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

Abstract

BackgroundExposure 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.ResultsFollowing direct exposure in zebrafish embryos from 0 to 6 days post-fertilization, 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0126-4) contains supplementary material, which is available to authorized users.

Environmental epigenetics in zebrafish

“…In a coeval study performed by a distinct group, the authors assessed the transgenerational effects of two distinct compounds, the well-known Dnmt1 inhibitor 5-azacytidine [ 106 , 107 ] and the plasticizer derivative mono(2-ethylhexyl)phthalate (MEHP), which is ubiquitously present in the environment [ 108 ]. It is worth mentioning that in this study only fertilized eggs of the F0 generation were exposed once in a lifetime, until 6 days post-fertilization, to 5-azacytidine or MEHP at concentrations unable to elicit detectable adverse effects on development [ 109 ]. Despite this, both compounds altered dnmt gene expression and DNA methylation level to a different extent in the directly exposed individuals.…”

Treatment with a DNA methyltransferase inhibitor feminizes zebrafish and induces long-term expression changes in the gonads

“…As it occurs in mammalian cells [ 17 , 29 ], treatment of zebrafish with 5-aza-CR results in global hypomethylation in embryonic cells [ 30 ] as well as in adult hepatocytes [ 31 ]. In female zebrafish fed by 5-aza-dC during 32 days a decrease in global DNA methylation was observed [ 32 ], likewise, in larvae treated with 10 or 25 µM during 0–6 dpf [ 33 ]. However, transgenerational effects up to the F2 generation were only observed in the latter study [ 33 ].…”

Functions and mechanisms of epigenetic inheritance in animals

“…Other notable differ ences in early embryonic reprogramming also exist between zebrafish and mammals, including the absence of early TET expression and of active demethylation in zebrafish 127,129,130 . The lack of global embryonic demethyl ation and remethylation makes zebrafish a potentially useful model organism for MEI stud ies in vertebrates 131 . However, whether such princi ples of embryonic reprogramming are common to teleosts and other anamniotes is currently unclear.…”