Fig 1: Widespread modulation of epigenetic landscape for differentiated organoids is driven by Hnf4g Heatmaps showing epigenome dynamics on peaks with significant changes in DNA accessibility and histone modifications between CV, ENR, and EN organoids. Heatmaps are split on their location near a transcription start site (< 500 bp from TSS, top heatmaps) or other location. Heatmaps are further subdivided based on the dataset it was found significantly changing in (shown at the left). Shown P-values are corrected for multiple testing hypotheses with the BH method.Clustered genomic regions from (A) are shown. Regions were associated with their single closest TSS and corresponding mRNA expression changes are shown in the most left heatmaps. The best match score for the HNF4G motif is shown in the middle heatmap. Hnf4g binding is shown in the most right heatmaps.Scatterplot with the transcription factors that are most likely to explain the dynamics shown in (A). The x-axis is the linear correlation between the best motif score and the log2-fold change EN over ENR under each significantly changing peak for a given dataset as shown in (A). The y-axis is the feature importance of the motif in a random forest model in percentage increase in mean-squared error (MSE), where motifs that are important for the model to accurately predict the given fold changes get a high error increase. Different datasets are color-coded. For motifs with more than one transcription factors associated to it, we show the transcription factor name with the most significant change in protein expression. The vertical line over each dot represents two times the standard deviation of the MSE.Heatmaps showing change in protein (left) and mRNA (right) expression of selected transcription factors, compared to ENR organoids.Data information: In panels (A and B), relative changes between different organoids are shown as log2 row mean subtractions (RMS) where the signal is compared to the row mean of all samples.
Fig 2: Hnf4g drives differentiation of enterocytes Gene Set Enrichment Analysis (GSEA) of Hnf4g KO organoids compared to WT organoids. Significantly changing intestinal cell-type gene sets from Haber et al (2017) are shown (FDR < 0.05).Hematoxylin and PAS (periodic acid–Schiff) staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using hematoxylin and goblet cells stain positive for PAS.Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian blue.Strand-specific RNA-seq data, Hnf4g ChIP-seq in EN, and promoter-specific histone modification H3K4me3 over the Hnf4a locus are shown. Data from different organoid cultures are color-coded. RNA-seq reads mapping to the positive strand (sense strand) are plotted in the top window of every sample, while RNA-seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample. The two isoforms of Hnf4a that are expressed are shown in the bottom, together with the antisense transcript Hnf4aos. The “phyloP30way” track from the UCSC genome browser is plotted at the bottom to show sequence conservation.Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine. Dots represent the amount of goblet cells counted per villus. (n = 7–25 villi from 11 WT mice and 3–17 villi from 14 Hnf4g KO mice). P-value is from two-tailed Mann–Whitney U-test. The central line in each boxplot represents the median, the notch around this line is the approximate 95% confidence interval, the hinges are the first and third quartile, and the whiskers extend to the lowest and highest values within 1.5× the interquartile range from the hinges.
Fig 3: Hnf4g dynamics and importance in the different cell‐type‐enriched organoids Western blot for Hnf4g on whole cell extract of CV, ENR, and EN cultured WT and Hnf4g KO organoids. β‐Actin is used as a loading control.ChIP‐seq and RNA‐seq profiles of CV, ENR, and EN WT organoids at the Alpi locus.Scatterplot showing the percentage of peaks inside a TAD that have fold change in H3K27ac signal in the same direction as the average fold change in H3K27ac of the whole TAD. Striped line demarcates the empirical 0.05 FDR cutoff. Significantly changing TADs that contain a known marker of intestinal homeostasis are highlighted.Number of identified peaks of the Hnf4g ChIP‐seq in the different cell‐type‐enriched WT organoids.Pie chart showing all significantly upregulated genes in EN organoids. Genes with a Hnf4g motif in their promoter are highlighted in pink. Normalized enrichment score (NES) and motif association were determined in iRegulon (Janky et al, 2014). KEGG pathways that were significantly overrepresented (FDR < 0.01) in upregulated genes with a Hnf4g motif are listed.
Fig 4: Hnf4g importance in human colon cancer organoids and regulation of the Hnf4a locus S-curve scatterplot with genes ranked by their average fold changes between STem cell Ascl2 Reporter (STAR) negative over STAR positive is plotted. HNF4G is highlighted in red. Average fold change is calculated from the colorectal tumor progression models and colorectal cancer patients reported in Oost et al (2018).Transcription factors ranked by their normalized enrichment score in the promoters of significantly downregulated genes (P < 0.001) in the STAR-positive samples from (A) were shown. Normalized enrichment score (NES) and motif association were determined in iRegulon (Janky et al, 2014).Genome browser windows over the complete Hnf4a locus showing all data and all used transcript databases.
Fig 5: HNF4G promotes NRP1 transcription by binding its promoter region in glioma cells. (A) The HNF4G binding site in the NRP1 promoter was analyzed using the UCSC Genome Browser. (B) Chromatin immunoprecipitation-reverse transcription-quantitative PCR confirmed that HNF4G bound to the promoter region of NRP1 in LN229 and U251 cells. (C) Luciferase activity in LN229 and U251 cells 2 days after transfection with pGL3-NRP1-luc. Luciferase activity in LN229 and U251 cells after co-transfection with (D) pGL3-NRP1-luc and HNF4G overexpression vector, and (E) pGL3-NRP1-luc and HNF4G siRNA. (F) NRP1 mRNA expression was significantly upregulated in glioma samples compared with that in normal tissues. (G) A notable positive association was detected between HNF4G and NRP1 mRNA expression in glioma tissues. (H) The Cancer Genome Atlas data revealed that HNF4G expression was positively associated with NRP1 expression in glioma. (I) NRP1 mRNA expression in glioma cells transfected with HNF4G overexpression vector or siRNA. (J) NRP1 mRNA expression in xenograft tumors. NRP1 protein expression in glioma cells transfected with (K) HNF4G overexpression vector and (L) HNF4G siRNA. (M) NRP1 protein expression in xenograft tumors. *P<0.001 vs. respective control, n=3. HNF4G, hepatocyte nuclear factor 4?; NRP1, neuropilin-1; luc, luciferase; ov, overexpression; siRNA, small interfering RNA; NC, negative control; sh-Ctrl, short hairpin control; shRNA, short hairpin RNA.
Supplier Page from MilliporeSigma for Anti-HNF4G antibody produced in rabbit