Normal and Pathologic Functions of CTCF and Its Distinct Classes of DNA-targets

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CTCF, a highly conserved DNA binding protein, serves as a global organizer of chromatin architecture. CTCF is involved in the regulation of transcriptional activation and repression, gene imprinting, control of cell proliferation and apoptosis, chromatin domain insulation, X-chromosome inactivation, prevention of oligonucleotide repeat expansion, and other chromatin resident processes. The multiple functionality of CTCF is based on its ability to bind a wide range of diverse DNA sequences as well as on the intrinsic capacity to interact with a partner protein through the combinatorial usage of eleven C2H2 Zinc Fingers (11 ZFs). With the advance of next generation sequencing techniques, CTCF binding sites have been identified across fly, mouse, and human genomes. Reflecting the multitude of CTCF functions, CTCF Target Sites (CTSes) were found to be associated with the genomic regions engaged in the long-range chromatin interactions, including enhancers, promoters, insulators and boundary elements. It remains obscure, however, how the DNA sequences of given CTSes are related to the specific CTCF functions at these sites. This year we have made advances in the direction of understanding of CTCF multiple functionality. By mapping of CTCF and BORIS occupancy genome-wide, we uncovered two classes of CTCF binding regions that are pre-programmed and evolutionary conserved in DNA sequence.We found that 70% of CTCF bound regions enclose a single CTCF binding sites, aka "1xCTSes" while other 30% of CTCF-binding regions detected by ChIP-seq as single peaks are, in fact, shown to contain the dual CTCF binding sites, aka "2xCTSes". Occupancy of both DNA sites within bipartite 2xCTS-regions constrains 2 adjacent CTCF proteins to form homodimers in normal somatic cells and to assemble heterodimers of CTCF and BORIS co-bound at the same DNA spot in germ and cancer cells expressing BORIS. The recent breakthrough discovery of 2xCTS-regions (unresolved by a standard CTCF ChIP Seq alone) has unable us, for the first time, to address the long-standing question how CTCF can serve in the context of the same nucleus as a bona fide transcription factor, while maintaining a substantial presence at putative insulator/boundary sites that bear no indications of transcriptional activity. Indeed,only 20 % of all CTCF binding regions are located in promoter regions in any given cell type, while the rest of CTSes are not associated with transcriptional start sites. The obvious candidates for the determinants of such distinct functional roles would be DNA sequences themselves and/or differential identity of chromatin at these two types of sites. In our study we presented genome-wide evidence that DNA sequences underlying the two types of CTCF target sites are structurally different. The structural difference between two classes of CTCF binding sites is connected to the functional difference: 2xCTSes are preferentially located at active promoters and enhancers, and are associated with retained histones in human and mouse sperm, in stark contrast to genomic regions harboring a single CTCF binding site. Our study is challenging the perception in the current literature that all CTCF sites are equal and characterized by a single CTCF motif. Next, mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncovered extensive chromatin reorganization during lineage specification. We observed that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we found that widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results provided a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.