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Alcohol Alters DNA Methylation Patterns and Inhibits Neural Stem Cell Differentiation

Authors

  • Feng C. Zhou,

    1. From the Department of Anatomy and Cell Biology (FCZ, YB, RPS); Stark Neuroscience Research Institute (FCZ); Department of Medical and Molecular Genetics (MT, YL); Center for Computational Biology and Bioinformatics (MT, YL); Center for Medical Genomics (MT, YL); Medical Sciences Program and Department of Cellular & Integrative Physiology (KPN); Institute of Psychiatric Research (YB), Indiana University School of Medicine, Indianapolis, Indiana; and School of Computer Science and Technology (MT), Harbin Institute of Technology, Harbin, Heilongjiang, China.
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  • Yokesh Balaraman,

    1. From the Department of Anatomy and Cell Biology (FCZ, YB, RPS); Stark Neuroscience Research Institute (FCZ); Department of Medical and Molecular Genetics (MT, YL); Center for Computational Biology and Bioinformatics (MT, YL); Center for Medical Genomics (MT, YL); Medical Sciences Program and Department of Cellular & Integrative Physiology (KPN); Institute of Psychiatric Research (YB), Indiana University School of Medicine, Indianapolis, Indiana; and School of Computer Science and Technology (MT), Harbin Institute of Technology, Harbin, Heilongjiang, China.
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  • MingXiang Teng,

    1. From the Department of Anatomy and Cell Biology (FCZ, YB, RPS); Stark Neuroscience Research Institute (FCZ); Department of Medical and Molecular Genetics (MT, YL); Center for Computational Biology and Bioinformatics (MT, YL); Center for Medical Genomics (MT, YL); Medical Sciences Program and Department of Cellular & Integrative Physiology (KPN); Institute of Psychiatric Research (YB), Indiana University School of Medicine, Indianapolis, Indiana; and School of Computer Science and Technology (MT), Harbin Institute of Technology, Harbin, Heilongjiang, China.
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  • Yunlong Liu,

    1. From the Department of Anatomy and Cell Biology (FCZ, YB, RPS); Stark Neuroscience Research Institute (FCZ); Department of Medical and Molecular Genetics (MT, YL); Center for Computational Biology and Bioinformatics (MT, YL); Center for Medical Genomics (MT, YL); Medical Sciences Program and Department of Cellular & Integrative Physiology (KPN); Institute of Psychiatric Research (YB), Indiana University School of Medicine, Indianapolis, Indiana; and School of Computer Science and Technology (MT), Harbin Institute of Technology, Harbin, Heilongjiang, China.
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  • Rabindra P. Singh,

    1. From the Department of Anatomy and Cell Biology (FCZ, YB, RPS); Stark Neuroscience Research Institute (FCZ); Department of Medical and Molecular Genetics (MT, YL); Center for Computational Biology and Bioinformatics (MT, YL); Center for Medical Genomics (MT, YL); Medical Sciences Program and Department of Cellular & Integrative Physiology (KPN); Institute of Psychiatric Research (YB), Indiana University School of Medicine, Indianapolis, Indiana; and School of Computer Science and Technology (MT), Harbin Institute of Technology, Harbin, Heilongjiang, China.
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  • Kenneth P. Nephew

    1. From the Department of Anatomy and Cell Biology (FCZ, YB, RPS); Stark Neuroscience Research Institute (FCZ); Department of Medical and Molecular Genetics (MT, YL); Center for Computational Biology and Bioinformatics (MT, YL); Center for Medical Genomics (MT, YL); Medical Sciences Program and Department of Cellular & Integrative Physiology (KPN); Institute of Psychiatric Research (YB), Indiana University School of Medicine, Indianapolis, Indiana; and School of Computer Science and Technology (MT), Harbin Institute of Technology, Harbin, Heilongjiang, China.
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Reprint requests: Feng C. Zhou, PhD, Department of Anatomy & Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, MS 508, Indianapolis, Indiana 46202; Tel.: 317-274-7359; Fax: 317-278-2040; E-mail: imce100@iupui.edu

Abstract

Background:  Potential epigenetic mechanisms underlying fetal alcohol syndrome (FAS) include alcohol-induced alterations of methyl metabolism, resulting in aberrant patterns of DNA methylation and gene expression during development. Having previously demonstrated an essential role for epigenetics in neural stem cell (NSC) development and that inhibiting DNA methylation prevents NSC differentiation, here we investigated the effect of alcohol exposure on genome-wide DNA methylation patterns and NSC differentiation.

Methods:  Neural stem cells in culture were treated with or without a 6-hour 88 mM (“binge-like”) alcohol exposure and examined at 48 hours, for migration, growth, and genome-wide DNA methylation. The DNA methylation was examined using DNA-methylation immunoprecipitation followed by microarray analysis. Further validation was performed using Independent Sequenom analysis.

Results:  Neural stem cell differentiated in 24 to 48 hours with migration, neuronal expression, and morphological transformation. Alcohol exposure retarded the migration, neuronal formation, and growth processes of NSC, similar to treatment with the methylation inhibitor 5-aza-cytidine. When NSC departed from the quiescent state, a genome-wide diversification of DNA methylation was observed—that is, many moderately methylated genes altered methylation levels and became hyper- and hypomethylated. Alcohol prevented many genes from such diversification, including genes related to neural development, neuronal receptors, and olfaction, while retarding differentiation. Validation of specific genes by Sequenom analysis demonstrated that alcohol exposure prevented methylation of specific genes associated with neural development [cut-like 2 (cutl2), insulin-like growth factor 1 (Igf1), epidermal growth factor–containing fibulin-like extracellular matrix protein 1 (Efemp1), and SRY-box-containing gene 7 (Sox 7)]; eye development, lens intrinsic membrane protein 2 (Lim 2); the epigenetic mark Smarca2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2); and developmental disorder [DiGeorge syndrome critical region gene 2 (Dgcr2)]. Specific sites altered by DNA methylation also correlated with transcription factor binding sites known to be critical for regulating neural development.

Conclusion:  The data indicate that alcohol prevents normal DNA methylation programming of key neural stem cell genes and retards NSC differentiation. Thus, the role of DNA methylation in FAS warrants further investigation.

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