Weighted gene co-expression network analysis was used to identify genes that are affected by ethanol using both differentiated (embryoid bodies) and undifferentiated stem cells (Khalid et al. 2014). Unsupervised hierarchical clustering noted two clusters (labeled as turquoise and light yellow in the study) that had similar gene expression in differentiated cells with ethanol as in undifferentiated with or without ethanol, providing evidence for repression. Following a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the turquoise gene group associated with methyltransferase and the light yellow group with RNA polymerase, which may be repressed, suggesting mechanisms affected by ethanol exposure. To confirm that these patterns were due to methylation, a sample set of four of the top-ten downregulated genes was assayed after treatment with ethanol or 5-azacytidine (5-azaC), which blocks methylation. All four genes exhibited a reversal of inhibition and increased expression over untreated control levels. While the sample size for validation was small, the high rate of agreement substantiates the analysis methods used for interpreting methylome-wide effects. Furthermore, this not only confirms that ethanol-induced methylation caused gene repression but also that basal levels of methylation may have been present.
