Fig 2: 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 3: Aberrantly accessible chromatin in day 2 GATA6−/− populations is enriched at sites of transient GATA6 binding(A–H); Western blot analysis for GATA6 and β-actin during early endoderm formation, quantification Figure S2A; (B) Schematic representation of doxycycline supplementation experimental plan; (C) Western blot analysis for GATA6 and β-actin at day 2 of differentiation in GATA6+/+ and GATA6−/− cells ± doxycycline; (D) Heatmaps depicting ATAC-seq signal intensity at subsets of GATA6 binding in GATA6+/+ and GATA6−/− cells ± doxycycline during early endoderm formation; (E) Heatmaps depicting GATA6 ChIP- and ATAC-seq signal intensity during early endoderm formation in GATA6+/+ and GATA6−/− cells at high confidence GATA6 occupied regions of differentially accessible chromatin identified between GATA6+/+ and GATA6−/− cells ± doxycycline at day 2 of differentiation; (F) Pie chart showing the percentage of sites with increased chromatin accessibility in GATA6+/+ or GATA6−/− cells at day 2 of differentiation that are bound by GATA6 at 1) day 2 only, 2) day 4 only, or 3) both day 2 and day 4 in wild-type cells; (G) Hypergeometric motif enrichment analysis of DNA regions enriched in chromatin that is uniquely accessible GATA6−/− cells; (H) Histograms showing density of GATA6 and EOMES motifs and their proximity to the regions of chromatin with differential accessibility between GATA6+/+ and GATA6−/− cells at day 2 of differentiation.

Fig 4: 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.

Fig 5: RIME analysis of GATA6 interacting proteins(A) Volcano plot of RIME-detected proteins with more than one spectral count and more than one unique peptide fragment. GATA6 interactome: 5-fold enriched over IgG controls, p < 0.05, n = 2. See Table S5.(B) Validation of RIME proteins by western blot analysis of GATA6, CHD4, SMARCA4, and KDM1A following immunoprecipitation of GATA6-3× FLAG protein in GATA6Ex4?1/?2;ind GATA6 mesendoderm cultures differentiated with and without doxycycline.(C) Venn diagram depicting overlap of peaks from EOMES ChIP-seq completed in GATA6+/+ and GATA6Ex4?1/?2;ind GATA6 cells without doxycycline differentiated to mesendoderm (upper panel). Heatmaps of EOMES and GATA6 ChIP-seq intensity in GATA6+/+ and GATA6Ex4?1/?2;ind GATA6 cells without doxycycline at regions of rapid GATA6-dependent chromatin remodeling (day 2) (lower panel).(D) Published KDM1A ChIP-seq (Vinckier et al., 2020) from similar differentiation stage (day 5; foregut) overlapped with definitive endoderm GATA6 binding sites. GATA6 (blue), KDM1A (green), H3K4me1 (red), and ChIP-seq and ATAC-seq (purple) intensity displayed at subgroups of GATA6-bound sites with different endoderm accessibility profiles.(E) Temporal profile of ATAC-seq data fragment depth during the hepatic specification at sites of GATA6-dependent chromatin accessibility. See Table S6.(F) Proposed model of GATA6-dependent chromatin modulation during endoderm formation and specification. End transcription factors (TFs): EOMES, SOX17, and FOXA2.