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

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。

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.