From 10-01-05 to 9-01-06, we continued our efforts on a genome wide characterization of various functions mediated in chromatin of normal and cancer cells by the 50-bp-long DNA sequences capable of specific binding to the highly conserved 11 Zinc Finger (11ZF) DNA-binding domain (DBD) shared in two unique nuclear factors, CTCF and BORIS. CTCF is highly conserved, multifunctional, multivalent, nuclear factor with the properties of a tumor suppressor at 16q22. It is ubiquitously expressed in all somatic cells, and involved in promoter activation and repression, hormone-inducible gene silencing, and creation of constitutive or methylation-sensitive chromatin boundaries. An exciting breakthrough in understanding the epigenetic mechanisms was our discovery of a paralogue of CTCF termed BORIS (for Brother Of Regulator of Imprinted Sites). Normally, only CTCF but not BORIS is expressed in all somatic cells. During male germ cell differentiation, the pair displays a mutually exclusive expression pattern that correlates with the patterns for: (1) genome-wide re-establishment of DNA-methylation marks, and (2) expression of certain testis-specific chromatin-remodeling factors and DNMTs. BORIS is normally expressed only during male germ cell differentiation in a pattern mutually exclusive to that of CTCF, which is tightly coupled with epigenetic reprogramming. We showed earlier that CTCF and BORIS share identical exons encoding the 11 ZF DBD to interact with the same spectrum of CTCF/BORIS-binding sites, but diverge at the amino- and carboxy-termini. Both genes were originally identified and molecularly cloned by V. Lobanenkov and collaborators. Thus, somatic CTCF and testis-specific BORIS could serve as counteracting genes by manifesting and reprogramming paternal epigenetic states, respectively. The sites for maternal epigenetic marking require a third factor with the same DBD but expressed in embryonic ovaries. Indeed, we discovered a female germ cell-specific counterpart of BORIS, that we have named """"""""Natasha"""""""" to highlight the differences from the male germ cell-specific BORIS and from somatic CTCF. This year, studies of partial cDNA clones of Natasha revealed the same 11ZF DBD that was earlier shown to be common to BORIS and CTCF. Identification of Natasha has completed our search of all three factors required for re-establishing and reading parentally-determined epigenetic marks at chromatin regions with DNA sequences recognized by the the same 11ZF DBD shared by these three unique factors. We called such sequences CTCF/BORIS/NATASHASHA-target sites, or """"""""CBN-sites"""""""". Our working model of how these factors operate suggests; 1) that regulation of access to CBN-sites in vivo is highly cell type dependent; 2) that different functional outcomes are to be expected from the in vivo occupancy of the same genomic sites by each of these factors; and 3) that unscheduled co-expression of these genes would lead to competition for common binding sites leading to genome-wide changes in epigenetic status that have been found to characterize immortalized and transformed cells. Indeed, studies from our laboratory and others have shown that BORIS is aberrantly expressed in a high proportion of cancers of many different types that uniformly express CTCF as well. In accord with this hypothesis, we have shown that BORIS and CTCF do compete with one another at shared target sites. Occupancy by BORIS of sites normally occupied by CTCF can result in biallelic expression of genes normally expressed from only one allele, such as imprinted genes in association with altered methylation of DNA flanking the site. The importance of understanding the fine details of CTCF/BORIS/NATASHA interactions with DNA became strikingly apparent in studies of genetically determined abnormalities in humans. The first revolved around families with Beckwith-Wiedemann syndrome (BWS). BWS is a disorder characterized by fetal and perinatal growth abnormalities and increased risk of childhood solid tumors. It was found two families in which BWS was associated with a small deletion that eliminated two CTCF target sites in a critical imprinting control region (ICR) and other families in which these sites were methylated and thus unable to bind CTCF. The ability of CTCF to bind to these target thus appears to be a major determinant of normal gene expression at this locus. The second relates to aberrant regulation of X chromosome inactivation. In normal women, the chance that either one of the two X chromosomes ? one from the father and one from the mother - will become physiologically inactivated is essentially random The binding of CTCF to a critical region on just one chromosome is known to mark that chromosome for inactivation. Two abnormal variants of this process were known to occur: 1) cases in which essentially only the X from one parent was silenced; and 2) cases in which the chances that either X would be inactivated were markedly diminished. We found that the first scenario was cased by a point mutation that markedly increased the binding affinity of CTCF for the site, making it essentially irreversible. The second was caused by a different base mutation at the same exact site but in this instance essentially abolished CTCF binding. Finally, we are working to understand the ability of CTCF and BORIS to discriminate among functionally distinct targets in vivo. Current studies suggest that the dissimilar flanks to the central 11ZF DBD are responsible for differential partnering with other proteins including factors involved in chromatin modification.
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