Methylation Mediated Repression of Selected Genes in Prostate Cancer

摘要

The present project is focused on understanding the exact mechanism by which methylation silences gene expression in prostate cancer cell lines. Certain proteins bind preferentially to methylated DNA and these proteins have been shown to repress gene expression. In order to determine which of these protein or proteins interact with methylated genes inside the cells, we plan to use using chromatin immunoprecipitation assay. Two important requirements for this assay include an optimal sonication of the fixed chromatin and quantitative PCR assay for the gene under study. We have determined the linear PCR conditions for the proposed genes as well as the optimal fixation and sonication conditions for the prostate cancer cell lines. Using chromatin immunoprecipitation assay, we have shown that as compared to the AR promoter, the GSTPl promoter is enriched in deacetylated histones H3 and H4 in LNCaP prostate cancer cells. We have also found that MeCP2 but not MBDl or MBD2 interacts with GSTPl and CD44 promoters in LNCaP cells. These results are consistent with the recruitment of histone deacetylase containing complexes by methylated DNA, resulting in a localized deacetylation.