Dose dependent impact of epimutagenesis measured in iPSCs exposed to 1, 50, and 100 μM BPS.

a) Ideogram plots displaying chromosomal distribution of genome-wide changes in DNA methylation caused by BPS exposure. b) Mean difference (MD) plots of changes in gene expression following exposure to increasing doses of BPS. Exposure to increasing doses of BPS induced increasing, although plateauing numbers of DMCs, DMRs, and DEGs. Blue horizontal lines = hypomethylated DMCs, red horizontal lines = hypermethylated DMCs, black squares = DMRs.

Chromosomal distributions and annotations of BPS-induced epimutations in pluripotent, somatic, and germ cell types.

a) Ideograms illustrating chromosomal locations of DMCs induced by exposure of each cell type to 1 μM BPS. Blue horizontal lines = hypomethylated DMCs, red horizontal lines = hypermethylated DMCs. b) Enrichment plots indicating feature annotations in genomic regions displaying prevalent BPS-induced epimutations in each cell type. Dot size = number of overlapping DMCs with specific annotation, dot color = enrichment score reflecting the relative degree to which epimutations occurring in a specific annotated class are overrepresented.

Treatment-specific differentially methylated sites (DMCs) (treated vs. control).

Correlation between cell-type specific expression of estrogen receptors and density of genomic EREs associated with BPS-induced epimutations.

a) Assessment of expression of ERα and ERβ by cell types co-stained for known cell-type specific markers. Somatic cell types express both receptors, pluripotent cells express ERβ but not ERα, and germ cells do not express either estrogen receptor. b) Motif plots displaying the full ERE consensus sequence and the more biologically relevant ERE half-site motifs found to be enriched from ERα ChIP-seq. c,d) Normalized density plots and box plots displaying the frequency of ERE half-sites identified c) within 500 bp of all BPS-induced DMCs genome-wide, or d) within 500 bp of the most enriched categories of BPS-induced DMCs in each cell type (= enhancer regions for somatic and pluripotent cell types and promoter regions in the germ cell type).

Direct comparison of BPS exposure-specific and cell-type specific features between cell types.

a) Assessment Venn diagrams indicating DMCs that are due either to BPS exposure (top, smaller ovals) or inherent cell-type specific differences (bottom, larger ovals). Numbers of apparent endocrine-signaling related DMCs are shown in the light orange arrow, and apparent endocrine-signaling independent DMCs are shown in the dark orange arrows. Enrichment plots indicating feature annotations in genomic regions displaying b) apparent endocrine-signaling related DMCs occurring predominantly in enhancer regions in somatic Sertoli and granulosa cell types or pluripotent cells expressing one or more estrogen receptors, or c) a smaller set of apparent endocrine-signaling independent DMCs occurring predominantly in promoter regions in all four cell types regardless of +/- expression of relevant endocrine receptors. d) Normalized density plots and box plots displaying the frequency of ERE half-sites identified within 500 bp of apparent endocrine-signaling related DMCs occurring predominantly in enhancer regions and apparent endocrine-signaling independent DMCs occurring predominantly in promoters.

Exposure-specific differentially expressed genes*

Correlation between the proximity of DMCs to promoters and dysregulation of gene expression.

a) Proximity plot displaying distances from exposure-specific DMCs to nearest promoter regions. Dotted lines indicate median points of the data for each cell type. b) Correlation plot displaying a negative relationship between the distance from DMCs to nearest promoters and resulting dysregulation of gene expression within each cell type.

Potential involvement of non-canonical estrogen signaling pathways in BPS-induction of epimutations.

Relative expression of genes a) enriched for apparent endocrine-signaling independent promoter-region DMCs found in all cell types or b) dysregulated in PGCLCs which lack expression of estrogen receptors. c) Heatmap of relative expression of estrogen receptor genes (Esr1 and Esr2) and G-coupled protein receptors (Gprc5a, Gpr107, Gprc5b, Gpr161, and Gpr89) in pluripotent, somatic, and germ cell types. Gprc5a, Gpr107, Gprc5b, Gpr161, and Gpr89 all have been shown to bind to BPA or 17β-estradiol in rat models and represent potential G-coupled protein receptors which could lead to the induction of endocrine-signaling independent DMCs.

Persistence of BPS-induced epimutations through recapitulation of early germline reprogramming in vitro.

a) Schematic illustrating derivation of PGCLCs from iPSCs in vitro. iPSCs are first induced to form EpiLCs which are then induced to form PGCLCs. iPSCs were exposed to either ethanol + 1 μM BPS or ethanol (carrier) only, then induced to undergo transitions to form EpiLCs and then PGCLCs. b) DNA samples from BPS-exposed or control iPSCs as well as subsequently derived PGCLCs were assessed for exposure-specific DNA methylation epimutations by EM-seq. BPS-treated iPSCs showed 38,105 DMCs and subsequently derived PGCLCs showed 28,169 DMCs. Of those, only 1,417 (3.7%) of the DMCs were conserved from the BPS-exposed iPSCs to the subsequently derived PGCLCs. c) RNA samples from BPS-exposed or control iPSCs and subsequently derived PGCLCs were assessed for global gene expression patterns by RNA-seq. BPS-treated iPSCs showed 1,637 exposure-specific DEGs and subsequently derived PGCLCs showed 1,437 exposure-specific DEGs. Of those, only 138 (8.4%) were conserved from the BPS-exposed iPSCs to the subsequently derived PGCLCs.