Fig. 6: Optimization of FOXA-binding motifs at an NKX6.1 enhancer redefines patterns of FOXA association and gene expression. | Nature Communications

Fig. 6: Optimization of FOXA-binding motifs at an NKX6.1 enhancer redefines patterns of FOXA association and gene expression.

From: Sequence logic at enhancers governs a dual mechanism of endodermal organ fate induction by FOXA pioneer factors

Fig. 6

a Schematic illustrating base editing strategy at NKX6.1 enhancer via CRISPR-Cas9. Degenerate FOXA-binding motifs and base edits are indicated in red. b ChIP-qPCR comparing FOXA1, FOXA2, H3K4me1, and H3K27ac ChIP-seq signal at the NKX6.1 enhancer in control and motif optimized hESC lines at GT stage. Plots show two independent primer pairs for NKX6.1 enhancer and one primer pair for a negative control region; n = 3 technical replicates. c UMAP representation of single-cell RNA-seq data from both control and motif optimized PP2 cells (integrated) and dot plot showing expression of marker genes in each population (bottom). NKX6.1 expression across populations in control and motif optimized cell lines (right). d Volcano plot comparing genes co-expressed with NKX6.1 in motif optimized compared to control PP2 cells. Wilcoxon rank sum test, 2-sided, corrected for multiple comparisons. e Representative flow cytometry analysis for PDX1 and NKX6.1, mean fluorescence intensity (MFI) of PDX1 signal in NKX6.1+ cells, and quantification of PDX1+ and NKX6.1+ cells in control and motif optimized PP2 cells (n = 3 independent differentiations; P < 1.0 × 10−4; student’s t-test, 2-sided). Bar graph shows mean ± S.E.M. f Schematic illustrating temporal patterns of FOXA recruitment and NKX6.1 expression at the PP2 stage in cells with degenerate and optimized FOXA motifs at the NKX6.1 enhancer.

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