CTCF介导不同类型染色质互作的机制、功能及有丝分裂遗传研究

The CCCTC binding factor (CTCF) is evolutionarily highly conserved, ubiquitously expressed, multivalent regulator and only one known insulator -binding protein in vertebrates. By the different combinations of 11 zinc finger domains, CTCF recognizes diverse sequences and exerts various physiological functions such as gene activation and repression, X chromosome inactivation and genomic imprinting, enhancer blocking and chromatin insulation, recombination and replication etc. CTCF's diverse functions partially due to it's ability to context- dependently interacts with different proteins and itself, and it's different types of post-transcriptional modifications. More recently, CTCF's function in mediating inter- and intra-chromatin interactions has been extensively studied, which strongly indicates CTCF might be the master waver of the genome architecture. But the detail molecular mechanism and function significance of CTCF-mediated chromatin interactions remain elusive, especially on the context of different types of CTCF binding sites and associated chromatin interaction sub-networks. Our previous studies have documented that CTCF mediated extensive chromatin interactions to form cell-type nonspecific imprinting genes network which regulates imprinting genes' expression and replication timing in liver tissue and ESC respectively. We also found CTCF can mediate cell-type specific intra- and inter- chromatin interactions to regulate cell-type specific expression of Kcanq5 etc. In this proposal, we would like: to globally profile, validate the CTCF-mediated chromatin interactome in mouse, human ESC, GM12878 cells by the combination use of 4CALLs(3C-ChIP-one-but-for-ALLs), high throughput sequencing, bioinformatics analysis, 3D DNA-FISH etc.; to delineate the cell-type specific vs. non-specific, species conserved vs. non-conserved sub-networks by the comparison within and across human and mouse cells; to explore the CTCF's role in genome nuclear architecture by the interrogation of different sub-networks with CTCF's occupancy, different epigenetic profiles such as histone modifications, DNA methylation, and large physical domains; to reveal the different sub-networks' biological functions, corresponding molecular mechanisms by comparison with gene transcription profile and the co-localization analysis of sub-networks with different nuclear compartments; to mine potential new genome-wide functions of CTCF. The mitotic inheritance of CTCF-mediated different chromatin interactions and its contribution to rapid reestablishment and maintenance of higher-order chromatin structure will also be exploited by synchronizing the cells to metaphase.

CTCF是一种高度保守的多功能蛋白，识别众多不同DNA序列，介导广泛的染色质之间、染色质之内互作，在X染色体失活与基因印迹、基因活化与沉默、DNA重组与复制等诸多生理过程发挥不同作用。但其介导不同染色质互作发挥不同功能的分子机制、生物学意义尚不清楚。我们拟在前期发现CTCF介导细胞类型非特异印迹基因网络的形成调节印迹基因的表达和复制、介导细胞类型特异性染色质作用调节Kcnq5等的细胞类型特异性表达等基础上，利用已建立的4CALLs技术，通过鼠、人ESC等细胞中CTCF介导的染色质互作的筛选、鉴定、比较及与不同类型表观遗传谱、转录谱、染色质大尺度结构域等的关联研究，获得CTCF介导的染色质作用谱，分析其细胞类型特异性和保守性，揭示CTCF介导染色质互作在基因组三维高级结构的形成及其有丝分裂后迅速重建中的作用，阐明CTCF介导不同类型染色质作用的生物学意义及分子机制，挖掘其潜在新功能。

CTCF是一种高度保守的多功能蛋白，是基因组三维高级结构的关键组织者，通过识别众多不同DNA序列，介导广泛的染色质互作，在X染色体失活与基因印迹、基因活化与沉默、DNA重组与复制等诸多生理过程发挥不同作用。但其介导不同染色质互作发挥不同功能的分子机制、在有丝分裂过程中的传递、生物学意义等尚不清楚。本研究中，通过对DNA超声随机断裂末端的生物素高效标记、微量DNA二代高通量测序文库的高效建立等技术的改进、优化，建立了鲁棒、特异、敏感的研究特定蛋白介导染色质互作的4CALLs-seq、研究微量样本全基因组三维高级结构的eHi-C技术及其配套的“一站式”生物信息学分析流程HBP。随后利用ChIP-seq、4CALLs-seq、eHi-C以及免疫荧光等技术，针对CTCF介导的染色质互作及其在有丝分裂中的传递这一科学问题展开了研究，结果发现：CTCF与不同类型的DNA结合位点结合，介导广泛的“一对多”、“多对多”等不同拓扑类型染色质互作子网络的形成；通过和Cohesin、ZNF143等不同转录因子协同，CTCF介导形成三种不同类型的染色质Loop结构，对基因表达发挥不同的调控作用；部分CTCF结合位点与RNAPII Foci共定位，提示CTCF通过介导互作网络与转录工厂共享高效调控基因的表达；那些高度保守、高结合强度的CTCF结合位点与CTCF的结合及其参与形成的部分染色质互作网络在细胞有丝分裂过程中维持不变，这部分CTCF结合位点常常位于染色质一级结构Synteny、三维高级拓扑结构TAD的边界处，表明CTCF与保守结合位点的结合、及其互作网络可以有丝分裂过程中稳定传递，发挥特殊的有丝分裂书签作用，这一结果将对高效iPS策略的建立提供一种全新视角。此外，还发现了基因组范围的、大尺度的、保守的GC3偏性结构域；对CTCF在包括CMG2、IFITM3等特定基因座位局部的基因表达的三维调节等也进行了研究。