In 2000-01, the Molecular Pathology Section (MPS) in the LIP NIAID continued to work on better understanding of the molecular mechanisms that underlie normal function of the transcriptional factor, called CTCF, in development, cell-cycle regulation, and gene imprinting; and of the molecular events causally linked with CTCF malfunction in cancer, and in other human diseases associated with abnormal site-specific DNA methylation at the variety of highly diverged CTCF-target sites (CTSs). The first comprehensive review on CTCF published in """"""""Trends in Genetics"""""""" in 2001 by V. Lobanenkov and collaborators, provided a summary of experimental results which show that CTCF is a uniquely versatile transcriptional regulator with diverse functions linked to epigenetics and disease. Our earlier results demonstrated that CTCF is the ubiquitously expressed gene upregulated during the S/G2-stage of the cell cycle. It encodes a nuclear factor containing three major functionally distinct regions with amino acid sequences that were maintained practically identical throughout vertebrate evolution: a DNA-binding domain composed of the 11 Zinc Fingers (ZFs), and two flanking trans-acting transcriptional repressor/activator regions that account for approximately two-thirds of the entire protein Recent review of the literature established CTCF as a true """"""""multivalent multifunctional"""""""" protein which utilizes different sets of ZF to form distinct complexes with varying ~50 bp CTCF-target sites (CTS) that mediate distinct functions in regulation of gene expression. Others and we have shown that these functions include context-dependent promoter repression or activation, creation of modular hormone-responsive gene silencers, and formation of diverse vertebrate enhancer-blocking elements (chromatin insulators or boundaries). Functions of varying CTCF/DNA complexes may be regulated by post-translational protein modifications; by physical interactions with other multifunctional nuclear proteins that include, among others, RNA/DNA binding factor YB-1 and the repression-associated mSin3A/HDACs; and by attenuation of the interactions with DNA via specific methylation of CpG pairs involved in recognition of specific CTS by the protein. For example, the latter class of conserved targets which require particular sets of CTCF ZF for formation of the very high-affinity complexes with CTCF, were characterized [see Trends in Genetics 17:520-7 (2001) for review] within the Imprinting Control Region (ICR) between growth-regulating gene IGF2 and a candidate tumor suppressor gene, H19. In collaboration with R. Ohlsson lab in Sweden, we showed that specific CpG methylation eliminates interaction of CTCF with the ICR, allowing the protein to distinguish normally differentially methylated maternal versus paternal IGF2/H19 alleles IN VIVO; and that methylation-regulated formation of CTCF/ICR complexes controls activity and conformation of the chromatin insulator that regulates imprinted IGF2 and H19 expression. In addition to the IGF2/H19 ICR CTSs, critical regulatory regions at the promoters of vertebrate MYC oncogenes have been shown to contain CTS that mediate negative transcriptional control by CTCF modulated by the carboxyterminal phosphorylation. Moreover, a number of novel functional CTS were identified; in respect to cell proliferation control, some of these are neutral while some others are important, for example the CTS in mouse/human PLK and p19ARF genes and mouse/human PIM1 oncogene among others. We have also found that disrupting the spectrum of functional CTCF/DNA complexes either (i) by selective ZF point-mutations observed in some tumors with frequent LOH at CTCF locus mapped on chromosome 16q22 (10) or (ii) by abnormal CpG-methylation of CTS that constitute insulator sites upstream of IGF2 observed in tumors with LOI, is associated with cancer development. We demonstrated recently that CTCF normally inhibits cell cycle progression. In three separate experimental systems, it was shown that the cell cycle profiles of growth arrested cells expressing ectopic CTCF did not noticeably differ from control populations in log phase growth. This ability of CTCF to apparently freeze cells at any stage of cell cycle progression seems to be unprecedented and suggests that CTCF may control expression of genes that arrest cells at each stage in this progression. This model implies function of CTCF as a universal coordinator of an intertwined network of genes in which, if considered separately one from another, each network may perform only strictly specialized tasks in cell cycle control. The same concept may also point to a driving force for exceptional conservation of CTCF, and for evolving of the unusual ability of CTCF ZFs to recognize diverse spectrum of binding sites. On the other hand, it also predicts a very complex multilevel mode for regulation of CTCF itself that may include regulation of CTCF promoter activity during cell cycle, posttranslational modifications including phosphorylation, interactions with multifunctional protein partners, and other mechanisms yet to be discovered. Furthermore, it is conceivable that effects of CTCF on cell cycle progression may depend not only on transcriptional regulation of a variety target genes by CTCF, but also on DNA-independent direct interactions of CTCF with certain components and regulators of the cyclin-CDK complexes, and with some of the proteins directly involved in replication machinery and in cytokinesis. Our preliminary studies of numerous CTCF-interacting functional partners identified by an affinity chromatography of total cell extracts on matrix-immobilized pure recombinant CTCF, by GST-CTCF-mediated protein """"""""pull-down"""""""" approach with nuclear extracts, and by the yeast two-hybrid method, have provided evidence that CTCF may indeed be a subject of such interactions. Thus, CTCF functions may go beyond of being just a transcription factor. However, it is clear that better understanding of cell growth regulation by CTCF will require an in depth evaluation of a complete repertoire of CTCF target genes and CTCF-interacting partners. To this end, the program """"""""Mechanisms of Transcriptional Regulation by CTCF"""""""" takes advantage of cancer-prone mouse CTCF knock-out models that we developed earlier, and of Drosophila genetics based on identification and cloning of the CTCF homologue in flies. Identification and functional characterization of critically important CTCF target genes and protein partners that are involved in regulation of CTCF function in somatic cells and in germline lead the Section well beyond studies of CTCF per se. By September 2001, some of these proprietary genes have already been characterized and are now being patented by NIAID NIH, and evaluated for diagnostic and therapeutic purposes. Several other genes encoding CTCF-interacting proteins are at the initial stages of deciphering their role(s).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000860-02
Application #
6521454
Study Section
(LIP)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2001
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Bougel, Stéphanie; Renaud, Stéphanie; Braunschweig, Richard et al. (2010) PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells. J Pathol 220:87-96
Renaud, Stephanie; Pugacheva, Elena M; Delgado, M Dolores et al. (2007) Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors. Nucleic Acids Res 35:7372-88
Kouprina, Natalay; Noskov, Vladimir N; Pavlicek, Adam et al. (2007) Evolutionary diversification of SPANX-N sperm protein gene structure and expression. PLoS ONE 2:e359
Renaud, S; Loukinov, D; Abdullaev, Z et al. (2007) Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene. Nucleic Acids Res 35:1245-56
Jaikumar, Sivakumar; Zhuang, Zhengping; Mannan, Poonam et al. (2007) Interspecies comparative genomic hybridization (I-CGH): a new twist to study animal tumor models. Cell Cycle 6:836-42
Fitzpatrick, Galina V; Pugacheva, Elena M; Shin, Jong-Yeon et al. (2007) Allele-specific binding of CTCF to the multipartite imprinting control region KvDMR1. Mol Cell Biol 27:2636-47
Kim, Tae Hoon; Abdullaev, Ziedulla K; Smith, Andrew D et al. (2007) Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128:1231-45
Chernukhin, Igor; Shamsuddin, Shaharum; Kang, Sung Yun et al. (2007) CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide. Mol Cell Biol 27:1631-48
Kurukuti, Sreenivasulu; Tiwari, Vijay Kumar; Tavoosidana, Gholamreza et al. (2006) CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci U S A 103:10684-9
Pugacheva, Elena M; Kwon, Yoo-Wook; Hukriede, Neil A et al. (2006) Cloning and characterization of zebrafish CTCF: Developmental expression patterns, regulation of the promoter region, and evolutionary aspects of gene organization. Gene 375:26-36

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