Establishment of a high-throughput detection system for DNA demethylating agents

Epigenetic alterations underlie various human disorders, including cancer, and this has resulted in the development of drugs targeting epigenetic alterations. Although DNA demethylating agents are one of the major epigenetic drugs, only two compounds—5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2'-deoxycytidine (5-aza-dC, decitabine)—have obtained clinical approval. Here, we aimed to establish a detection system for DNA demethylating agents suitable for a high-throughput screening (HTS) in mammalian cells. We inserted luciferase and <i>EGFP</i> reporter genes under the <i>UCHL1</i> promoter, which is methylation-silenced in human colon cancers and can be readily demethylated to drive strong expression. Methylated <i>UCHL1</i> promoter was introduced into HCT116 colon cancer cells, and transfectants with methylated exogenous <i>UCHL1</i> promoter were obtained. By screening subclones from each of the epigenetically heterogeneous transfectant clones, we finally obtained three optimal subclones that expressed luciferase and <i>EGFP</i> after 5-aza-dC treatment with high signal-to-noise ratios. Nucleosomes with H3K9me2 were present around the exogenous <i>UCHL1</i> promoter in all three subclones. Using one of the subclones (HML58-3), HTS was conducted using 19,840 small molecules. Two hit compounds were obtained, and these turned out to be 5-aza-dC and 5-aza-CR. The assay system constructed here demonstrates a robust response to DNA demethylating agents, along with high specificity, and will be useful for screening and biological assays in epigenetics.