Differential DNA methylation at conserved non-genic elements and transgenerational inheritance following developmental exposure to mono(2-ethylhexyl) phthalate and 5-azacytidine in zebrafish. Danio rerio

Background Exposure to environmental stressors during development may lead to latent and transgenerational adverse health effects. To understand the role of DNA methylation in these effects, we used zebrafish as a vertebrate model to investigate heritable changes in DNA methylation following chemical induced stress during early development. We exposed zebrafish embryos to non-embryotoxic concentrations of the biologically active phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP, 30 µM), and the DNA methyltransferase 1 inhibitor 5-azacytidine (5AC, 10 µM). Direct, latent and transgenerational effects on DNA methylation were assessed using global, genome wide and locus specific DNA methylation analyses. Results Following direct exposure in zebrafish embryos from 0 to 6 days post fertilization (dpf), genome wide analysis revealed a multitude of differentially methylated regions, strongly enriched at conserved non-genic elements for both compounds. Pathways involved in adipogenesis were enriched with the putative obesogenic compound MEHP. Exposure to 5AC resulted in enrichment of pathways involved in embryonic development, and transgenerational effects on larval body length. Locus specific methylation analysis of 10 differentially methylated sites revealed six of these loci differentially methylated in sperm sampled from adult zebrafish exposed during development to 5AC, and in first and second generation larvae. With MEHP consistent changes were found at 2 specific loci in first and second generation larvae. Conclusions Our results suggest a functional role for DNA methylation on cis-regulatory conserved elements following developmental exposure to compounds. Effects on these regions are potentially transferred to subsequent generations. Overall design: 15 RRBS samples, 5 Controls, 5 MEHP and 5 5AC exposed samples 94 BisPCR2 samples, F0 6 dpf samples (6x control, 6x MEHP and 6x 5AC 10 µM and 6x 5AC 25 µM), F0 15 dpf samples (5x Control, 5x MEHP, 5x 5AC), Control DNA (0, 25, 50, 75 and 100% methylated), Technical duplicate analysis (6 dpf F0 5AC1 and 5AC), F0 sperm (6x Control, 6x MEHP and 6x 5AC), F1 6dpf (5x control, 5x MEHP and 5x 5AC), F2 6dpf (5x control, 5x MEHP and 5x 5AC)