Fig 2: GATA6 and EOMES co-occupancy during early endoderm formation(A–D) Western blot analysis for GATA6 and β-actin during endoderm formation, quantification Figure S2A; (B) Western blot analysis for EOMES and β-actin during early endoderm formation, quantification Figure S2B; (C) Heatmap intensity of GATA6 and EOMES ChIP-seq signal depicting overlap of GATA6 and EOMES binding patterns at all GATA6 bound loci (left panel) and all EOMES bound loci (right panel); (D) Venn diagram of the overlap between GATA6 and EOMES peaks as determined by ChIP-seq (upper panel), heatmaps depicting GATA6 and EOMES ChIP-seq intensity within each subset (middle panel). Hypergeometric motif enrichment analysis of DNA regions uniquely enriched within each subset (lower panel).

Fig 3: Characterization of human cortical neurons generated in vitro from pluripotent stem cells. a Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. b Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. c GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. d Syn, a synaptic vesicle protein. Nuclei are stained with DAPI. e Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 µm

Fig 4: EOMES occupies regions of increased chromatin accessibility in GATA6−/− cells(A–F) RT-qPCR analysis of GATA6 and EOMES mRNA levels at day 2 of differentiation in GATA6+/+ and GATA6−/− cells ± doxycycline. Values normalized to the housekeeping mRNA RPL13a and shown relative to GATA6+/+ cells, n = 3 mean ± SD; (B) Heatmap intensity of GATA6 and EOMES ChIP-seq signal at subsets of EOMES binding that are GATA6-dependent and GATA6-independent; (C) Heatmaps depicting EOMES and GATA6 ChIP- and ATAC-seq signal intensity in GATA6+/+ and GATA6−/− cells at GATA6 occupied regions of differentially accessible chromatin identified between GATA6+/+ and GATA6−/− cells ± doxycycline during early endoderm formation; (D) GO biological process analysis. Selected significantly-enriched developmental pathways related to the genes that neighbor regions of chromatin uniquely accessible in GATA6−/− cells; (E) Genome viewer representation of the HAND1, TBXT, MESP1, and MIXL1 genomic loci aligned with GATA6 and EOMES ChIP-seq and ATAC-seq signal during early endoderm formation in GATA6+/+ and GATA6−/− cells; (F) mRNA expression of the genes that neighbor the selected examples of differentially accessible regions of chromatin, RNA-seq data displayed as counts per million (CPM), n = 2 mean ± SD.

Fig 5: EOMES interactome is altered in the absence of GATA6(A–E) Western blot analysis of GATA6, EOMES, and SOX17 following immunoprecipitation of GATA6-3xFLAG protein in GATA6−/− day 2 cultures differentiated ± doxycycline, quantification Figure S4A; (B) Correlation analysis of the spectral counts of proteins enriched within the EOMES interactome in GATA6+/+ and GATA6−/− cells at day 2 of differentiation, n = 2 immunoprecipitations; (C) Proteins differentially enriched within the EOMES interactome between GATA6+/+ and GATA6−/− cells, n = 2 immunoprecipitations; (D) Heatmaps representing the relative log2 fold change in protein enrichment and mRNA expression of proteins with p < 0.05 and −0.6 > log2 fold change >0.6 differential enrichment in GATA6−/− compared to GATA6+/+ cells at day 2 of differentiation., mean of n = 2 immunoprecipitations, or n = 2 RNA-seq samples; (E) Potential mechanisms of GATA6-dependent repression of chromatin opening non-endoderm-associated enhancers during early endoderm formation.