Gene expression signatures affected by alcohol-induced DNA methylomic deregulation in human embryonic stem cells.

Transcriptomic profiling of gene signatures in EtOH-treated or differentiating hESCs(A) WGCNA consensus cluster analysis of hESC genes comparing EtOH (20 mM) treatment of hESCs in their undifferentiated or EB states, as well as comparing undifferentiated and differentiated hESCs without EtOH treatment. Correlations of each respective comparison are shown below the module cluster. (B) EtOH dose-response (at 0, 20 and 50 mM) comparison between H1 and H9 cell lines using heatmaps of representative modules. We selected three representative modules that showed the same pattern of expression changes after EtOH treatment in both H1 and H9 hESCs. This is to demonstrate an example of EtOH-induced alterations in gene signatures independent of cell lines. (C) Cluster of the different modules in relation to differentiation and EtOH treatment. Turquoise and light-yellow modules that showed consistent gene expression correlations are indicated by asterisks. (D) A Volcano plot identified the number of the most significantly altered genes upon EtOH treatment between H1 and H9. (E) The top fourteen modules significantly affected by EtOH were analyzed by R package cluster Profiler and the resulted showed that the modules formed distinct ontological clusters without any overlap. (F) Molecular pathway analysis using KEGG database showed the dot plot of the gene ontology unique to each module of the most affected clusters in (E). Fisher Exact test was adopted to measure the gene-enrichment in annotation terms. When members of two independent groups can fall into one of two mutually exclusive categories, Fisher Exact test was used to determine whether the proportions of those falling into each category differ by group (Huang da et al., 2009). (G) Hive plot of the gene network associated with the turquoise consensus module. Red dots showed two top hub genes- SCUBE3 and SLC22A5. (H) Standard gene network representing the turquoise module. (I) The top hub genes, SCUBE3 and SLC22A5, and implicated genes based on maximal clique centrality.

Global DNA methylation changes at the transcription start site (TSS) and CpG islands (CGIs) upon EtOH treatment(A) EtOH increases DNA methylation in undifferentiated hESCs as measured by changes in % of 5-methylcytosine (5-mC). *, p < 0.05 versus 0 mM EtOH (one-way ANOVA). (B) DNA methylation changes after EtOH treatment in undifferentiated hESCs were assessed. Relative DNA methylation levels at the TSS (2.5 kb upstream and downstream) after treatment with 20 mM EtOH were plotted (left). It shows the status of DNA hypermethylation at the TSS resulted from EtOH treatment. Aligned probes plot of methylation showed that treatment of undifferentiated hESCs with 20 mM EtOH resulted in an increase in DNA methylation at the TSS compared to non-treated control (Middle). The Log plot for methylation at the TSS comparing control cells (x-axis) vs. cells treated with 20 mM EtOH (y-axis) showed greater level of DNA methylation after EtOH treatment (right). (C) DNA methylation changes at CGIs after EtOH treatment were assessed as described in (B). Relative DNA methylation plot at the CGIs after treatment with 20 mM EtOH showed an increase in DNA methylation on the DNA sequences upstream of CGIs (left). Aligned probes plot of methylation showed that treatment with 20 mM EtOH resulted in an increase in DNA methylation at the CGIs compared to non-treated control (Middle). The Log plot for methylation at the CGIs comparing control cells (x-axis) vs. cells treated with 20 mM EtOH (y-axis) showed greater level of DNA methylation after EtOH treatment (right).

EtOH-induced increases in DNA methylation in undifferentiated hESCs but not in embryoid bodies (EBs)Comparisons of average relative DNA methylation levels between (A) promoters of genes and (B) CpG islands of genes from undifferentiated hESCs (top panels) and hESC EBs (middle panels) treated with 0 mM or 20 mM EtOH. Bottom panel shows fold changes in DNA methylation for promoters and CpG islands of genes in EtOH-treated undifferentiated hESCs (blue bars) and EBs (red bars). Statistical analysis revealed that the EtOH-induced increases in methylation status of Promoter regions (and to a lesser extent CpG islands) were significantly different (p < 0.05, unpaired t-test) for undifferentiated hESCs vs. EBs at all 23 chromosomes.

Methylation vs. gene expression in EtOH-treated hESCsMethylation vs. gene expression of promoter regions during: (A) differentiation of hESCs, (B) EtOH treatment of undifferentiated hESCs and (C) EtOH treatment of EBs. Methylation vs. gene expression of CpG islands during: (D) differentiation of hESCs, (E) EtOH treatment of undifferentiated hESCs, and (F) EtOH treatment of EBs. (G) Summary of hyper and hypo methylated regions and their correlation to expression. (H) Rescue of genes downregulated by EtOH treatment after treatment with 5-azacytidine (5-Aza). Level of ACADY4, FGF17, HOXA1 and PHOSPHO1 gene was quantitatively assessed by qRT-PCR analysis with, without EtOH (20 mM), or after co- treatment with 20 mM EtOH and 1 μM of 5-Aza for 24 hrs. *, p < 0.05 vs. control; **, p < 0.05 vs. EtOH treatment (one-way ANOVA with Tukey's post hoc). (I) The level of hypo and hypermethylation at 2.5 kb upstream and downstream of the transcription start site (TSS) in EtOH-treated undifferentiated hESCs as shown in (B). The genes represent inverse correlation between methylation and expression- hypermethylated and downregulated (gold) vs. hypomethylated and upregulated (green). (J) A graph representing the average methylation found in I (green represents hypomethylated genes and gold represents hypermethylated genes). (K) Pie chart representing the level of hypo- and hypermethylation in genes both positively and negatively regulated. It shows how much hypo- or hypermethylation refers to either a decreased (negative) or increased (positive) gene expression.

Profiling of molecular signatures specifically affected by EtOH-induced DNA methylation in hESCsTo obtain a list of genes that are epigenetically altered by EtOH-induced DNA methylation changes, genes showing correlative changes in DNA methylation and in gene expression were identified. Genes showing β>0.5 in DNA methylation either on CpG islands or promoter regions with >2-fold change in gene expression were chosen from Fig. 4. Comparison was done to examine the effect of EtOH on the differentiation of hESCs. By combining the list of genes differentially regulated during hESC differentiation and the list of genes dysregulated by EtOH treatment in undifferentiated hESCs, we were able to identify genes that are potentially involved in the differentiation of hESC by EtOH-induced DNA methylation changes (common genes).

Quantitative RT-PCR validation of gene listThe expression levels of part of genes list obtained from Fig. 5 were validated. hESCs were treated with 0, 20, or 50 mM EtOH for 24 hrs and qRT-PCR performed as described in Materials and Methods. (A) Genes that were upregulated by EtOH-induced DNA hypomethylation. Note significantly increased expression in 3 of 8 genes, with none significantly decreased. (B) Genes that were downregulated by EtOH-induced hypermethylation. Note significantly decreased expression in 4 of 8 genes, with none significantly increased. Bars are mean ± SEM from triplicates; *, p < 0.05 vs. control; †, p < 0.05 vs. 20 mM EtOH (one-way ANOVA with Tukey post hoc).

Common motif analysis and EtOH-related transcription factor discoveryGenes that are affected by normal differentiation are shown in purple Venn diagrams (Up_Diff: Hypomethylated and upregulated during differentiation; Down_Diff: Hypermethylated and downregulated during differentiation). Genes that are significantly affected by EtOH are shown in yellow Venn diagrams (Up_EtOH: Hypomethylated and upregulated upon EtOH treatment; Down_EtOH: Hypermethylated and downregulated upon EtOH treatment). Subsets of genes that are affected by both EtOH and differentiation are shown in the middle. These genes are also represented in the middle orange box of Figure 5. Selected genes were further subjected to STAMP analysis to identify potential cis-regulatory elements that are present in the differentially regulated genes and transcription factors that could be potentially affected by EtOH treatment. Based on the common gene lists, common motifs and associated transcriptional factors are found for CpG and promoter respectively. E-value is calculated based on the Wasserman and Sandelin method (Wasserman and Sandelin, 2004).