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. Author manuscript; available in PMC: 2019 Oct 15.
Published in final edited form as: Nat Genet. 2019 Apr 15;51(5):777–785. doi: 10.1038/s41588-019-0384-0

Figure 2. HNF4 binding is required to activate enhancer chromatin and stimulate genes required for intestinal differentiation.

Figure 2

HNF4 factors function primarily to activate rather than repress transcription, as evidenced by (a) heatmaps (Kolmogorov-Smirnov test, one-sided for positive and negative enrichment scores, P < 0.001). The lower heatmap displays a log2 fold-change in gene expression (n = 3 biologically independent mice). The upper heatmap shows the corresponding frequency (yellow shading) of HNF4 binding sites located within 20 kb of the TSSs of the corresponding genes in 10-gene binned groups displayed in the expression heatmap. b, Volcano plot of gene expression changes (n = 3 biologically independent mice). Significant genes were called via Cuffdiff. c, Distribution of TSS distances in 10-kb windows from the nearest HNF4 binding sites. Genes with an FDR < 0.05 are determined as either decreased or increased in the Hnf4αγDKO duodenum compared to the controls (n = 3 biologically independent mice). Downregulated genes are more likely to have nearby HNF4 binding sites than upregulated genes. d, Genes that decrease in HNF4-deficient epithelium are more likely to harbor multiple HNF4 binding events than genes that show increased expression (n = 3 biologically independent mice). e, HNF4 bound genes are significantly downregulated in Hnf4αγDKO compared to unbound genes. The middle line represents the median; whiskers represent the 10th and 90th percentile (Mann-Whitney test, two-sided at P < 0.001 ***; n = 3 biologically independent mice). f, Functional annotation of HNF4 direct and indirect genes (log2 fold-change < −1, FDR < 0.05) by DAVID. Direct genes: HNF4 bound genes (genes within 30 kb of annotated TSSs nearby HNF4 binding sites). Indirect genes: HNF4 unbound genes. Benjamini P values were calculated using DAVID. g, MNase-ChIP-seq profiles (k-means = 4, MACS P ≤ 10−5) of H3K27ac. Statistical tests were embedded in the MACS package. h, Aggregate plots of H3K27ac ChIP-Seq signal are centered on the 5,952 enhancer sites that are bound by HNF4, and loss of HNF4 paralogs prominently reduces H3K27ac signal. i, Model of HNF4-control of active chromatin structures in the intestine.