Subsequently, we studied whether there was any sequence specificity to the genes that were being affected by EtOH treatment (Fig. 7). From the genes that are significantly affected by EtOH (shown in yellow Venn-diagram), we wanted to find a subset of genes that is also related to differentiation (shown in purple Venn-diagram). The common genes between genes affected by EtOH treatment and genes altered during differentiation represent genes that may be involved in differentiation profile due to EtOH treatment (middle in Venn-diagram). The common genes were found in all four conditions, CpG and promoter; as well as increased expression (upregulated) and decreased expression (downregulated). To examine if these common genes affected by EtOH share any sequence motifs for transcription factors, we have performed motif analysis using the regulatory sequence analysis tool (RSAT) (Thomas-Chollier et al., 2012). We discovered two major motifs represented within the genes that are commonly associated with differentiation of hESCs and also EtOH exposure. Thus, these motifs may represent potential binding sites of transcription factors that may play a role in differentiation changes that are occurring in
