Transgenerational inheritance of DNA methylation alterations at the H19 imprinting control region following maternal ethanol exposure in mice
Foetal Alcohol Syndrome (FAS) is characterised by growth retardation, craniofacial dysmorphology and neurodevelopmental deficits. Whilst, not all alcohol exposed offspring display alcohol-related developmental anomalies, the percentage of affected offspring is greatly underestimated. Common behavioural disorders, such as ADHD and anxiety, are likely to be linked to the transgenerational effects of in utero alcohol exposure. Epigenetics has been highlighted as a potential mechanism in the aetiology of alcohol teratogenesis due to alcohol’s disruptive effects on the folate pathway, and subsequently DNA methylation. The imprinted H19/Igf2 domain is critical in foetal growth and development. The locus is regulated by the methylation-sensitive CTCF binding protein which binds to the H19 imprinting control region (ICR) upstream of the H19 locus. CTCF binding allows for the reciprocal expression of H19 and Igf2 in an allele-specific parent of origin manner. Due to the monoallelic expression of imprinted genes, DNA methylation changes within their control regions can lead to altered gene expression and possibly disease. Furthermore, if these alterations occur in the germline, disease states or susceptibility to disease may be transmittable to future generations. A mouse model was used to investigate the potential transgenerational effects of F0 chronic maternal ethanol exposure on parturition, growth, locomotor activity and anxiety. Furthermore, the transgenerational inheritance of H19 ICR DNA methylation was investigated and its possible contribution to the aforementioned phenotypes was determined. Phenotypic analysis revealed significantly reduced F1 fertility following alcohol exposure (P = 0.003) but no other significant perturbations in parturition. Although not significant at all generations, alcohol’s effects on growth and behaviour were apparent. DNA was extracted from tail biopsies, bisulfite modified and the CTCF1 and CTCF2 regions of the H19 ICR amplified. DNA methylation quantification via Pyrosequencing revealed significantly reduced mean methylation profiles at CTCF1 and CTCF2 within the F1 EtOH exposed group (P = 0.021), with CpG sites 1, 2, 4 and 6 of CTCF1 and CpG sites 1, 2, 3 (P = 0.021) and 5 (P = 0.043) of CTCF2 displaying statistically significant differences. In contrast, the EtOH group of the F2 generation showed an increase in CTCF1 mean methylation that trended towards significance (P = 0.083) suggesting a potential recovery or compensatory mechanism within the epigenetic machinery. The F3 generation EtOH exposed group displayed decreased CTCF1 mean methylation levels (P = 0.083). The F2 and F3 generations showed no significant difference in CTCF2 methylation levels between treatment groups. The significant change in CTCF1 methylation at the F1 generation and the trend towards significance in the F2 and F3 generations indicated potential transgenerational inheritance of altered H19 ICR DNA methylation. Correlations between DNA methylation at the H19 CTCF1 and CTCF2 binding sites with growth rate and behaviour measures revealed no significant relationships. This dissertation supports the involvement of epigenetic mechanisms in alcohol teratogenesis. In addition it contributes to the growing field of transgenerational epigenetic inheritance, with implications for the treatment of those with Foetal Alcohol Syndrome and/or Foetal Alcohol Spectrum Disorders and their progeny.
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree in Master of Science (Medicine) in the Division of Human Genetics