Fig 2: AHR drives termination of colonic epithelial regeneration.a Representative images showing Sca-1 (green) expression and proliferative (EdU+, red) cells in regenerative foci in the colon of DSS-treated Vil-cre AHRfl/fl and WT control mice. Regenerative foci are identified as regions with epithelial Sca-1+ expression and significant crypt dysplasia as well as proliferating cells (EdU; 2 h post-i.p injection). b Measurement of mucosal and submucosal thickness and c Sca-1 MFI at d0, d12 and d30 post-DSS treatment. (d) Representative images showing expression of differentiation markers Muc2 (goblet cell, red) and Krt20 (pan-differentiation marker, red) in epithelial cells co-stained with Sca1 (green) and DAPI (blue) within regenerative foci in the colon of Vil-cre Ahrfl/fl mice and WT controls at d30 post-DSS challenge. Quantifications for (b, c) were done measuring mean ± SD distances or mean MFI ± SD per mouse. The data-points represent n = 3 (d0), n = 4 (d12, d30) WT control mice and n = 3 (d0), n = 5 (d12), n = 7 (d30) Vil-cre AHRfl/fl mice). Statistical significance between genotypes per timepoint was determined by Multiple t-test. P value of >0.05 was considered not significant (n.s.). Data-points for (d) represents n = 3 mice per genotype and quantifications graphed are mean MFI ± SD per mouse; an unpaired t-test (two-tailed) was used to assess statistical significance. Representative images show Sca-1 (green) expression and proliferative (EdU+, red) cells in regenerative foci found in the colon of Vil-cre R26LSL-Cyp1a1 and WT control mice (e) during the course of DSS. DAPI staining (blue) was used to identify nuclei in all images. Data is representative of at least 2–3 independent experiments. Scale bars: 100 µm. Source data for (1b–d) are provided with this paper in the source data file.

Fig 3: AHR restricts genomic accessibility to Yap/Tead targets.a Deeptools heatmap showing accessibility data of differentially expressed peaks identified in d4 ENR AHR KO vs WT organoids across samples (FDR < 0.05) (b) GO gene ontology (biological process) was conducted on genes that overlap between differentially accessible targets identified by ATAC-seq and differentially upregulated or downregulated genes in AHR KO vs WT organoids grown in D4 ENR conditions as determined by RNA-seq (FDR < 0.05). Statistical significance was determined by permutation testing of the overlap between the datasets (p value <0.00001). c IgV images of accessibility peaks (open regions) of known Yap/Tead targets (Ctgf, Cyr61, Amotl2) and fetal-like genes (Clu, Tacstd2, Ly6a) in d4 ENR WT and AHR KO organoids (d) Pairwise comparison of TF activity between differentiated AHR KO and WT organoids (d4 ENR). The volcano plots show the differential binding activity against the –log10 (p value) of all investigated TF motifs represented by a dot. Only the top 5% (both by fold-change, -log10 p value) of TF motifs identified are highlighted in red. TFs belonging to Tead and Ets-1 like family of factors are bolded. e Motif sequences of some identified TFs belonging to Tead (Tead 3, Tead4, Tead1) and Ets-1 family are shown, and the dotted line highlights the similarity in the binding sequence of these factors (f) Top 5% of identified motifs were aligned to each other and matched to an archetypal consensus motif. The color represents the fold-change (Red to grey – highest to lowest) in differential binding score between d4 AHR KO vs WT organoids. Source data for (4b, d) are provided with this paper in the source data file.

Fig 4: Tipe0-/- mice have inappropriate basal activation of the injury-response stem cell program, but an inability to induce the program to regenerate.a Sca-1 staining in Tipe0-/- spheroids generated by depriving growth factor withdrawal; bars = 130 µm; insert 3× magnified. Representative of 3–4 Tipe0-/- spheroids/experiment, with three independent replicates. b Flow cytometry for Sca-1+/Epcam+ cells. N = 3, representative of 2 independent experiments. p = 0.0005 two-tailed t-test with Welch's correction vs WT. c IF for Sca-1 and E-cadherin in healthy WT and Tipe0-/- tissue. E-cadherin was used as an enterocyte counterstain. Images representative of the mean intensity in 4 mice/group, with two fields of view/mouse randomly selected; bars = 130 µm; arrows mark enterocytes staining for Sca-1; insert 3× magnified. Sca-1 staining linearly enhanced in both images for easier merged channel visualization. d Quantification of enterocyte Sca-1 fluorescence intensity from c; N = 4 mice/group; p = 0.0286. e Sca-1 and E-cadherin staining from mice given DSS, with quantitation (f). E-cadherin is an used as an enterocyte counterstain. Quantification of total Sca-1 signal was by relative fluorescence intensity (RF), normalized to WT. N = 7 crypts/per mouse from 4 mice/group. Images representatives 3/4 mice evaluated for each group; bars = 70 µm; insert 3× magnified. g Colonic Sca-1 levels by RT-PCR. N = 7\(WT and Tipe0-/- healthy), N = 8 (WT DSS), N = 6 (WT and Tipe0-/-). Multiple group comparisons were by Kruskal–Wallis one-way ANOVA. Two-tailed Mann–Whitney U test was used to confirm ANOVA findings. h YAP staining in mice given DSS; bars = 70 µm, insert bars = 20 µm. Arrows show areas of high nuclear colocalization. Images representative of eight WT and five Tipe0-/- mice evaluated. i Quantification of nuclear YAP from h by Intensity Correlation Quotient (ICQ). N = 8 (WT), 5 (Tipe0-/-); p = 0.0025. j WB for pYAPS127 and YAP, with densitometry (N = 4/group); p = 0.0102 for YAP, p = 0.0021 for pYAPS127 by one-sample two-tailed t-test. For all graphs, *p < 0.05; **p < 0.01; p < 0.001; ****p < 0.0001 vs WT, or as indicated; error bars show mean ± SEM. Analyses done by two-tailed Mann–Whitney U test unless otherwise specified. Source data are provided as a source data file.

Fig 5: Enrichment for Yap/Tead targets in d4 ENR AHR KO organoids.a Enrichment plot for transcriptional signature of d4 ENR AHR KO organoids compared to the Cordenonsi Yap1 conserved signature dataset (GSEA C6: Oncogenic datasets - NES: 2.95, FDR q value: 0.00) (b) Heatmap of select Yap1 targets and fetal-like genes differentially expressed in d4 ENR AHR KO organoids; expression of YAP1 target Ctgf, Cyr61 and Ankrd1 validated by qPCR. c expression of Lgr5, Ascl2 and Sox9 by qPCR. d Representative flow cytometry plots and percentage of cells expressing high levels of surface Sca-1 (e) qPCR expression of Yap targets in WT and AHR KO organoids in WENR conditions after 24 h and 4 h of FICZ-stimulation, respectively. f qPCR expression of Ctgf and colonocyte differentiation marker Slc26a3 in organoids grown in matrigel containing either 0% or 60% Collagen I. Cells were stimulated with either DMSO (vehicle) or FICZ and expression was assessed 24 h post-treatment. g qPCR expression of Ctgf and Ankrd1 in WT and AHR KO organoids grown in 60% Collagen I (d4 ENR) treated with either 1 µM or 10 µM of ROCK-inhibitor (Y-27632). Organoids used for experiments were generated from either n = 3 (i.e. Lgr5, Sox9, Ankrd1, Ctgf) or n = 4 (i.e. Cyr61, Ascl2) mice per genotype. Data-points for the collagen and Y-27632 experiments are generated from n = 4 and n = 3 mice per genotype respectively. Statistical significance was determined by (a, b) unpaired t-test (two-tailed) (c–f) two-way ANOVA with Sidak’s multiple comparison test, (g) statistical differences between treatment and DMSO control per genotype was done using an ordinary one-way ANOVA with Dunnett’s multiple comparison test. P value of >0.05 was considered not significant (n.s.). Error bars displayed on graphs represent the mean ± SD of at least two independent experiments. Source data for (3b–g) are provided with this paper in the source data file.