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Identification of DNA sequence elements that direct CpG methylation of epigenetically‐regulated genes

Aberrant DNA methylation of promoter CpG islands leads to gene silencing in cancer. The mechanisms that account for aberrant methylation in cancer remain unknown. We hypothesize that the instructions that govern DNA methylation‐dependent regulation of gene expression will be contained within the promoter DNA sequence (cis elements) of methylation‐sensitive genes. We have developed a functional assay for identification of cis elements that direct methylation‐dependent gene silencing in breast cancer cell lines using the methylation‐sensitive gene promoter of CST6. CST6 promoter deletion variants were assembled into luciferase reporter gene constructs and transfected into breast cancer cell lines that differentially methylate the promoter. Methylation of the CST6 promoter after transfection into methylator cell lines results in silencing of the reporter gene, whereas CST6 promoter activity persists in non‐methylator cell lines. CST6 promoter truncations that uncouple an instructional sequence from the methylation target (CpG island) result in loss of methylation‐sensitivity (methylation resistance in methylator cell lines). Identification of cis elements that diminish CpG island methylation will advance our understanding of mechanisms governing aberrant DNA methylation in breast cancer.