Fig. 3: RNA sequencing and CHIP-qPCR analysis reveal genes and pathways regulated by ETV1, ETV4, and ETV5 transcription factors in hPSCs.

A A volcano plot was generated to visualize differentially expressed genes between KO and WT hPSCs. Red dots indicate 133 upregulated genes, and blue dots indicate 95 downregulated genes, showing log2 fold change (log2 FC) and −log10 adjusted p-value (−log10 adjp). B A volcano plot was generated to visualize differentially expressed genes between tKO and WT hPSCs. Red dots indicate 1201 upregulated genes, and blue dots indicate 2027 downregulated genes, showing log2 fold change (log2 FC) and −log10 adjusted p-value (−log10 adjp). C Volcano plot of differentially expressed genes between tKO and KO hPSCs shows log2 fold change (log2 FC) and −log10 adjusted p-value (−log10 adjp) for 679 downregulated (blue) and 400 upregulated (red) genes. D The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases were identified among the differentially expressed genes between KO and WT hPSCs. Enriched functional terms associated with upregulated and downregulated genes in KO compared to WT hPSCs are marked by red and blue triangles, respectively. E The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases among the differentially expressed genes between tKO and WT hPSCs. Enriched functional terms associated with upregulated and downregulated genes in tKO compared to WT hPSCs are marked by red and blue triangles, respectively. F The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases among differentially expressed genes between tKO and KO hPSCs. Enriched functional terms associated with upregulated and downregulated genes in tKO compared to WT hPSCs are marked by red and blue triangles, respectively. G Heatmaps demonstrate the top significant differentially expressed genes in KO compared to WT associated with cell–cell and cell-ECM adhesion, PI3K/AKT pathway (left panel), or pluripotency and cell fate (right panel). Diff. – differentiation. H Heatmaps demonstrate the top significant differentially expressed genes in tKO compared to WT associated with cell–cell and cell-ECM adhesion, and PI3K/AKT pathway (left panel) or pluripotency and cell fate (right panel). Diff. – differentiation. I ATAC-seq tracks highlight the ITGAX locus in WT (yellow), KO (green), and tKO (maroon) hPSCs. The peaks represent normalized and combined biological replicates (N = 2). J ATAC-seq tracks highlight the VAV1 locus in WT (yellow), KO (green), and tKO (maroon) hPSCs. The peaks represent normalized and combined biological replicates (N = 2). K Western blot demonstrates the ETV1 presence in both the chromatin-bound (CH) and soluble protein fraction (SF) in WT hPSCs. pHH3 was used as a positive control of fractionation, and total cell lysate (TL) was used as the positive control for ETV1. N = 2 biological replicates. L ChIP-qPCR analysis (antibody against ETV1) confirmed ETV1 binding to the promoter region of selected differentially expressed genes connected to cell adhesion, i.e., ITGA5, VCL, PDGFRB, and COL4A or pluripotency, i.e., JUN and CDKN1A. IgG – control (blue); ChIP ETV1 – target (red). Data are presented as the mean ± SDs. N = 2 biological replicates. For plots (A–C), the DESeq2 package and the Wald test were used to determine significance. DAVID online software was used for biological term enrichment analysis of the DEGs, with the Fisher Exact statistics to list annotation terms and their associated genes. The p-values are adjusted for multiple comparisons using the Benjamini and Hochberg approach. Genes with non-significant change in expression are depicted in gray (−log10 (adjp) <2; log2 FC between 0.5 and −0.5). Highlighted dots indicate selected genes in the enriched pathways, pictured in plots (D–F).