Fig. 6: HNF4 loss results in an increased stress fiber formation.
a, b Yolk sac data of WT vs Hnf4αKO. Schematic of experimental design is shown in Fig. 5e. a GSEA of RNA-seq data from E18.5 yolk sac reveals that stress fiber assembly (Kolmogorov–Smirnov test, one-sided for positive and negative enrichment scores, P < 0.001) and focal adhesion assembly (Kolmogorov–Smirnov test, one-sided for positive and negative enrichment scores, P = 0.0039) gene signatures are all elevated upon HNF4A loss. b IHC staining of stress fiber/focal adhesion-related proteins (n = 3 biological replicates) in E17.5 yolk sac. c–i Intestinal epithelium data of WT vs. Hnf4αγDKO. c Heatmap of RNA-seq data shows upregulated stress fiber/focal adhesion transcripts upon HNF4 loss in the intestinal epithelium (FDR < 0.05, n = 3 biologically independent mice). d Increased chromatin loops were observed at stress fiber/focal adhesion gene loci upon HNF4 loss. H3K4me3 HiChIP-seq was done in villus cells of Hnf4αγDKO and their littermate controls. Differential loops (DEseq2 P < 0.05) are visualized by Sushi package for the loops with q ≤ 0.0001 and counts ≥8 (combined 2 replicates). Bar charts show transcript levels of brush border genes. The data are presented as mean ± SEM (RNA-seq: n = 3 biological replicates, ***Cuffdiff FDR < 0.001 and *FDR < 0.05). H3K4me3 HiChIP-seq: n = 2 biological replicates; H3K27ac ChIP-seq: n = 2 biological replicates; HNF4 ChIP-seq: n = 2 biological replicates. e IHC staining of stress fiber/focal adhesion-related proteins (n = 3 biological replicates). f Increased chromatin loops at Flnb locus were observed upon HNF4 loss. Elevated protein levels of Filamin A were observed by g western blotting (n = 2 biologically independent mice) and h immunofluorescence staining (n = 3 biologically independent mice). i Loss of HNF4 factors results in downregulated brush border genes and upregulated stress fiber genes.
