Fetal alcohol syndrome (FAS) is a significant cause of mental retardation and birth defects worldwide. However, the mechanisms by which ethanol causes the developmental anomalies and neurotoxicity characteristic of FAS are poorly understood. We hypothesize that chronic exposure to ethanol during gestation via maternal intake during pregnancy causes specific epigenetic changes in the developing fetus, particularly in the nervous system. These stable changes in the normal regulation of a subset of critical genes would contribute to the developmental abnormalities seen in FAS. We further postulate that the epigenetic changes in response to ethanol exposure in utero also contribute to long-term health problems of the offspring later in life. The goal of this project is to identify a subset of genes that undergo epigenetic changes in response to ethanol exposure in utero and result in aberrant gene expression in the brain. We will expose pregnant mice to ethanol via a liquid diet throughout gestation, then compare brain tissue from experimental and control (non-ethanol exposed) pups for changes in DNA methylation patterns at promoter regions in the mouse genome. This strategy will employ methylated DNA immunoprecipitation (meDIP) in conjunction with commercially available tiling arrays from Affymetrix in meDIP-on-chip assays of brain DNA. First, a fraction of brain DNA highly enriched in methylated DNA sequences will be generated by immunoprecipitation of sheared genomic DNA from ethanol-exposed and control mouse pups using antibodies against 5-methylcytosine. This DNA fraction then will be amplified, labeled, and hybridized to Affymetrix microarrays containing all known promoter regions in the mouse genome. Any changes in DNA methylation detected at specific promoters due to prenatal ethanol exposure will be correlated with changes in mRNA expression levels of the corresponding genes by parallel expression microarray studies of changes in brain mRNA levels across the genome in response to ethanol. Genes that exhibit changes in both DNA methylation and mRNA expression levels in response to prenatal ethanol exposure will be candidate genes whose aberrant epigenetic regulation in the brain may contribute to the FAS phenotype. Our proposed studies should provide novel insight into the effects of ethanol at a molecular level not previously explored, and potentially identify genes that contribute to the pathology of prenatal ethanol exposure.
Research Statement Fetal alcohol syndrome (FAS) is a major cause of birth defects and mental retardation in the human population. The incidence of children born in the United States with FAS has risen dramatically, nearly seven fold, between 1979 and 1993. The detrimental effects of maternal ethanol ingestion during pregnancy are numerous, and include severe growth impairment, and morphological, behavioral and neurological abnormalities. However, the molecular basis for FAS remains unclear. This project will explore the hypothesis that epigenetic changes in the fetus in response to ethanol exposure in utero, particularly in the brain, contribute to the clinical phenotype of FAS. An understanding of the underlying mechanisms of FAS may permit the development of novel therapies for treatment of FAS.
