The proto-oncogene c-myc is implicated in the regulation of such fundamental cellular processes as growth, differentiation and programmed death. De-regulation of c-myc expression is associated with neoplasia in humans and experimental animals. Relatively little is known about genes which regulate c-myc expression. Considering the apparent role of myc in molecular biology of cancer, it is reasonable to assume that critical myc regulators could in turn be proteins of major biological importance like oncogenes and tumor suppressor genes. The general goal of this proposal is to obtain an understanding of the molecular biology of a novel candidate tumor suppressor gene, Dr. Lobanenkov have discovered, called CTCF, which encodes an exceptionally highly conserved 11-Zn-finger transcriptional repressor specifically binding to several variant DNA sequences in the promoter-proximal regions of the c-myc proto-oncogene of different vertebrate species by utilizing different combinations of individual fingers. Several additional lines of evidence suggest that CTCF appears to be an important regulatory gene: (1) It is exceptionally highly conserved transcriptional repressor, (2) Its binding to diverged promoter sequences of vertebrate c-myc genes represses their transcription; (3) CTCF locus is rearranged in some F-MuLV-induced mouse erythroleukemia cell lines; (4) CTCF gene is localized at the locus of chromosome abnormalities frequently found in several human cancers. It is rearranged in some breast cancer cell lines, primary breast tumors and in certain Wilms' tumors. To investigate the mechanism of CTCF activity and to gain insight into CTCF's role in neoplastic change and normal development this project will: (1) Characterize structure-function of the CTCF polypeptide and determine whether specific binding of CTCF to the human c-myc promoter is involved in regulation of polymerase II initiation and/or processivity; (2) Characterize alterations of CTCF gene in certain breast cancer tumors and cell lines and in some Wilms' tumors which have already found to have an aberrant structure of the CTCF locus. Expand mutational analysis of CTCF to a larger group of breast tumors to characterize in detail an association of CTCF mutations and breast tumorigenesis. Determine whether CTCF has aberrant structure and/or expression in a number of other cancers associated with abnormalities of chromosome 16q22, the locus of human CTCF; (3) Define structure of the altered CTCF gene locus and sequence of abnormal CTCF-containing mRNA(s) which Dr. Lobanenkov have found in some Friend leukemia virus (F-MuLV)-induced cell lines; (4) Characterize the consequence of deletion of CTCF in transgenic mice and the pattern of CTCF expresion in normal development; (5) Determine additional CTCF-binding DNA sequences and identify other CTCF-target genes besides c-myc.
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