Transcriptional Regulation in hMLH1-Silenced Colon Cells

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DESCRIPTION (provided by applicant): The studies in this proposal will dissect conditions leading to escape from aberrant methylation in a cohort of genes localized on chromosome 3 in a cell line representative of a major subgroup of colon cancers following treatment of cells with 2-deoxy-5- azacytidine [AzadC]. Detailed microarray analyses in our laboratory of a human colon tumor cell line in which the mismatch repair gene, hMLH1 is aberrantly silenced for expression have defined a set of genes on chromosome 3 that are co-induced for expression with the hMLH1 gene. Proposed experiments will utilize rolling circle probes to localize these genes in interphase nuclei. Proposed studies will determine if there is a structural basis for co-expression of these genes that are widely separated from each other on chromosome 3 and will probe how their regulation differs from other AzadC responsive genes. Further studies will dissect the fine structure of the promoter regions of each of these genes for their CpG methylation patterns in gone silenced cells and after transient AzadC-induced release from this repression, and exploit a group of constitutively expressing subclones isolated after AzadC exposure. These studies will test the hypothesis that these genes are representative of a co-regulated gene cohort resulting from the aberrant methylation process. They will further contrast conditions leading to abrogation of regulatory mechanisms in this subset of genes with those required for AzadC-induced modulation of other gene sites. The mechanisms leading to gene silencing and escape from these gene repression processes will be further examined by analyzing the intersection of genes altered for expression in cells treated with AzadC versus antisense RNA to the three major human DNA methyltransferase enzymes, DNMT1, 3a and 3b. Mechanistic differentiation of methylation-related gene expression regulation will also be further dissected employing antisense RNAs to specific histone deacetylases. The cell lines that are a focus of the proposed studies are representative of 15-20% of human colon cancers that exhibit microsatellite instability [MSI], which is almost universally synonymous with defects in DNA MMR. 2/3 of these MSI cancers are classified as cancers of sporadic origin because their occurrence does not correlate with a known familial defect.