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. 2014 Jan 17;15:30.
doi: 10.1186/1471-2164-15-30.

Perinatal Bisphenol A Exposure Promotes Dose-Dependent Alterations of the Mouse Methylome

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Free PMC article

Perinatal Bisphenol A Exposure Promotes Dose-Dependent Alterations of the Mouse Methylome

Jung H Kim et al. BMC Genomics. .
Free PMC article

Abstract

Background: Environmental factors during perinatal development may influence developmental plasticity and disease susceptibility via alterations to the epigenome. Developmental exposure to the endocrine active compound, bisphenol A (BPA), has previously been associated with altered methylation at candidate gene loci. Here, we undertake the first genome-wide characterization of DNA methylation profiles in the liver of murine offspring exposed perinatally to multiple doses of BPA through the maternal diet.

Results: Using a tiered focusing approach, our strategy proceeds from unbiased broad DNA methylation analysis using methylation-based next generation sequencing technology to in-depth quantitative site-specific CpG methylation determination using the Sequenom EpiTYPER MassARRAY platform to profile liver DNA methylation patterns in offspring maternally exposed to BPA during gestation and lactation to doses ranging from 0 BPA/kg (Ctr), 50 μg BPA/kg (UG), or 50 mg BPA/kg (MG) diet (N = 4 per group). Genome-wide analyses indicate non-monotonic effects of DNA methylation patterns following perinatal exposure to BPA, corroborating previous studies using multiple doses of BPA with non-monotonic outcomes. We observed enrichment of regions of altered methylation (RAMs) within CpG island (CGI) shores, but little evidence of RAM enrichment in CGIs. An analysis of promoter regions identified several hundred novel BPA-associated methylation events, and methylation alterations in the Myh7b and Slc22a12 gene promoters were validated. Using the Comparative Toxicogenomics Database, a number of candidate genes that have previously been associated with BPA-related gene expression changes were identified, and gene set enrichment testing identified epigenetically dysregulated pathways involved in metabolism and stimulus response.

Conclusions: In this study, non-monotonic dose dependent alterations in DNA methylation among BPA-exposed mouse liver samples and their relevant pathways were identified and validated. The comprehensive methylome map presented here provides candidate loci underlying the role of early BPA exposure and later in life health and disease status.

Figures

Figure 1
Figure 1
Experimental and bioinformatics pipeline. Next generation sequencing was performed after methylation-sensitive enzyme-based enrichment using the Illumina Genome Analyzer IIx (GAIIx) platform with single-end read length of 80 bp on 12 genomic liver DNA samples obtained from isogenic day 22 a/a mice, exposed to BPA through maternal dietary intake (control, 50 ug/kg diet, or 50 mg/kg diet; n = 4 per group). To regions of altered methylation (RAMs) associated with BPA exposure group, the number of aligned sequencing reads per 100 bp window with 50 bp moving shifts was obtained for each sample. edgeR analysis was performed after applying filters to remove the regions with low reads. Top candidates were identified, and candidate RAMs were validated using EpiTYPER.
Figure 2
Figure 2
Characterization of genome-wide methylation in BPA-exposed offspring. The genomic distributions of regions of altered methylation (RAMs), delineated as genome-wide or within promoter regions, from the edgeR analysis with p-value & 0.05 are displayed. (A) The bar graph identifies the number of RAMs in genome-wide scale. (B) The Venn diagram displays the overlap of RAMs among control (Ctr), 50 μg (UG) and 50 mg (MG) diet groups. (C) The bar graph and (D) Venn diagram of RAMs within promoter regions is displayed. (E) For each exposure comparison and for the full M-NGS library, pie charts display the proportion of RAMs in number of windows at CpG islands (CGIs), CpG shores, CpG shelves, and the remainder of the genome. In the Ctr vs. MG and in the UG vs. MG comparisons, approximately half of the RAMs occur in CpG shores (0-2 kb from CGIs). (F) The genomic distributions of RAMs (p-value & 0.05) at 5′UTRs, TSSs, CDSs, and 3′UTRs are delineated in bar graphs. The percentage of RAMs at CDS exons in the Ctr vs. UG comparison is decreased by more than half compared to other group comparisons. At the TSSs, the percentage of RAMs is comparable among the three group comparisons.
Figure 3
Figure 3
Regions of Altered Methylation (RAMs) in gene promoters. RAMs within ±1.5 kb flanking transcription start sites (TSSs) are represented. Each column separated by gray lines represents a single sample (4 per group), and each row represents a unique gene promoter region, ±1.5 kb from the TSSs (magenta dotted line). The known BPA-interacting genes queried from the Comparative Toxicogenomic Database are also indicated on the right. Two arrows pointing to the Myh7b and Slc22a12 gene promoters (in blue text) show candidate RAMs validated in a larger subset of animals.
Figure 4
Figure 4
Enriched biological processes among genes harboring differential methylation. The genes harboring differential methylation within their promoters (FDR adjusted p-value & 0.05) were subjected to GO term enrichment analysis. (A) A total of 198 genes from Ctr vs. MG comparison, (B) 76 genes from Ctr vs. UG comparison, and (C) 371 genes from UG vs. MG comparison are used for the analysis, and enriched pathways are involved in basic biological processes, including basic cellular, metabolic, and immune and stimulus responses, as well as some binding activities. The enriched biological processes are graphed using REViGO software. The colors of the circles represent the various levels of statistical significance, where the darker shade represents more significance in p-values than the lighter one. The various sizes of the circles represent the number of genes in given GO terms. A complete list of GO terms including cellular components and molecular functions is available in Additional file 2: Table S4.
Figure 5
Figure 5
Candidate region validation using Sequenom EpiTYPER. RAMs from candidate regions including (A)Myh7b and (B)Slc22a12 promoters and (C, D) two intergenic regions from 29 mouse liver samples including the 12 original samples sequenced for M-NGS were quantitatively validated via Sequenom EpiTYPER and shown as box plots.

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