The first US patent issued for CTCF in October 1999 included full length cDNAs sequences coding for exceptionally conserved gene ubiquitously expressed in somatic cells from a wide range of evolutionary divergent species, including Drosophila, birds, mice, and humans. CTCF is unique multifunctional nuclear factor with multiple sequence-specificity which controls global genome architecture and many aspects of allele-specific epigenetic regulation including direct repression and/or activation of transcription, pausing-associated splicing, replication timing, long-range enhancer-promoter communications and hormone-induced silencing. Initially demonstrated by us (MCB 2004) in vitro dimerization between distinct CTCF/DNA complexes was recently shown to mediate universal site-specific intergenic, intra-chromosomal, and inter-chromosomal linking-looping in vivo through interactions between distinct classes of regulatory elements containing varying DNA sequences of CTCF-target-sites (CTSes). We discovered that sequence-specific DNA-bound CTCF-complexes are capable of dimerization in vitro which could serve in vivo to bridge different CTCF-bound sites across nuclear chromatin. Next, we have first demonstrated that upon binding to dissimilar DNA-sites CTCF can thereby function through two major mechanisms: either by direct regulation of a gene downstream of CTSes or by indirect regulation via the formation of chromatin loops stabilized upon CTCF/DNA-dimerization. that affects allele-specific relationships between the promoter, enhancer, and/or an imprinted control region (ICR) on either same or different chromosome. Dimerization of DNA-bound CTCF may potentially be at the core of its activity as a versatile chromatin-bridging and chromatin-looping agent, underlying its fundamental biological functions. The loop-forming activity of CTCF can be naturally extended to include formation of localized somatic inter-chromosome pairing sites that may acquire potential for epigenetic co-regulation through allele-specific transcription factories, DNA replication factories, 5-mC/5-hmC excisions, and DNA repair foci. Many other chromatin-anchored functions, such as the establishment of imprinting marks and their reading, X-chromosome inactivation, and apoptosis, are also regulated by CTCF. CTCF has emerged as a key facilitator of 3D organization of interphase chromatin, as well as a major player in cell proliferation control. In some cases, the loop-forming activity of CTCF was found to be accompanied/complemented by the more direct regulation of a particular gene. This mixed mode regulation is likely the most appropriate representation of a native gene regulation framework. In addition to phosphorylation and poly(ADP)rybozylation of CTCF, we also identified CTCF-interacting partner proteins, Sin3A and YB-1, which have recently gained increased attention in course of ongoing interpretation of the NGS data generated by MPS (in collaboration with Dr. B. Ren) with regard to the genomewide CTCF and CTCFL occupancy, Pol 2 initiation, transcription pausing coupled with splicing, and rewiring regulatory elements of CTCFL positive cancer cells by mobile retrotransposones with variable length tandem repeats (VNTR) affected often by a gain of CTCF site specific SNPs. Moreover, we expanded our previous studies of important CTCF activity that directly links CTCF to transcriptional machinery: the binding of CTCF to the Pol II. This novel pathway is sensitive to external signals that affect post-translational modifications of critical CTCF amino acids and provides either a mechanism for opening loop-independent transcription start sites downstream of the promoter-determined +1 site (at intron/exon sequences)and it may have specifically evolved to induce non-coding transcripts throughout the genome depending on the presence of BORIS in a particular cell type under study. Mechanistically regulated recruitment and the subsequent release of Pol II from a DNA-bound CTCF complex indicates that the CTCF site itself could act as an attenuator and/or promoter in some locations in the genome. At imprinted genes, CTCF likely works together with BORIS (Brother of the Regulator of Imprinted States), the testis/cancer-specific CTCF-paralog that we discovered and characterized (PNAS, 2002). In addition, initiated earlier (Cell 2007) genomewide mapping of CTCF targets by ChIP-Seq with our 9 CTCF Mabs lead to understanding fundamental role of CTCF in spatiotemporal coordination of major cellular functions (Nature, 2011;2012). By virtue of having so many vital functions CTCF became an essential gene in vertebrates, as CTCF KO mice are non-viable, and early lethality occurs at the very early embryonic stages (PloS One 2012). With respect to human disease, CTCF is a candidate tumor suppressor gene (TSG);several functional point mutations in the 11ZF DBD of CTCF have been characterized in primary cancers, in combination with the LOH of the CTCF locus. In the past year, we studied several genes and their associated regulatory sequences in an effort to elucidate the contributions of CTCF and CTCF binding sites to the regulation of gene expression. These studies included genes important for immune responses, mono-allelic multigene families of sensory receptors, as well as genes with a potential for a breakthrough in the development of new approaches for cancer treatment. Unlike somatic cells, testicular germ cells undergo meiosis rather that mitosis, - whereby CTCF is normally likely to work together with BORIS. Our recent results have identified BORIS as an anti-silencing component of undifferentiated ES and cancer cells that directly interacts with CTCF. We found earlier that functionally important dimerization between two different CTCF/DNA complexes is based on the capability of CTCF to interact with itself that requires Zn-fingers (MCB, 2004), and showed that literally same fingers are uniquely duplicated and preserved in evolution of mouse and human BORIS proteins (PNAS, 2002). Since formation of homodimeric CTCF/CTCF complexes on DNA underlie site-specific long-range interactions that serve for chromatin linking-folding in normal somatic cells (which do not express BORIS), we expect that formation of heterodimeric CTCF/BORIS complexes observed in chromatin of germ/stem and in cancer cells is likely to have many important structure-functional implications for understanding functional consequences of abnormal CTCF/BORIS dimerization, including an altered chromatin packaging mode normally driven by CTCF alone. While CTCF is mostly known as a regulator of gene expression, our data point to its potential functions in nuclear and nucleolar compartmentalization and heterochromatinization, as well as in in formation of centrosomes. We have also characterized an unusual form of CTCF protein in condensed mitotic chromosomes pointing to its roles in mitosis and meiosis, and thereby suggesting a significant housekeeping role of CTCF in spatiotemporal coordination of genome organization and chromosome segregation in dividing diploid and haploid cells. CTCF was previously shown to undergo a variety of post-translational modifications and we expanded these studies to characterize novel modifications. Another pathological aspect of the misregulated CTCF occupancy on promoter target sites is aberrant alteration of DNA methylation pattern at CTCF sites that can loose protection by DNA-bound CTCF in cancer. Both of these novel biological roles of CTCF are subjects of ongoing studies in the MPS. We also have obtained direct in vivo evidence that at least one role of BORIS is to facilitate DNA re-methylation and Pol 2 recruitment at certain intergenic CTS-sequences found in intrones and promoters of two cancer/testis genes as we published recently.

Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2013
Total Cost
$765,770
Indirect Cost
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Kemp, Christopher J; Moore, James M; Moser, Russell et al. (2014) CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer. Cell Rep 7:1020-9
Nakahashi, Hirotaka; Kwon, Kyong-Rim Kieffer; Resch, Wolfgang et al. (2013) A genome-wide map of CTCF multivalency redefines the CTCF code. Cell Rep 3:1678-89
Mendez-Catala, Claudia Fabiola; Gretton, Svetlana; Vostrov, Alexander et al. (2013) A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells. Neoplasia 15:898-912
Kim, Jung-Hyun; Ebersole, Thomas; Kouprina, Natalay et al. (2009) Human gamma-satellite DNA maintains open chromatin structure and protects a transgene from epigenetic silencing. Genome Res 19:533-44
Heintzman, Nathaniel D; Hon, Gary C; Hawkins, R David et al. (2009) Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459:108-12
Smith, Sheryl T; Wickramasinghe, Priyankara; Olson, Andrew et al. (2009) Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization. Dev Biol 328:518-28