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An Epigenetic Basis to the Etiology of Fetal Alcohol Spectrum Disorders
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Observations from a number of independent laboratories indicate that ethanol has the capacity to act as a powerful epigenetic disruptor and potentially derail the process of cellular differentiation. The aim of this dissertation was to determine the epigenetic effects of alcohol on chromatin structure, the heritability of these effects in vitro and in vivo, and whether the severity of these alterations is tied to the differentiation state of the cell. First, we investigated the epigenetic impact of ethanol exposure in a murine neural stem cell model using chromatin immunoprecipitation, quantitative polymerase chain reaction (ChIP-qPCR) and RNA analysis. We found that two widely-studied histone modifications, trimethylated histone 3 lysine 4 (H3K4me3) and trimethylated histone 3 lysine 27 (H3K27me3), were disrupted at promoters of a panel of homeobox genes involved in neural development in the presence of alcohol, and that these disruptions do not correlate with changes in the expression of the examined genes. Second, we determined whether the disruption of chromatin structure caused by alcohol is heritable through cell division after an acute exposure in vitro. We monitored changes in H3K27me3, H3K4me3, and acetylation/demethylation of histone 3 lysine 9 (H3K9ac and H3K9me2, respectively) at the promoters of our candidate homeobox genes using ChIP-qPCR. We found that alterations in these marks persist beyond the window of exposure, and do not retain the same levels compared to controls after a recovery period in which ethanol is withdrawn. Furthermore, changes in the expression of these genes often occurred after recovery and again do not correlate with histone modifications present at their respective promoters. These alterations occur despite no indication of cell stress, but are associated with increased expression of genes involved in cell proliferation and neural lineage markers after recovery. A decrease in many oxidative stress pathway genes was also observed upon exposure that was rectified after recovery. Importantly, changes in the gene expression of histone methyltransferases and DNA methyltransferases were observed, with a concurrent change in DNA methylation. We next chose to determine if the observed alterations in chromatin structure also appear in vivo using a mouse model of early acute ethanol exposure. Pregnant dams injected with 2.9 g/kg ethanol at gestational day (GD) 7 were sacrificed at GD17, and the fetuses scored for ocular and forebrain defects. Levels of H3K27me3 were low at the promoters of many of the candidate genes in affected mice, and high levels of H3K9me2 specifically identified ethanol-affected mice, suggesting its potential as a marker for FASD phenotypes. Finally, we determined whether the epigenetic effects of ethanol are dependent on the differentiation state of the cell using a murine embryonic stem cell (ESC) model. Acute ethanol exposure resulted in oscillating changes in levels of histone modifications for a long as 10 days post-exposure. Despite these changes in chromatin structure, no lasting changes in expression of our candidate genes or chromatin modifiers was detected. Acute ethanol exposure also did not impact the capacity of the ESCs to differentiate along a neural lineage. While alcohol has the capacity to act as an epigenetic disruptor, its effects differ depending on the differentiation state of the cell and whether it is encountered in conditions maintaining stemness or during the execution of the developmental program. Furthermore, the alcohol-induced alterations in histone marks seem to be more of a byproduct of teratogenic insult rather than associated with a functional role in the transcriptional regulation of the cell. This study highlights the complexity of ethanol’s teratogenic effects and suggest the histone code may not be a direct regulator of transcriptional control, at least in the context of an environmental exposure. None-the-less, alcohol-induced alterations in chromatin structure persist beyond the window of exposure and strongly correlate with the development of FAS birth defects. This study provides a platform for new hypotheses in fetal alcohol epigenetics and possibly the establishment of a mechanism of alcohol’s effects on chromatin structure.
Veazey, Kylee Jordan (2015). An Epigenetic Basis to the Etiology of Fetal Alcohol Spectrum Disorders. Doctoral dissertation, Texas A & M University. Available electronically from