ieCtnnb1 is an intestinal enhancer of Ctnnb1.

(A) Schematic representation of the upstream region of mouse Ctnnb1 gene and the location of ieCtnnb1 (6,399 bp, pink shading), which is marked by H3K27ac and H3K4me1 peaks, and DNase I hypersensitivity in small intestine and large intestine of 8-week-old mice. The sequence conservation of the indicated species is shown at the bottom as vertical lines. Data were obtained from ENCODE. Locations of single-guide RNAs (sgRNAs) for generating ieCtnnb1 knockout mice were marked. (B) Top left: a schematic illustration showing that the knock-in reporter construct carries the Shh promoter, ieCtnnb1 core region sequences (2,153 bp) and the LacZ reporter gene. Top right: X-Gal staining (blue) of the gastrointestinal (GI) tract of an 8-week-old H11i.enh mouse. Bottom: X-Gal staining (blue) of the small intestine (left) and colon (right) of an 8-week-old H11i.enh mouse. Boxed areas were enlarged at top-right corners. (C) Representative images of small intestinal crypts co-labelled by X-gal with OLFM4 (left), and X-gal with Lysozyme (right). (D-E) Representative images of whole body (d) and GIs (e) of 8-week-old male wildtype (WT) and Ctnnb1Δi.enh mice. (F) Comparison of the body weight of 8-week-old male WT (n = 13) and Ctnnb1Δi.enh (n = 13) mice. (G-H) Measurements of small (G) and large (H) intestine length of 8-week-old male WT (n = 6) and Ctnnb1Δi.enh (n = 6) mice. (I-J) Relative mRNA levels of Ctnnb1 in small (I) and large (J) intestinal crypts of WT (n = 6) and Ctnnb1Δi.enh (n = 6) mice. (K-L) Left: immunoblotting of nuclear (K) and cytoplasmic (L) β-Catenin, GAPDH and H3 of small intestinal crypts of WT (n = 3) and Ctnnb1Δi.enh (n = 3) mice. Right: histograms showing protein levels of β-Catenin normalized to H3 (K) or GAPDH (L) levels. Values of WT were set as ‘1’. (M) Heat map of indicated Wnt target genes and GSEA analysis of Wnt signaling pathway according to transcriptome profiles of small intestinal crypts of WT (n = 3) and Ctnnb1Δi.enh (n = 3) mice. (N) RT-qPCR showing relative mRNA levels of indicated Wnt target genes (Axin2, Lgr5 and Mmp7) in small intestinal crypts of WT (n = 3) and Ctnnb1Δi.enh (n = 3) mice. Scale bars, 1 cm (B, top; D and E), 100 μm (B, bottom), 10 μm (B, magnified views; C). Quantification data are shown as means ± SEM, statistical significance was determined using an unpaired two-tailed Student’s t-test (F-L). Quantification data are shown means ± SD, statistical significance was determined using Multiple t-tests - one per row (N). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant. NES: Normalized Enrichment Score.

ieCtnnb1 knockout altered cellular composition and expression profiles of small intestinal crypts.

(A) Schematic illustration of single-cell sequencing. Crypts were extracted from small intestines followed by fluorescence activated cell sorting (FACS) to enrich EpCAM+DAPI-epithelial cells. Cells of two 10-week-old female mice for each genotype were pooled together to perform single cell transcriptome analyses. (B) UMAP were used to visualize the clustering of 11,824 single cells from H11i.enh mice and 8,094 single cells from Ctnnb1Δi.enh mice. Cell types were assigned according to expressions of marker genes. CBC, crypt base columnar cell; TAC, transit amplifying cell; EEC, enteroendocrine cell; imEC, immature enterocytes; mEC, mature enterocytes; GC, goblet cell; PC, Paneth cell; TC, tuft cell. (C) Expression and distribution of Ctnnb1 in small intestinal crypt cells of H11i.enh and Ctnnb1Δi.enh mice. (D) Violin plots showing the expression of Ctnnb1 in CBC, TAC, PC, GC, imEC, mEC; and the expression of Axin2 in CBC and TAC, of H11i.enh and Ctnnb1Δi.enh mice. (E) Comparison of the proportion of indicated small intestinal crypt cell types in H11i.enh and Ctnnb1Δi.enh mice. (F) Immunohistochemistry (left and middle) and quantification (right) of PCs in small intestines of H11i.enh (n = 6) and Ctnnb1Δi.enh (n = 6) mice. (G) RT-qPCR showing relative mRNA levels of PC marker genes (Lyz1, Pla2g2a, Wnt3, Math1) in small intestinal crypts of H11i.enh (n = 3) and Ctnnb1Δi.enh (n = 3) mice. (H) Distribution of G2M cells in H11i.enh and Ctnnb1Δi.enh small intestinal crypts, based on the expression of cell cycle marker gene Mki67. (I) Comparison of the proportion of G2M cells in CBC and TAC of H11i.enh and Ctnnb1Δi.enh small intestinal crypts. (J) Violin plots showing the expression of Mki67 in CBC and TAC of H11i.enh and Ctnnb1Δi.enh small intestinal crypts. (K) Immunohistochemistry (left and middle) and quantification (right) of Ki67+ cells in small intestines of H11i.enh (n = 6) and Ctnnb1Δi.enh (n = 6) mice. (L) Immunofluorescence (left and middle) and quantification (right) of EdU+ cells (red) in small intestines of H11i.enh (n = 6) and Ctnnb1Δi.enh (n = 6) mice after 4 hours EdU injection. Nuclei were labeled with DAPI (blue). (M-N) Violin plots showing expressions of marker genes for secretory cells (Lyz1, Ang4, Muc2, Mmp7) and absorptive cells (Fabp6, Apoa1, Fabp2, Reg3b) in secretory(M) and absorptive lineages (N) of H11i.enh and Ctnnb1Δi.enh small intestinal crypts. Scale bars, 50 μm (F, K, and L). Quantification data are shown as means ± SEM, statistical significance was determined using an unpaired two-tailed Student’s t-test (D, F, J, K, L, M and N). Quantification data are shown means ± SD, statistical significance was determined using Multiple t-tests - one per row (G). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Knocking out ieCtnnb1 inhibits tumorigenesis of colorectal cancer.

(A) Survival of ApcMin/+ (n = 15) and Ctnnb1Δi.enh;ApcMin/+ (n = 17) mice. (B) Colon images of 5-month-old ApcMin/+ (n = 9) and Ctnnb1Δi.enh;ApcMin/+ (n = 9) mice. (C) Weight statistics of 5-month-old ApcMin/+ (n = 20) and Ctnnb1Δi.enh;ApcMin/+ (n = 20) mice. (D) The statistical analysis of tumor counts in colons of 5-month-old ApcMin/+ (n = 9) and Ctnnb1Δi.enh;ApcMin/+ (n = 9) mice. (E) Representative H&E staining images of colon sections of 5-month-old ApcMin/+ and Ctnnb1Δi.enh;ApcMin/+ mice. (F) The statistical analysis of colon tumor area in 5-month-old ApcMin/+ (n = 6) and Ctnnb1Δi.enh;ApcMin/+ (n = 6) mice. (G-H) Immunohistochemistry (G) and quantification (H) of Ki67+ cells in colon tumors of 5-month-old ApcMin/+ (n = 6) and Ctnnb1Δi.enh;ApcMin/+ (n = 6) mice. (I-J) Immunohistochemistry (I) and signal intensity statistics (J, red dashed boxes of I) of β-Catenin in colon tumors of 5-month-old ApcMin/+ (n = 6) and Ctnnb1Δi.enh;ApcMin/+ (n = 6) mice. (K-L) Immunohistochemistry (K) and quantification (L) of MUC2+ cells in colon tumors of 5-month-old ApcMin/+ (n = 6) and Ctnnb1Δi.enh;ApcMin/+ (n = 6) mice. (M) The heat map showing relative expressions of Wnt signaling pathway genes of colon tumors from 5-month-old ApcMin/+ (n = 3) and Ctnnb1Δi.enh;ApcMin/+ (n = 3) mice. (N-O) KEGG analyses of downregulated (N) and upregulated (O) genes in colon tumors of 5-month-old ApcMin/+ (n = 3) and Ctnnb1Δi.enh;ApcMin/+ (n = 3) mice. Scale bars, 1 cm (B), 4 mm (E, G, I and K), 200 μm (magnified views in G and K), 100 μm (magnified views in I). Quantification data are shown as means ± SEM, statistical significance was determined using an unpaired two-tailed Student’s t-test (C, D, F, H, J and L) or log-rank analysis (A). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

ieCTNNB1 is the intestinal enhancer of human CTNNB1.

(A) Schematic representation of human CTNNB1 gene and the location of ieCTNNB1 (3,000 bp, pink shading), which is marked by H3K27ac and H3K4me1 peaks, and DNaseⅠ hypersensitivity in human small intestine (30-year-old female) and colon (34-year-old male). Data were obtained from ENCODE. (B) Top: a schematic illustration showing that the knock-in construct containing the Shh promoter, ieCTNNB1 sequences (3,000 bp), and the LacZ reporter gene. Bottom: X-Gal staining (blue) of the gastrointestinal tract, and sections of the proximal small intestine, distal small intestine, and large intestine in 8-week-old H11hi.enh mice. (C) Left: ieCTNNB1 is marked by enrichment of H3K27ac in human small intestine (30-year-old female) and colon (34-year-old male). Data were obtained from ENCODE. Locations of sgRNA target sites were indicated. Five subregions of ieCTNNB1 were shown. Right: luciferase reporter assay in HCT116 cells transfected with indicated plasmids for 48 hours. (D) RT-qPCR showing relative mRNA levels of CTNNB1 in HCT-15 cells transfected with indicated CRISPR activation or CRISPR interference vectors for 48 hours. Scale bars, 1 cm (whole mount in B), 100 μm (sections in B), 10 μm (magnified views in B). Quantification data are shown as means ± SEM, statistical significance was determined using one-way ANOVA analysis (C) and an unpaired two-tailed Student’s t-test (D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

ieCTNNB1 is activated in colorectal cancer and its activity positively correlates with the expression of CTNNB1.

(A) Schematic representation of ieCTNNB1 (pink shading) and CTNNB1 promoter (yellow shading), which is respectively marked by H3K27ac and H3K4me3 peaks, and mRNA signals in native and tumor tissues of a patient with colorectal cancer. The location of risk mutation site was indicated. (B) Comparison of CTNNB1 expression levels in native and tumor tissues of colorectal cancer patients (n = 68). (C) Left: comparison of H3K27ac signals at ieCTNNB1 in native and tumor tissues of colorectal cancer patients (n = 64). Right: comparison of H3K4me3 signals at CTNNB1 promoter in native and tumor tissues of colorectal cancer patients (n = 42). (D) Left: correlation between H3K27ac signals at ieCTNNB1 and CTNNB1 expression in native and tumor tissues of colorectal cancer patients (n = 55). Right: correlation between H3K4me3 signals at CTNNB1 promoter and CTNNB1 expression in native and tumor tissues of colorectal cancer patients (n = 38). (E) Left: comparison of CTNNB1 expression in esophagus between subjects with common sequence (C/C, n = 428) and variant sequence (C/T, n = 37). Right: comparison of CTNNB1 expression in transverse colon between subjects with common sequence (C/C, n = 343) and variant sequence (C/T, n = 25). (F) Luciferase reporter assay in HCT116 and HeLa cells transfected with indicated plasmids for 48 hours. Quantification data are shown as means ± SEM, statistical significance was determined using a paired (B, C and D) or unpaired (E) two-tailed Student’s t-test and Two-way ANOVA analysis (F). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant. R: Pearson correlation.

HNF4α and p-S133-CREB1 associate with ieCTNNB1 to regulate CTNNB1’s transcription.

(A) ChIP-seq tracks of indicated trans-acting factors enriched at ieCTNNB1 (pink shading) and CTNNB1 promoter (grey shading) in indicated colorectal cancer cell lines. Shaded regions were enlarged in C (pink) and D (grey) respectively. (B) RT-qPCR showing relative mRNA levels of CTNNB1 in HCT-15 cells transfected with indicated shRNA-expressing plasmids for 48 hours. The expression level of CTNNB1 in cells transfected with scramble (scr) shRNA was set to ‘1’. (C-D) Top: schematic diagram showing the enrichment of p-S133-CREB1 at ieCTNNB1 (C) and CTNNB1’s promoter (D). Locations of HNF4α and CREB1 binding motif sites were indicated. Middle and bottom: ChIP-qPCR showing enrichment of HNF4α (middle), CREB1 and p-S133-CREB1 (bottom) at ieCTNNB1 (C) and CTNNB1’s promoter (D) in HCT-15 cells. Locations of ChIP regions were indicated. (E) Comparison of expression levels of CREB1 and HNF4A between native and tumor tissues in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) tumors. (F) Correlations between the expression level of CTNNB1 and those of CREB1 or HNF4A in COAD tumors. (G-H) ChIP-qPCR showing enrichment of HNF4α and p-S133-CREB1 (H) at Ctnnb1 promoter in native colon tissues of WT (n= 3) mice, tumor tissues of ApcMin/+ (n= 3) mice and Ctnnb1Δi.enh;ApcMin/+ (n= 3) mice. Quantification data are shown as means ± SEM, statistical significance was determined using one-way ANOVA analysis (B), unpaired two-tailed Student’s t-test (E and F) and Multiple t-tests - one per row (C, D, G and H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant. R: Pearson correlation.