The effect of alcohol on the methylation status of the imprinting control regions contained within three developmentally significant loci

MSc(Med), Human Genetics, Faculty of Health Sciences, University of the Witwatersrand, 2009Imprinted loci are critical in foetal development and most are regulated by the methylation-specific CTCF binding protein which binds imprinting control regions (ICRs). The ICR is located between two genes that comprise imprinted loci, which are reciprocally expressed in a parent-of-origin specific manner. Maternally hypomethylated ICRs allow CTCF binding, creating a boundary element which prevents downstream enhancers from acting on the paternally expressed gene upstream of the ICR. Conversely, the hypermethylated (imprinted) paternal ICR prevents CTCF binding, allowing downstream enhancers to act on the gene upstream of the ICR, while suppressing the downstream maternally expressed gene. Alcohol and its metabolites are able to reach the testes via the blood supply and are known to reduce global DNA methylation by disrupting the folate, methyl group and homocysteine pathway. This may therefore affect gene expression at imprinted loci, whose parental alleles are discriminated by the imprinting status at the ICR. The effect of pre-conception paternal alcohol exposure on the DNA methylation of three paternally imprinted ICRs (H19, Rasgrf1, IG-DMR) as well as the maternally imprinted Snrpn ICR was examined in mouse sperm and their offspring. Male mice were gavaged with ethanol or sucrose. DNA was extracted from sperm of treated males and tail biopsies from offspring. Samples were bisulphite modified and the ICRs PCR amplified. DNA methylation patterns of ICRs were analysed by sequencing and quantitatively via pyrosequencing. Sperm samples of ethanol treated males did not show significant demethylation when compared to sucrose treated mice, with the exception of H19 CpG 7, Rasgrf1 CpG 26 and Snrpn CpG 10 (p=0.024, 0.014 an