| Referred to CTCF, the CCCTC-binding factor is a ubiquitously expressed and multi-functional zinc finger protein. It is highly conserved and considered as the only known insulator protein in vertebrates. Initially discovered as a repressor of the chicken c-myc gene, CTCF is reported to be involved in many biological processes, including transcription activation and repression, chromosome insulation, imprinting control, Xchromosome inactivation, m RNA alternative splicing, nucleosome positioning, and DNA replication, etc. The abnormal expression of CTCF usually leads to the occurrence and development of cancers and other diseases.Using different combinations of its 11 zinc fingers, CTCF can bind tens of thousands of DNA sites which are widespread across the genomes. The dynamic balance between CTCF binding to target sites and the DNA methylation status results in the formation of cell type specific binding sites. The sequence conservation and composition features are different among these sites. Through the intrinsically disordered N and C termini and different post-translational modifications, CTCF can interact with a variety of proteins, even RNAs. In addition, CTCF have been shown to bind to chromosomes during mitosis, and might act as a mitotic book marker. But the detailed molecular mechanism and potential functions remain elusive.As the master weaver of the genome, relying on widespread binding sites and combination with other proteins and RNAs, CTCF can mediate extensive intra- and inter-chromatin interactions, help to establish and maintain a higher-order chromatin conformation, and thus exert multiple functions. So far, the research about the relationship between different types of CTCF-binding sites, CTCF-mediated interactions and functions only focuses on the individual phenomenon or local data. It needs comprehensive and systematic analysis on the intrinsic logic and its biological significance.In this paper, we used multi-level omics data from ENCODE, including human CTCF Ch IP and Ch IA-PET data, Drosophila CTCF Ch IP data during interphase and mitosis, as well as other transcription factors, epigenetic modifications profiles. In combination with numerous gene information database, bioinformatics algorithms and tools, we did a comprehensive and detailed systematic analysis on genome-wide CTCF profiles from three aspects: CTCF-binding sites, CTCF-mediated interactions and mitotic inherence.In the first part, we collected 162,209 CTCF-binding sites across 70 human cell types from ENCODE database. We identified the three-part motif in CTCF-binding sites, and found a correlation between the cell type specificity, significance of motif enrichment and sequence conservation of the binding site, i.e. the higher the cell type non-specific, the stronger the motif, the higher the conservation. It was possible that cell type non-specific binding sites, i.e. constitutive sites, might act as the key nodes in the genome, and maintained the high conservation under the evolutionary selection pressure. Moreover, the stronger motif provided higher affinity for CTCF, resulting in stable binding.Next, the functional analysis on CTCF target genes showed that while cell type specific CTCF-binding sites were usually related with the regulation of cell type specific genes, non-specific sites targeted genes associated with basic biological processes. Furthermore, from a new point of view, we reported for the first time that 160 transcription factors could be clustered into four categories according to their colocaliaztion profiles with CTCF-binding sites. The result suggested that CTCF might bind to different types of sites and recruited different types of partners, and then played different biological roles.The second part focused on CTCF-mediated chromatin interactions in human genome. We first concluded that the cell type non-specificity(constitutive) was the necessary and sufficient condition for the ability to establish chromatin interactions, i.e. the cell type non-specific binding sites tended to participate in the interactions and sites involved in interactions were mostly highly non-specific. Further, from the perspective of topology, cell type non-specific binding sites were found at the center positions of the chromatin interaction network, playing key roles in maintaining the network’s connectivity, stability and robustness. We believed these sites might serve as backbones of the three-dimentional structure of chromatin. In particular, we found CTCF could establish a special “many to many” chromatin interaction module for the first time, which might be involved in the mouse-specific chromatin conformation, but its role in human genome need further investigation.Through the integration of different transcription factors’ Ch IP data, epigenetic modifications profiles, chromatin states and other data, we categoried CTCF-mediated chromatin interactions into several classes, and found that cohesin complex and ZNF143 might help CTCF establish and maintain chromatin interactions. The role of cohesin has been reported, while the molecular mechanism of ZNF143 remained unclear. Based on above results, we guessed CTCF might act as the chassis of the network, selectively recognized target sites, and established different classes of chromatin interactions with the help of cohesin and ZNF143. Part of these interactions served as backbones of the three-dimentional structure of the genome, another part further recruited other transcription factors, resulting in different mechanisms of transcriptional regulation.In the third part, we focused on the role of CTCF in the establishment and maintenance of cell identity. We discussed the distribution of Drosophila CTCF during the cell cycle and separated d CTCF-binding sites into three categories: interphasemitisis-common sites(IM), interphase-only sites(IO), and mitosis-only sites(MO). Compared with the motif enrichment, GC content and sequence conservation, we found MO sites were different from IM and IO sites. Due to the condensation of the chromatin during mitosis, whose environment and physical characteristics might be totally different from that in interphase, there could be a new molecular mechanism of d CTCF binding to MO sites. Moreover, IM sites showed significantly stronger motif enrichment, Ch IP signal intensity and higher conservation, and target genes were related with general biological processes, indicating the important role of d CTCF in the establishment and maintenance of cell identity.We further found that d CTCF-binding sites of the same class preferentially localized closer to each other, and were highly enriched at chromatin syntenic and topological associated domains boundaries, which also showed similar “like attracts like” phenomenon. It was possible that d CTCF helped establish and maintain TAD and chromatin syntenic domains throughout the whole cell cycle.Based on these results, we speculated that CTCF-binding sites remained associated with mitotic chromosomes might have following functions: CTCF bound to specific sites associated with genes which were necessarily functioned during mitosis, and facilitated the re-activation of gene regulation network during the M/G1 phase transition. Meanwhile, CTCF helped to establish cell cycle phase specific chromatin domains and facilitated the rapid re-establishment of chromatin structures when entering the interphase.In conclusion, we did a comprehensive research from the points(CTCF-binding sites) to the lines(CTCF-mediated chromatin interactions), then to dynamic changes throughout the whole cell cycle. We characterized the function of CTCF in detail, and built multi-level profiles, providing important experiment resources and theoretical basis for the further study. |
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