From 10-01-04 to 9-01-05, 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 mutually exclusive expression pattern that correlates with the patterns for (1) genome-wide re-establishing of DNA-methylation marks and (2) certain testis-specific chromatin-remodeling factors, and DNMTs. BORIS is normally expressed only during MALE-germ-cell differentiation, in a mutually exclusive with CTCF pattern that 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 have originally been identified and molecularly cloned by V. Lobanenkov with collaborators and co-workers, who first submitted to GenBank the full-length cDNA sequences of CTCF in April 1993 (Acc. No. Z22605) and of BORIS in January 2001 (Acc. No. AF336042). Any later submissions of BORIS cDNA and/or protein sequences under """"""""CTCF-L"""""""" name and other accession numbers (for an EST, or a hypothetical """"""""CTCF-like"""""""" protein) are incorrect and misleading, because there are ABSOLUTELY NO SIMILARITIES between CTCF and BORIS amino acid sequences in the 11ZF-flanking N-terminal and C-terminal parts that account for the two-thirds of each polypeptide of similar total length, thereby making BORIS to be a factor, which UNLIKE CTCF can direct a totally distinct from CTCF function to a shared set of target DNA-sequences recognized by the 11ZF-domain. By 9-01-04, two reviews on CTCF and BORIS genes, written by V. Lobanenkov and co-workers, are available. Thus, somatic CTCF and testis-specific BORIS could serve as counteracting genes by manifesting and reprogramming PATERNAL epigenetic states, respectively, by DNA-sequences binding to the 11 ZF DBD shared by the two already-published genes CTCF and BORIS. The sites for MATERNAL epigenetic marking require a third factor with the same DBD to be expressed in embryonic ovaries. Indeed, we discovered a FEMALE-germ-cell-specific counterpart of BORIS, named """"""""Natasha"""""""" to highlight the difference both with the MALE-germ-cell-specific BORIS and with somatic CTCF. This year, we have proven the stunning presence in the ORF of the partial Natasha cDNA clones of the same 11ZF DBD that was earlier described in the unique CTCF & BORIS PAIR. Identification of Natasha has completed our search of all three factors required for the re-establishing of parental epigenetic marks at chromatin regions with DNA-sequences recognized by the same 11ZF DBD shared by these three unique factors. We called such sequences CTCF/BORIS/NATASHASHA-target sites, or """"""""CBN-sites"""""""". Our working hypothesis suggests that regulation of the in vivo access to CBN-sites, and functionally different outcomes from the in vivo occupancy of the same genomic sites by competing against each other distinct factors equipped with the same 11ZF-DBD, may serve to target, to maintain, or to alter local status of epigenetic modifications genome wide in immortalized/transformed cells. Indeed, unlike normal somatic cells with CTCF, the vast majority of cancer cells are found to abnormally co-express CTCF and BORIS together. In accord with this hypothesis, several studies of the IGF2/H19 LOI by altering mono-allelic CTCF-binding in tumors with aberrant bi-allelic hypo- and/or hyper- methylation of at least one of seven CTCF-sites in the H19 ICR, suggested that CTCF-binding sequences, or CBN sites, are taking the central stage in human cancer epigenetics. In some human families, Mother Nature herself performed some of the most revealing experiments on the role of CBN-sites, for example, by showing that a narrow deletion of only two CTCF-binding ICR sites in two different families (or abnormal bi-allelic methylation of the same sites in several other families) results in the Beckwith-Wiedemann syndrome (BWS) including prenatal death on maternal transmission with the signs of BWS. Various CBN 11ZF-DBD-targets can incorporate additional binding sites for other (lineage-specific) transcriptional factors, because formation of various 11ZF/DNA-complexes generates 50-60-bp-long footprints with different contexts of ZF-contacting nucleotides on both DNA strands of a CBN site, which explains how CTCF and BORIS are capable of discriminating functionally distinct targets, form so many distinct geometry complexes with DNA, and result in a SELECTIVE interactions of the complexes one with another. The ability of CTCF and BORIS to discriminate in vivo functionally distinct targets suggested that the functional outcomes from changing a particular mode of the in vivo occupancy at the 11ZF-DBD-binding sites can differ and be cell-type-specific. Indeed, for example, an ectopic tet- expression of BORIS (but not the 11ZF-DBD alone) in primary normal human fibroblasts results in the selective demethylation and re-activation of the CBN target promoters normally regulated by the CTCF-BORIS-switching in germ cells. Such targets include promoters of so called """"""""cancer-testis"""""""" genes, such as MAGEs, LAGEs, NY-ISO-1 and other downstream BORIS-targets like the Oct-4 promoter, for which the change of the in vivo occupancy at CTCF/BORIS-sites, initially mapped by gel-shifts, was directly shown by the ChIP approach. On the other hand, an ongoing mapping and verifying each novel site for methylation-sensitive binding in vivo, strongly supports an idea that misdirected epigenetic regulation in tumors is mediated through a misuse of varying sequences recognized by the CBN 1ZF DBD, because no exceptions have been yet found for the presence of such sequences in promoters reported in the literature for aberrant hypermethylation, with near 100 of such regions tested, and CBN-sites arrayed for ChIP-to-chip assays. Finally, we showed that Natasha-BORIS-genes are located within the region on 20q13 commonly involved in copy-gain in breast, ovarian, brain and many other cancers, and in fact are abnormally activated in those tumors, thereby making these factors an exceptionally attractive new anti-cancer target and a super-sensitive early diagnostic tool.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000860-06
Application #
7196660
Study Section
(LIP)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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