In conjunction with biochemical and computational methods, we found that the location and relative orientation of genome-wide CTCF-binding sites (CBSs) determine the specificity of long-distance chromatin looping interactions in mammalian genomes, suggesting that 3D genome topology can be predicted based on CTCF/Cohesin directional binding to CBSs and the controlling elements can be engineered by CRISPR-Cas9 DNA fragment editing. Finally, we found that protocadherins function in regulating dendritic development and Globus Pallidus connectivity. In this grant application, we will dissect the long-distance DNA interactions between promoters and enhancers by molecular genetics and 3C/4C/5C methods. In addition, we will investigate the signaling mechanisms by which protocadherins function in neurodevelopment in gene-targeted and modified mouse models. These studies have important implications regarding the establishment and maintenance of cell-surface codes for trillions of specific neuronal connections in the brain.

项目申请人具有20多年的基因组结构、表达和功能研究经验，首次发现了三个紧密相联的原钙粘蛋白基因簇，在哺乳动物中总共有大约六十个新基因形成串联阵列，具有可变区和恒定区基因结构，非常类似于Ugt，Ig和Tcr基因簇，所编码的多样性原钙粘蛋白以细胞“克隆特异性”形式组合表达在神经细胞突触。申请人课题组最近开发了CRISPR DNA片段编辑新技术方法，发现绝缘子结合蛋白CTCF和染色体粘联蛋白Cohesin介导的特异性DNA环化对于原钙粘蛋白细胞“克隆特异性”表达的启动子选择非常重要，阐明了原钙粘蛋白在神经元树突发育和突触连接功能，解决了长期悬而未决的增强子和绝缘子方向性问题（CELL，2015）。我们将利用课题组开发的DNA片段编辑方法并结合染色体构象捕获技术研究启动子和增强子远程交互的特异性，阐明原钙粘蛋白细胞“克隆特异性”组合表达机制，通过基因打靶和基因编辑小鼠表型分析研究原钙粘蛋白功能。

项目负责人曾在哺乳动物中发现了三个紧密相联的原钙粘蛋白基因簇，共有约六十个基因形成串联阵列，具有可变区和恒定区基因结构，非常类似于Ugt、Ig和Tcr基因簇，所编码的多样性原钙粘蛋白以细胞“克隆特异性”形式组合表达在神经细胞突触。项目团队最近开发了CRISPR DNA片段编辑新技术方法，发现绝缘子结合蛋白CTCF和染色体粘联蛋白Cohesin介导的特异性DNA环化对于原钙粘蛋白细胞“克隆特异性”表达的启动子选择非常重要，阐明了原钙粘蛋白在神经元树突发育和突触连接功能，解决了长期悬而未决的增强子和绝缘子方向性问题。该项目利用自主开发的DNA片段编辑方法并结合染色体构象捕获技术研究启动子和增强子远程交互的特异性，研究原钙粘蛋白细胞“克隆特异性”组合表达机制，通过基因打靶和基因编辑小鼠表型分析研究原钙粘蛋白的功能。项目揭示了染色质重排接头处特定碱基加入的规律，通过调控细胞修复系统实现了精准DNA片段编辑技术。发现了原钙粘蛋白和细胞粘附激酶通过对肌动蛋白细胞骨架的动态调控参与大脑皮层神经元的长距离迁移和神经突生长。揭示了CTCF/cohesin调控基因组的非对称性阻断机制。发现了绝缘子具有拓扑性，近端的CTCF位点与近端的CTCF位点互作，远端的CTCF位点与远端的CTCF位点互作。揭示了REST通过串联排列的锌指结构域，以反平行的方式、碱基特异性识别基因组位点，调控神经系统基因的表达。项目研究成果对于解析建立和维持人类大脑中数以万亿的特异性神经连接的细胞表面编码具有重要意义。