Fig 2: Single-cell transcriptional analysis of GATA6 mutant hiPSC-CMs during differentiation.(A) tSNE of single-cell RNA-Seq of day 4 GATA6 mutant hiPSC-CMs identified four clusters. Labels reflect marker gene expression: SC, Stem cell; CPC, cardiac progenitor cells; E-CPC, cardiac progenitors enriched with endoderm markers; ECM, endodermal-like cells enriched for extracellular matrix proteins. (B) Examples of marker gene expression in clustered hiPSC-derived cells. CPC cells express mesodermal factors (MESP1, EOMES) as well as cardiac transcription factors (GATA6, GATA4, ISL1, NKX2.5, HAND1, and HAND2). SCs expressed OCT4 (POU5F1). E-CPCs expressed GATA6, SOX17, HNF4A, and FOXA2. (C) tSNE clustering of single-cell RNA-Seq of day 8 GATA6 mutant hiPSC-CMs identified five clusters. Labels reflect marker gene expression: CM, cardiomyocytes; CM (‘RA’), cardiomyocytes with increased RA-signaling pathway genes; SC, Stem Cell; CM (‘Endo’), cardiomyocytes with enrichment in endothelial genes; FB; fibroblast-like cells (D) Examples of marker gene expression in clustered cells. SC expressed OCT4 (POU5F1). CMs expressed sarcomere protein genes (MYH6, MYH7), SMYD1, a CM-specific histone methyl-transferase, and HAND2, a second heart field transcription factor. CMs (RA) also expressed retinoic acid pathway genes (SOX6, HOXB1). CMs (Endo) have upregulated endothelial cell gene expression (MEF2C, BMPER), while FB cells expressed ECM markers (IGFBP7).

Fig 3: Pancreatic TF Bindings Are Enriched in Less Accessible Regions in FOXA2 KO(A) Cumulative plot of opening (19,363) and closing (21,925) peaks from the ES-to-PP1 transition.(B) Tornado plot of less accessible peaks in FOXA2 KO shown for the DE, FG, and PP1 stages. FOXA2 and GATA6 ChIP-seq in less accessible peaks is shown in the same order as ATAC-seq. See also Table S6.(C) Venn diagram showing the overlap between FOXA2 and GATA6 binding sites at the PP1 stage in WT cells.(D) Average of normalized counts visualized as a metapeak. Metapeaks show FOXA2 binding in WT cells and GATA6 binding in WT and FOXA2 KO cells at less accessible peaks. The maximum value of each y axis is annotated in TPM.See also Figures S3 and S4.

Fig 4: Different Stages during Pancreatic Differentiation Are Associated with Distinct TF Motifs(A) Pancreatic differentiation efficiency was verified by examining lineage markers using flow cytometry. ES, embryonic stem cells; DE, definitive endoderm; FG, posterior foregut; PP1, primary pancreatic progenitor; d, day(s). See STAR Methods for a detailed differentiation protocol.(B) Principal-component analysis (PCA) of ATAC sequencing (ATAC-seq) for the top 3,000 variable peaks.(C) TF motif enrichment during stage transitions. The numbers of opened and closed peaks during stage transitions are indicated. Opened or closed peaks between successive stages were compared with the total atlas to examine the TF motif enrichments using the one-sided Kolmogorov-Smirnov (KS) test. The KS test effect size is shown on the y axis, and the proportion of peaks associated with the TF motif is plotted on the x axis. The size of each circle represents the odds ratio (OR), which was defined as the frequency of the TF in an opened or closed group divided by its frequency in the entire atlas. TF motifs with a KS test effect size = 0.05 (indicated by the dashed lines) and OR = 1.2 are shown; when there were more than 10 such motifs, only the top 10 are shown. TF motifs derived from species other than Homo sapiens are marked with brackets. GATA motifs (GATA2, GATA3, GATA4, and GATA6) are marked in red, the HNF1B motif is in blue, and FOXA motifs (FOXA1, FOXA2, and FOXA3) are in green. See also Table S1.(D) Proportion of ATAC-seq peaks with the GATA6, FOXA2, and HNF1B motif are shown.(E) HOMER motif analysis was performed during the stage transition. The top 5 known motifs are shown after removing redundant motifs.

Fig 5: Model for GATA6 transcriptional regulation of cardiac and pancreatic gene expression (see Discussion).ChIP-seq and ATAC-seq data of WT hiPSCs identified GATA6 bound to closed chromatin in intergenic regions without a GATA-binding motif. Moreover, GATA6 binding was associated with temporal activation of transcription in nearby genes that activate cardiomyocyte and endoderm gene network. These findings indicate that GATA6 engages chromatin and fosters a competent state for transcription factor binding and transcriptional activation, supporting the conclusion that GATA6 is a pioneer factor, as is suggested for other GATA proteins (Fisher et al., 2017). Notably, nonsense-mediated decay of GATA6+/- transcripts reduced GATA6 protein levels, altered chromatin accessibility and decreased gene transcription, implying that intergenic sites are sensitive to GATA6 dosage. In addition, GATA6 functions as a traditional transcription factor, binding GATA motifs in promoters and activating transcription. These functions were relatively insensitive to half normal GATA6 levels.