Finding genetic loci that are sensitive to demethylation to assist in the development of DNA methylation-based in vitro assay for the identification of carcinogens

Purpose: To use reduced representation bisulfite sequencing and RNA-seq to identify demethylated promoters of genes that were derepressed following treatment with demethylating agent, 5-Aza-2'-deoxycytidine (5aCdR, CAS# 2353-33-5). Primary Syrian hamster fetal cells were exposed to benzo[a]pyrene for 7 days and developed morphologically transformed (MT) colonies, from which one colony was picked and reseeded and maintained in culture until it bypassed senescence. For this experiment, these immortalized cells were treated with vehicle alone or with one of two concentrations of 5aCdR (4 technical replicates for each group). Genome wide DNA methylation and mRNA expression were measured using reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNA-seq) respectively. Genes differentially induced by 5aCdR treatment whose promoters concurrently exhibited differential demethylation were considered epigenetically labile loci for further study. All cells were confirmed male using a rapid XY qPCR assay.