WHOLE-GENOME BISULFITE SEQUENCING REVEALS GENOMIC REGIONS VULNERABLE TO EPIGENETIC PERTURBATION
Martos, Suzanne Nicole
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Environmental exposures that alter epigenetic marks can have phenotypic consequences for multiple generations. Prenatal nutrition and environmental stressors have been shown to frequently alter DNA methylation at retrotransposon-associated metastable epialleles and imprinted germline differentially methylated regions (gDMRs). Such frequent alterations suggest that these loci may be especially susceptible to epigenetic perturbation. Previously, we used whole-genome bisulfite sequencing (WGBS) of wildtype (WT) mouse embryonic stem cells (mESCs) and three mutant mESCs with altered DNA methyltransferase (DNMT) enzymes: loss of “maintenance” DNMT1 (1KO; Dnmt1-/-), loss of “de novo” DNMT3a/3b (DKO; Dnmt3a-/-/Dnmt3b-/-), and loss of “maintenance” and “de novo” DNMTs (TKO; Dnmt1-/-/Dnmt3a-/-/Dnmt3b-/-) to demonstrate a division of labor between DNMT1 and DNMT3a/3b to suppress distinct types of retrotransposons (i.e., DNMT1-maintained or DNMT3a/3b-maintained regions). Here we show that DNA methylation maintenance at imprinted gDMRs requires DNMT1 and DNMT3a/3b. Characterization of rescued-1KO mESCs (r1KO; Dnmt1-/- with exogenous Dnmt1 cDNA) revealed that loss of methylation at known imprinted loci in 1KO cells was not rescued by exogenous DNMT1. We developed a computational method, Non-Rescued DMRs (NORED), to identify 2500 NORED regions (FDR = 0.02) genome-wide. We implemented a second computational approach, MethylMosaic, to identify 2500 genomic regions (FDR = 0.2) with bimodal read-level methylation, a characteristic that arises at gDMRs from one parental allele being fully methylated and the other being unmethylated. Overlap of the two methods, identified 207 regions, named as bimodal NORED, including 19 of 21 known gDMRs. To determine whether DNMT1-maintained or bimodal NORED regions are more susceptible to environmental perturbation than other genomic features, we compared fetal liver methylomes from male C57BL/6J mice that exposed in utero to Bisphenol A (BPA) through maternal supplemented diet (0 μg/kg bw/d, 10 μg/kg bw/d, or 10 mg/kg bw/d). We then identified DNA methylation-associated pathway perturbation to discover potential molecular mediators of metabolic abnormalities observed in male mice. In summary, distinct DNA methylation maintenance paradigms and epigenetic characteristics found at germline imprints and retrotransposons influenced susceptibility to altered methylation from prenatal exposure to BPA and DNA methylation-mediated pathway perturbation provided insight into adult phenotypes.