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. A 2002 review by Klenova et.al. (see refs) 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. 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 WT1, 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. Moreover, 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. Our unexpected discovery of the gene termed BORIS has turned every known CTCF-target site into target for BORIS because it shares with CTCF the same 11 Zn-finger DNA-binding domain. CTCF and BORIS are expressed in a mutually-exclusive pattern that correlates with re-setting of methylation marks during male germ cell differentiation thus suggesting that BORIS directs epigenetic reprogramming at CTCF target-sites. Human BORIS maps to the cancer-associated region 20q13. We showed that BORIS belongs to the -cancer-testis- gene family because it is aberrantly activated in substantial proportions of different cancers. BORIS and CTCF compete for site-specific binding to DNA. Thus, BORIS and CTCF may normally act successively to govern epigenetic states in male germ cells. The sibling rivalry occasioned by aberrant expression of BORIS in cancer may interfere with normal functions of CTCF including growth suppression, and contribute to epigenetic dysregulation. Our current efforts are directed to understanding of how CTCF and BORIS can be regulated in such a unique coordinated fashion titgtly coupled with DNA re-methylation, and how this regulation is disturbed in cancers with LOH at 16q22 (locus of CTCF) and amplifications at 20q13 (locus of BORIS). We have mapped first critical elements in the promoters of both genes, and apply bisulfite-sequencing methods to elucidate effects of mutually-dependent methyaltion on transcription of CTCF versus BORIS.
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