Extended Data Fig. 9: HNF4A mediates Atrx loss phenotypes.

a) PCA plot of ATAC-seq data comparing AKP Control vs AKP Atrx^KO organoids, n = 3 vs 3 independent experiments. (b) Representative IGV browser tracks of ATAC peaks at colonic epithelial genes. Significantly altered peaks are outlined with a red box. Blue track refers to AKP control, while red track refers to AKP Atrx^KO organoids. (c) Representative IGV browser tracks of ATAC peaks at mesenchymal genes. Significantly altered peaks are outlined with a red box. Blue track refers to AKP control, while red track refers to AKP Atrx^KO organoids. (d) Representative IGV browser tracks of ATAC peaks at squamous genes. Significantly altered peaks are outlined with a red box. Note lack of significantly different peaks at Krt5 and Ly6d despite altered expression. Blue track refers to AKP control, while red track refers to AKP Atrx^KO organoids. (e) Heatmap showing transcription factor binding sites enriched in regions that lose accessibility in AKP Atrx^KO organoids. Brown shade gradient indicates regions where accessibility is lost in AKP Atrx^KO organoids. (f) PCA plot of H3K27ac CUT&RUN comparing AKP Control vs AKP Atrx^KO organoids, n = 3 vs 3 independent experiments. (g) RT-qPCR analysis of colonic epithelial cell marker expression in AKP Control, AKP Atrx^KO and AKP Atrx^KO Hnf4a overexpressing organoids, n = 3 vs 3 independent experiments. (h) RT-qPCR analysis of Hnf4α in AKP Control vs AKP Hnf4a^KO organoids, n = 3 vs 3 independent experiments (i) Western-blot analysis of AKP Control and AKP Hnf4a^KO organoids for HNF4A and β-actin. n = 4 vs 4 technical replicates. (j) Volcano plot depicting differentially expressed genes in AKP Control vs AKP Hnf4a ^KO organoids following RNAseq analysis. Red dots represent genes expressed at higher levels in the AKP Hnf4a ^KO organoids, while blue dots represent genes expressed at higher levels in the AKP Control, n = 3 vs 3 technical replicates. (k) Representative H&E images of subcutaneous tumours in mice subcutaneously injected with AKP Control or AKP Hnf4a^KO organoid cells, n = 5 vs 5 mice. Magnification highlights the distinct morphological features. Scale bars, 1 mm overview and 250 µm zoom. (l) Quantification of non-glandular morphology area of AKP Control vs AKP Hnf4a^KO subcutaneous tumours, n = 5 vs 5 mice. For (e) p values were calculated using one-sided Fisher’s exact test. For (g) and (h) gene expression was normalised to Actb and levels relative to AKP Control calculated using the ΔΔCt method. Data are mean ± SD. For (g) p values were calculated using one-way ANOVA with multiple comparisons test. For (h) p value was calculated using two-tailed student’s ttest. For (l) data are mean ± SD. P values were calculated using two-sided Mann-Whitney test.

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