Methylation Mediated Repression of Selected Genes in Prostate Cancer
Annual rept. 1 Apr 2002-31 Mar 2003
DEPARTMENT OF VETERANS AFFAIRS WASHINGTON DC
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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 most of the sonicated fragments should be around 500bp 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. We have performed chromatin immunoprecipitation assay for the GSTP1 and AR promoters in LNCaP prostate cancer cell lines. As compared to the AR promoter, the GSTP1 promoter was enriched in deacetylated histones H3 and H4. These results are consistent with the recruitment of histone deacetylase containing complexes by methylated DNA, resulting in a localized deacetylation.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research