Epigenetic inheritance of aberrant DNA methylation signatures as a consequence of chronic paternal alcohol exposure and the effect on embryonic gene expression in mice

Abstract

Epigenetic mechanisms regulate gene expression, a particularly important activity during foetal development. DNA methylation contained within promoter and regulatory intergenic regions influence gene activity. In utero alcohol exposure as a result of maternal consumption during pregnancy has been associated with disruption of foetal DNA methylation and gene expression, leading to neurological dysfunction, growth retardation and facial anomalies. While similar phenotypes in offspring have been associated with chronic preconception paternal alcohol exposure, the mechanisms underlying these effects remain largely unexplored. This study aimed to: (1) validate significant changes in sperm DNA methylation in a list of ten candidate genes in male mice chronically exposed for ten weeks to ethanol (n=10) compared to a calorie-equivalent sucrose solution (n=10); (2) validate significant changes in gene expression in candidate genes in the brain, liver and placenta of E16.5 embryos sired by ethanol (n=24) compared to sucrose (n=24) treated male mice; (3) quantify DNA methylation changes in candidate genes in the three embryonic tissues. (4) Lastly, previously generated microarray data were reanalysed using bioinformatics tools to generate a top ranked candidate differentially expressed gene list that was used to identify and analyse biological functions or pathways significantly over represented among these genes using PANTHER and DAVID. This study was unable to provide validation for most of the significant differences observed in the sperm DNA methylome in the original study, most likely because of the low sperm DNA concentration. Significant methylation differences were however observed at individual CpG sites in three candidate genes (Igf1r, Odc1, Depdc1b) in specific tissues of embryos sired by ethanol-exposed males relative to embryos sired by sucrose-treated males. There was concordance in the direction of altered gene expression between the cases and controls using the microarray and real-time PCR approaches for two genes in the brain (Grm7 and Zfp317), three genes in the liver (Igf1r, Vwf and Depdc1b) and one gene in the placenta vii (Vwf). However, none of the candidate genes selected for validation showed statistically significant changes. This may be a result of the modest fold changes observed in the microarray experiment that as shown in many cases, often do not replicate. The remainder of the genes showed no changes in expression in the test embryos relative to the control. The functional enrichment analysis revealed biological processes that were over represented in the brain and liver indicating that they may be more vulnerable to the effects of alcohol, compared to the placenta. Overall, the study could not provide a statistically significant correlation between methylation changes in the sperm that were inherited by the offspring which subsequently dysregulated gene expression in the embryo. However, as trends toward significance and significant DNA methylation changes were observed in the embryonic tissues, this study supports the idea that preconception paternal alcohol exposure can induce epigenetic alterations in a locus and organ specific manner within offspring.