Sasha Burrowes

Fetal Alcohol Syndrome (FAS) and Fetal Alcohol Spectrum Disorders (FASD) are leading non-genetic causes of neurodevelopmental disabilities in the US and worldwide¹. Acting as a teratogen, ethanol has been shown to drastically change the developmental course of maturing neural stem cells in treated mouse NSCs via alterations in DNA methylation, histone modifications, and microRNA regulation.^2,3,4 It has been previously shown, that alcohol does not kill fetal neural stem cells (NSCs), but instead, promotes aberrant maturation.^2,3,4 However, the mechanisms underlying premature maturation and loss of renewal capacity are poorly understood. Investigation of ethanol-induced alterations in the epigenetic profiles of neural stem cells may further the development of necessary practices in education and crafting novel treatment interventions. METHODS To give a global epigenetic perspective, human embryonic stem cells were treated with ethanol, and RNA and genomic DNA were extracted. Changes in methylation of transcription start sites (TSS) and CpG islands were quantified as percent changes in DNA methylation, and pluripotent gene expression profiles were evaluated.⁵ In effort to further investigate neural specific targets of ethanol teratogenesis, Methyl CpG Binding protein 2 (Mecp2) was examined in murine neural stem cells (mNSCs). An ethanol treatment paradigm was utilized in cultured neurospheres to model binge and chronic ethanol exposure as well as withdrawal. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and methylated DNA immunoprecipitation (MeDIP) reflected region specific changes in methylation on Mecp2 regulatory elements (REs). Additionally, DNA dot blot assays were used to identify global alterations in DNA methylation.⁶ RESULTS In hESCs despite each chromosome showing an overall increase in methylation status, a large number of gene promoters showed hypomethylation as a result of ethanol exposure.⁵ This indicates that the effects of ethanol exposure may result in widespread variance in the transcriptional regulation of hESC genes. DNA methylation analysis of Mecp2 gene loci in differentiating NSCs revealed increased 5-hydroxymethylcytosine (5hmC) and decreased 5-methylcytosine (5mC) enrichment at specific REs were associated with upregulated Mecp2/MeCP2 following continuous ethanol exposure. Withdrawal treatment showed significant hypermethylation, which corresponded to a significant decrease in Mecp2 transcript and protein.⁶ CONCLUSION A combination of ethanol induced epigenetic dysregulation during exposure and withdrawal may potentiate alterations in patterns of neural maturation and differentiation leading to FAS neuropathology. Investigation of epigenetic modulation of Mecp2 gene loci may not only give valuable insight into a mechanism of ethanol teratogenesis, but also a potential avenue for novel treatment of FAS in the future.

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2. Veazey KJ, Carnahan MN, Muller D, Miranda RC, Golding MC. Alcohol-Induced Epigenetic Alterations to Developmentally Crucial Genes Regulating Neural Stemness and Differentiation. Alcoholism: Clinical and Experimental Research. 2013;37(7):1111-1122. doi:10.1111/acer.12080.
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4. Kleiber ML, Diehl EJ, Laufer BI, et al. Long-term genomic and epigenomic dysregulation as a consequence of prenatal alcohol exposure: a model for fetal alcohol spectrum disorders.Frontiers in Genetics. 2014;5. doi:10.3389/fgene.2014.00161.
5. Khalid O, Kim JJ, Kim H-S, et al. Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells. Stem cell research. 2014;12(3):791-806. doi:10.1016/j.scr.2014.03.009.
6. Liyanage V, Zachariah R, Davie J, Rastegar M. Ethanol deregulates Mecp2/MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements. Experimental Neurology. 2015;265:102-117. doi:10.1016/j.expneurol.2015.01.006.