Fig. 3

Transcriptome interactions between key transcription factors that regulate hiPSC-CM populations. ChIP-seq was performed on transcription factors (NR2F2, TBX5, and HEY2) enriched within three hiPSC-CM populations. a ChIP-seq peak density demonstrates enrichment of the ChIP-seq signal around the transcriptional start sites (TSS). b GO biological functions were graphed corresponding to peaks called within each ChIP-seq dataset (GREAT analysis, hypergeometric test, p < 0.05). NR2F2 was highly enriched for terms within the nervous system, TBX5 for cardiac tissue, and HEY2 for neural and enteric terms. c Venn diagram demonstrates the number of unique and common genes called (using HOMER) within each ChIP-seq dataset. d Coverage of the reads demonstrating a peak near the MYL2 locus in the TBX5 and HEY2 ChIP-seq datasets, but not the NR2F2 dataset. e RNA-seq was performed on cardiomyocytes from each gene-edited line [NR2F2^GE (NR2F2^GE1 and NR2F2^GE2 lines), HEY2^GE (HEY2^GE1 and HEY2^GE2), and TBX5^GE (TBX5^GE1 and TBX5^GE2)], as well as cardiomyocytes over expressing NR2F2 (NR2F2^OE), TBX5 (TBX5^OE) and HEY2 (HEY2^OE). Biological terms associated with genes significantly different (GO-Elite algorithm AltAnalyze) between these lines were annotated (left panel–blue text), as well as the corresponding ChIP-seq peaks identified (right panel). Gene sets associated with each of the single-cell populations from Fig. 1b are highlighted in orange. Genes associated with the enriched GO terms and single-cell populations are displayed to the right of the heatmap. f GO biological analysis was performed on significant genes upregulated or downregulated in hiPSC-CMs overexpressing NR2F2, TBX5, or HEY2 with corresponding NR2F2, TBX5, or HEY2 gene-edited lines. g Interactions among genes regulated by NR2F2, TBX5, and HEY2 were visualized by combining the significant genes called within each NR2F2^GE1, TBX5^GE1, and HEY2^GE1 with genes called within each ChIP-seq dataset. Nodes (blue circles) represent individual genes and the links represent the genes that interact with each transcription factor. Red indicates an upregulation and green represents a downregulation observed in the RNA-seq of each gene-edited line. h ChIP-seq/RNA-seq interactions using a selected set of cardiac-specific genes. MYL2 is highlighted to be positively regulated by TBX5 and HEY2 expression