title: Establishment of a high-throughput detection system for DNA demethylating agents.
abstract: 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 EGFP reporter genes under the UCHL1 promoter, which is methylation-silenced in human colon cancers and can be readily demethylated to drive strong expression. Methylated UCHL1 promoter was introduced into HCT116 colon cancer cells, and transfectants with methylated exogenous UCHL1 promoter were obtained. By screening subclones from each of the epigenetically heterogeneous transfectant clones, we finally obtained three optimal subclones that expressed luciferase and EGFP after 5-aza-dC treatment with high signal-to-noise ratios. Nucleosomes with H3K9me2 were present around the exogenous UCHL1 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.
authors.affiliation: a Division of Epigenomics , National Cancer Center Research Institute , 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 , Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Okochi-Takada
authors.affiliation: a Division of Epigenomics , National Cancer Center Research Institute , 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 , Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Hattori
authors.affiliation: b Chemical Genetics Laboratory , RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 , Japan.|c Chemical Genomics Research Group , RIKEN Center for Sustainable Resource Science , 2-1 Hirosawa, Wako, Saitama 351-0198 , Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ito
authors.affiliation: a Division of Epigenomics , National Cancer Center Research Institute , 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 , Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Niwa
authors.affiliation: a Division of Epigenomics , National Cancer Center Research Institute , 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 , Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Wakabayashi
authors.affiliation: a Division of Epigenomics , National Cancer Center Research Institute , 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 , Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Kimura
authors.affiliation: b Chemical Genetics Laboratory , RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 , Japan.|c Chemical Genomics Research Group , RIKEN Center for Sustainable Resource Science , 2-1 Hirosawa, Wako, Saitama 351-0198 , Japan.|d Seed Compounds Exploratory Unit for Drug Discovery Platform , RIKEN Center for Sustainable Resource Science , 2-1 Hirosawa, Wako, Saitama 351-0198 , Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: a Division of Epigenomics , National Cancer Center Research Institute , 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 , Japan.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Ushijima
mesh_terms: D018899:CpG Islands; D000073399:DNA Demethylation / Q000187:drug effects*; D000077209:Decitabine / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D054852:Small Molecule Libraries / Q000494:pharmacology*; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism
chemical_list: D007364:Intercalating Agents; D054852:Small Molecule Libraries; C482860:UCHL1 protein, human; D049452:Green Fluorescent Proteins; D000077209:Decitabine; D043222:Ubiquitin Thiolesterase
keywords: DNA demethylating agent; Epigenetics, DNA methylation; high-throughput screening; luciferase; mammalian cells