Crypt density and recruited enhancers underlie intestinal tumour initiation

Abstract

Oncogenic mutations that drive colorectal cancer can be present in healthy intestines for long periods without overt consequence^1,2. Mutation of Apc, the most common initiating event in conventional adenomas³, activates Wnt signalling, thus conferring fitness on mutant intestinal stem cells (ISCs)^4,5. Apc mutations may occur in ISCs that arise by routine self-renewal or by dedifferentiation of their progeny. Although ISCs of these different origins are fundamentally similar^6,7, it is unclear whether both generate tumours equally well in uninjured intestines. It is also unknown whether cis-regulatory elements are substantively modulated upon Wnt hyperactivation or as a feature of subsequent tumours. Here we show in two mouse models that adenomas are not an obligatory outcome of Apc deletion in either ISC source, but require proximity of mutant intestinal crypts. Reduced crypt density abrogates, and aggregation of mutant colonic crypts augments, adenoma formation. Moreover, adenoma-resident ISCs open chromatin at thousands of enhancers that are inaccessible in Apc-null ISCs that are not associated with adenomas. These cis elements explain adenoma-selective gene activity and persist, with little further expansion of the repertoire, as other oncogenic mutations accumulate. Thus, cooperativity between neighbouring mutant crypts and new accessibility at specific enhancers are key steps early in intestinal tumorigenesis.

Extended Data Fig. 1 |. Atoh1^Cre(ER) (Sec-Cre) mice capture routine Sec dedifferentiation into conventional homeostatic Lgr5⁺ ISCs.

a, Crypt-villus units from a representative Sec-Cre;R26R^Tom (Apc^+/+) ileum 1 day PCI (N=8 mice), showing labelled villus Sec and crypt base Paneth cells. Ten days PCI, nearly every villus carried scattered goblet cells and strips of consecutive tdTom⁺ enterocytes, which represent the progeny of dedifferentiated Atoh1^Cre labelled ISCs competing neutrally with unlabelled ISCs (N=3 mice). Three weeks PCI, tdTom label persisted only in long-lived Paneth cells and occasional whole crypt-villus units (N=5 mice), reflecting clonal fixation of dedifferentiated Atoh1^Cre-marked ISCs. Scale bars 50 μm. b, Labelled clonal ribbons were present 100 days PCI, indicating that dedifferentiated and clonally fixed ISCs are long-lived. N=3 mice. Scale bars: left 1 mm, right 50 μm. c-d, Single-cell RNA analysis of ileal Sec-Cre;R26R^Tom crypt cells captured by tdTom flow cytometry 0.5 and 1.5 days (combined) PCI, showing a substantial fraction of tdTom⁺ cells that uniquely express classic ISC markers: Lgr5, Axin2, Olfm4, and others shared with replicating Atoh1⁺ Sec progenitors (Sec-pro): Top2a and Mki67. The findings confirm that Sec-cell dedifferentiation is a routine homeostatic event. Ent-pro, enterocyte progenitors; Gob/Pan (GP), goblet/Paneth precursors; EE, enteroendocrine. e, Higher labelling of whole crypts in ISC-Cre (N=4 mice) than in Sec-Cre (N=6 mice) Apc^+/+ (WT) ilea. Bars, mean ±SD; unpaired t test with Welch’s correction, one-tailed). f, Adenomas carpeted ISC-Cre duodenum 3 weeks PCI, necessitating euthanasia (N=12 mice). Scale bar 1 mm.

Extended Data Fig. 2 |. Different tumour loads in ISC-Cre and Sec-Cre Apc^Fl/Fl ileum.

a, Distributions of clonal events in ISC-Cre and Sec-Cre Apc^+/+ and Apc^Fl/Fl ilea. N, mouse numbers. P values from comparisons between Apc^Fl/Fl ilea in ISC-Cre vs. Sec-Cre derived using unpaired t test with Welch’s correction (one-tailed). b, Photomicrograph of an Apc^−/− Sec-Cre ileum 16 weeks PI, showing a single diminutive adenoma (dashed rectangle, magnified in inset). Scale bar 1 mm, inset 100 µm. c, ISC-Cre Apc^−/− ileal adenoma showing that S-phase (EdU⁺) cells are not confined to crypts, as in neighboring Apc^+/+ tissue, but present throughout the tumour. N=4 mice, scale bar 50 μm. d, Illustrative tdTomato⁻ microadenoma (dashed oval, 1 example from N=2 mice) in Sec-Cre ileum, reflecting Cre-induced recombination at the Apc but occasionally not at the Rosa26 locus. Bar 50 μm. e, PCR genotyping of genomic DNA for Apc exon 14 shows the excised (floxed, 240 bp) product in FACS-purified ISCs (Tom⁺ cells) from ISC-Cre or Sec-Cre mice, but not in Tom⁻ cells. A larger non-specific PCR product is amplified in all samples. N=2 independent isolates. Whole gel is shown. f, Axin2 RNA in situ hybridization shows expression as a marker of active Wnt signaling in proliferative normal crypt cells and elevated levels in scattered cystic structures (arrows) in Sec-Cre;Apc^fl/fl ileum. N=3 intestines. Scale bar 1 mm; boxed area is magnified below, scale bar 50 μm. g, PCR genotyping of genomic DNA for Kras^G12D (top, N=3 mice) and null Smad4 (right, N=2 mice) alleles verifies Cre-mediated recombination in FACS-purified ISCs (Tom⁺ cells) but not in DNA extracted from toes (top N=3 mice, bottom N=2 mice), which lack Cre recombinase. Source gels shown in Supplementary Figure 1.

Extended Data Fig. 3 |. Gene deregulation in Apc-null ISCs and organoids.

a, Representative FACS plots for purification of Apc^−/− ISCs (GFP⁺ Tom⁺) from ISC-Cre;R26R^Tom mice (left, n >10) and from Sec-Cre;R26R^Tom mice that also carry the Lgr5^Dtr-Gfp allele for fluorescent ISC marking (right, n >10). ISCs reflecting dedifferentiation of Atoh1^Cre labelled Sec cells represent 1.7% of viable cells in Sec-Cre mice 6 weeks PCI. Supplementary Fig. 2 shows the FACS gating strategy. b, Apc-null adenomas in ISC-Cre contain a high proportion of GFP⁺ (Lgr5⁺) ISCs, extending well beyond the crypt base. N=8 mice. Left, merged fluorescent signals; right, isolated GFP (Lgr5) signal. Scale bar 50 μm. c, Aberrant expression of 3,286 genes (RNA-seq, DESeq2 analysis, two-tailed Wald test corrected for multiple comparisons, q <0.01, log₂ fold-change >2, base mean >20 counts) in Apc^−/− ISCs isolated from ISC-Cre mice compared to Apc^+/+ (WT, from mice lacking Cre) ISCs, both isolated 14 days PCI. d, Additional example of Notum expression (RNA in situ hybridization) in Sec-Cre ileal clones (N=4 mice, scale bar 50 μm). e, Wild-type (Apc^+/+) organoids are budding structures, distinct from the spheroidal morphology observed in Apc-null organoids. N=5 independent cultures, scale bar 1 mm. f, Aberrant gene expression in Apc^−/− organoids in vitro overlaps with genes dysregulated in Apc^−/− ISCs isolated from ISC-Cre mice. Table shows examples of genes dysregulated (average DESeq read counts from N=2 mice each) in ISCs from ISC-Cre mice and their relative expression in ISCs isolated from WT mice, Apc^−/− ISCs isolated from Sec-Cre mice (in vivo), and organoids cultured from each source of small intestine crypts (N=2 independent cultures from each). g, RNA in situ hybridization shows Notum (top, N=2 mice) and Spock2 (bottom, N=2 mice) expression predominantly in ISC-Cre adenomas. Scale bars 50 μm; dashed lines demarcate crypt bottoms. h, Context-dependent consequences of Apc loss. In gene activity and absence of adenomatous features, ISCs from Sec-Cre behave like WT ISCs in vivo but their expansion in organoid medium lacking RSPO phenocopies their ISC-Cre counterparts.

Extended Data Fig. 4 |. Monte Carlo simulations of crypt clustering.

a, Variegated Lgr5^Cre expression in ISC-Cre mice and sporadic dedifferentiation in ISC-Cre mice result in areas with variably dense (top, 5 days PCI, N=6 mice) and sparse (bottom, N=15 mice) tdTom labelling, respectively. Scale bars 50 μm. b, Top: the relation between crypt clustering (y-axis, values ±SD) and basal rates of crypt labelling (x-axis) is non-linear. Bottom: derivatives of the clustering rates. The largest change in clustering rate occurs between the labelling rates observed in ISC-Cre and Sec-Cre ileum. c, Simulation of crypt clustering in silico. Using normal distributions fit to the observed rates of crypt labelling, sequential crypts with randomly selected cells were represented by vectors, given the prescribed rate of labelling, and simulated in silico. Distributions of clustered crypts were generated from 100,000 iterations for each dataset. d, The cluster distribution of Sec-Cre crypts (top) fell at the low end of the range expected from random labelling. In contrast, ISC-Cre mouse ilea (bottom) showed more clustering than predicted from random basal crypt labelling, as expected from the known patchiness of variegated Lgr5^Cre expression. Dark grey: 5^th to 95^th percentile range, light grey: maximum range for the simulated data. Cumulative frequencies displayed together for comparison in Fig. 2f. e, Whole mount fluorescence micrograph of the largest crypt cluster detected in a Sec-Cre ileum (14 weeks PCI, example from N=4) and showing adenomatous features: extension beyond the tissue plane and effaced crypt boundaries. Scattered isolated crypts appear on the periphery. Whole-mount fluorescence image from Apc^Fl/Fl ISC-Cre ileum 16 days PCI shows an adenoma (dashed yellow structure: extra-planar growth, fused dysmorphic crypts) among variably sized patches of untransformed Apc-null crypts with distinct borders. N=4 mice, scale bars 1 mm.

Extended Data Fig. 5 |. Perturbations of crypt clustering in Sec-Cre mice.

a, Whole-mount images of representative ISC-Cre ilea 5 days and 15 days PCI demonstrate dense, patchy labelling correlated with adenomas (15 days PCI), compared to diffusely scattered singletons observed in Sec-Cre as late as 14 weeks PCI. Bars 1 cm. Below, tdTom fluorescence signals in ISC-Cre whole mounts (5 days PCI, N=4 ilea) are greatest in the distal 2 cm, correlating with tumour size and abundance at euthanasia (2–3 weeks PCI, N=4 ilea). b, Increased rates of clonal crypt marking in Sec-Cre;Apc^Fl/Fl mice with 12 injections of Tam or by ablating Lgr5^DTR-Gfp ISCs with Diphtheria toxin (DT), hence increasing Sec cell dedifferentiation. N, mouse numbers; first two columns repeated from Fig. 1d; unpaired t test with Welch’s correction, one-tailed. c, Upon elevation of basal crypt labelling with 12 doses of Tam (top) or DT treatment (bottom), Monte Carlo simulations showed more doublets than expected from random crypt distributions (dark dots in right graph), but larger clusters remained rare. Cumulative frequencies displayed together for comparisons in Fig. 3b; dark grey shading: 5^th to 95^th percentile range, light grey: maximum range for simulated data. d, High TdTom labelling in Sec-Cre colon, owing to large number of native Sec cells available to dedifferentiate. N=6 colons, scale bar 1 mm. e, High adenoma burden after 12X Tam in a representative Sec-Cre;Apc^Fl/Fl colon from N=8 mice. Scale bar 1 mm, boxed area magnified below (scale bar 150 μm).

Extended Data Fig. 6 |. Perturbation of crypt clustering in ISC-Cre mice.

a, PCR genotyping of genomic DNA for Apc exon 14 shows the excised product (240 bp) in FACS-purified ISCs (Tom⁺ cells) from ISC-Cre mice treated with 0.1 mg Tam (N=2 mice) but not in ISCs purified from animals that did not receive Tam (N=2 mice). Purified Tom⁻ and Tom⁺ crypt cell fractions from mice treated with 1 mg Tam (Extended Data Fig. 2g) serve as negative and positive controls. Whole gel is shown; a larger non-specific PCR product is amplified in all samples. b, In situ hybridization revealed Notum expression in clonal structures in ISC-Cre;Apc^fl/fl ileum after 2 doses of 0.1 mg Tam (N=2 mice). Top: low magnification, bottom (2 images): high magnification; all scale bars 50 μm. c, Monte Carlo simulations revealed high clustering after ISC-Cre mice were treated with 0.1 mg Tam, as expected from the underlying patchy variegation, but cluster sizes were substantially reduced compared to mice treated with 1 mg Tam. Cumulative frequencies compared with others in Fig. 3g; dark grey: 5^th to 95^th percentile range, light grey: maximum range for simulated data. Note different y-axis ranges for 1 mg and 0.1 mg treatments; circled dots: measured clustering frequencies. d, Sparse crypt labelling detected in ISC-Cre Apc^Fl/Fl ileum whole mounts 5 days PCI with 0.1 mg Tam. N=3 mice, scale bar 1 cm. e, Whole ISC-Cre;Apc^Fl/Fl ileum 8 weeks (N=4 mice) and 23 weeks (N=2 mice) PCI after 0.1 mg Tam, showing small discrete adenomas (dashed ovals) in one of the latter ilea. Scale bars 1 cm.

Extended Data Fig. 7 |. Altered enhancer accessibility in adenoma-resident Apc-null ISCs.

a, Aberrant accessibility at 7,298 enhancers (DESeq2 analysis, log₂ fold-change ≥2, two-tailed Wald test corrected for multiple comparisons, q <0.01) in Apc^−/− duodenal ISCs from ISC-Cre mice, compared to Apc^+/+ (WT, from mice lacking Cre) ISCs, both isolated 14 days PCI. N=2 mice each. b, DNA at enhancers newly accessible in ISCs from adenoma-bearing ISC-Cre mice are hypomethylated (range 0% to 100% methylation in sites containing ≥5 CpGs) relative to the same sites in Apc^+/+ ISCs. Signals representing the full spectrum of DNA methylation are shown for comparison: at 5,000 arbitrary active H3K27ac⁺ enhancers (unmethylated in ISCs), at all CpGs (largely methylated), and at 5,000 neuronal enhancers (unmethylated in neuronal cells but methylated in WT and Apc^−/− ISCs). c, DNA sequence motifs appreciably enriched among enhancers newly accessible in Apc^−/− ISCs match transcription factors from the AP-1, TCF/LEF and FOX0 families. P values derived using the Fisher Exact test (one-tailed, corrected for multiple comparisons). d, Additional representative PyGenome tracks illustrate newly accessible enhancers (asterisks) in a locus (Spock2) with elevated RNA expression in Apc^−/− ISCs. e, Top: differential analysis of enhancers in Apc^−/− ileal ISCs from ISC-Cre mice treated with 1 mg or 0.1 mg Tam, showing differences similar to those across ~100,000 called ATAC-seq peaks (DEseq2, two-tailed Wald test corrected for multiple comparisons, q <0.01, log₂ fold-change ≥2) between WT and adenoma-resident duodenal ISCs. Bottom, alternative display of volcano plot shown in panel a. f, Pearson correlations among called peaks in ATAC-seq analysis of WT or adenoma-resident Apc^−/− duodenal ISCs and of WT or Apc^−/− ileal ISCs from Sec-Cre mice and from ISC-Cre mice treated with 1 mg or 0.1 mg Tam. Black boxes demarcate like samples: WT ISCs (bottom left, showing regional differences); adenoma-resident ISCs (top right, duodenal and ileal); ISCs not associated with adenomas (center, Sec-Cre mice treated with 1 mg Tam and ISC-Cre mice treated with 0.1 mg Tam). g, Differential analysis of enhancers in Apc^−/− ISCs from Sec-Cre mice treated with 1 mg Tam and from ISC-Cre mice treated with 0.1 mg Tam, showing few differences across ~100,000 called ATAC-seq peaks, in contrast to the modulated chromatin access in adenoma-resident Apc^−/− ISCs from ISC-Cre mice (1 mg Tam, panel e). h, Additional PyGenome tracks from ATAC-seq samples represented in Fig. 4d, showing sites selectively accessible (shaded boxes) in adenoma-resident ISCs.

Extended Data Fig. 8 |. Lack of appreciable additional enhancer accessibility when oncogenic mutations are superimposed on Apc-null ISCs.

a, PCR genotyping of genomic DNA for Trp53 deletion reveals the 612-bp product (verified by DNA sequence) expected from Cre-mediated recombination in FACS-purified ISCs (Tom⁺ cells, N=3 independent isolates) but not in DNA extracted from toes (N=3 mice), which lack Cre recombinase. A non-specific PCR product (uninterpretable DNA sequence) was amplified in all samples. Source gel shown in Supplementary Figure 1. b, Minimal expansion of the enhancer repertoire (519 additional accessible sites, log₂ fold-change ≥2, DESeq2, two-tailed Wald test corrected for multiple comparisons, q <0.01,) in adenoma-associated Apc^−/− ISCs upon addition of Kras^G12D mutation (N=4 mice) and no further expansion upon Trp53 loss (N=2 mice) in vivo. Two samples shown for each genotype. c, Principal component (PC) analysis of open chromatin (MACS2 called peaks) in WT (N=4 independent organoid cultures), Apc^−/− (A, N=3 cultures), Apc^−/−;Kras^G12D (AK, N=4 cultures), Apc^−/−;Kras^G12D;Trp53^−/− (AKP, N=2 cultures), and Apc^−/−;Kras^G12D;Trp53^−/−;Smad4^−/− (AKPS, N=3 cultures) intestinal organoids. The largest distinction (PC1) is between WT and all Apc-null organoids. d, K-means clustering (k=3) of 7,574 sites with objectively differential chromatin access in the mutational series compared to WT ISCs. All mutant organoids gave consistent signals across strong (cluster 1: n=701) and moderate (cluster 2: n=2,376) sites. The volcano plot shows that 4,497 sites (cluster 3) with ostensibly enriched access in AKPS organoids fail stringent thresholds for significance (two-tailed Wald test corrected for multiple comparisons). Control sites are 4,000 arbitrary enhancers accessible in all organoids, including WT. e, Significant overlap between enhancers differentially accessible in Apc-null ISCs in vivo and Apc^−/− organoid cultures. Sites are clustered by whether peaks were called only in vivo, only in organoids, or in both. At sites called only in one, signals are evident in the other, but not in WT ISCs or organoids. f, PyGenome tracks for ATAC-seq signals at the Sox17 locus in Apc^−/− ISCs isolated from ISC-Cre crypts in vivo and in organoids cultured from WT or Apc^−/− intestinal crypts. Boxed areas are magnified below to highlight chromatin access in those locations.

Fig. 1 |. Consequences of direct or indirect Wnt activation in ISCs.

a, Apc-null ISC clones (crypts), labelled after Cre expression from the Lgr5 (ISC, direct) or Atoh1 (Sec, indirect by dedifferentiation) locus, engender distinct clonal outcomes: adenomas, small cysts, or crypt-villus ‘ribbons.’ Images represent hundreds of examples (Extended Data Fig. 2a, scale bars 50 μm. b, Cross-sectional and gross images of representative ISC-Cre (21 days PCI, N=8 mice) and Sec-Cre (6 weeks PCI, N=15 mice) Apc^Fl/Fl ileum (center: proximal, periphery: distal). Labelled Paneth cells persist in Sec-Cre, alongside fewer clonal features, including ‘ribbons’ (insets) than in ISC-Cre. Adenomas, abundant in ISC-Cre (especially in distal ileum), were rare in Sec-Cre. Scale bar 1 mm. c, Fractions of ilea with ≥1 adenoma (left, Fisher Exact test, two-tailed) and fractions of Apc-null (Tom⁺) clones (right, Mann-Whitney test, one tailed) in ISC-Cre <3 weeks PCI and in Sec-Cre 6 weeks and 16 weeks PCI. N, mouse numbers. d, Clonal labelling rates in ISC-Cre and Sec-Cre Apc^+/+ and Apc^Fl/Fl ileum. Sec-Cre labelling, increased owing to Apc^−/− fitness, remains lower than direct ISC-Cre labelling. Unpaired t test with Welch’s correction, one-tailed. e, Kras^G12D or Smad4^null mutations did not increase Sec-Cre;Apc^Fl/Fl ileal adenomas (Mann-Whitney test, one-tailed). f, Pearson correlations of transcriptomes in ISCs purified from ISC-Cre and Sec-Cre Apc^+/+ and Apc^Fl/Fl ileum. Tumour-bearing ISC-Cre samples cluster apart from others. g, Differential gene expression (log₂ fold-change >2, q <0.01, Wald test, two-tailed, corrected for multiple comparisons) in ISCs from ISC-Cre and Sec-Cre Apc^+/+ and Apc^Fl/Fl intestines (N=2 replicates each). Duod, duodenum. h, PyGenome tracks for Notum and Sox17 mRNA levels in purified ISCs of indicated genotypes and sources. Scale for Notum in ISC-Cre is 30 times larger than others. i, Comparable Notum expression (RNA in situ hybridization) in ISC-Cre and Sec-Cre ileal clones N=4 mice each, scale bars 50 μm. Bars in all graphs: mean ±SD.

Fig. 2 |. Environmental and spatial contexts for tumourigenesis.

a, Crypts from ISC-Cre or Sec-Cre Apc^Fl/Fl ileum 10 days PCI yielded abundant spheroidal organoids in RSPO-free cultures. Scale bars 1 mm. b, In contrast to transcriptional differences in vivo, Apc^−/− organoid transcriptomes from ISC-Cre and Sec-Cre were similar (Pearson correlations); both expressed 732 genes differently than WT organoids (log₂ fold-change >2, q <0.01, DEseq2, Wald test, corrected for multiple comparisons). c, PyGenome tracks for Notum and Sox17 mRNAs (same scale for all samples) in ISC-Cre or Sec-Cre Apc^+/+ (WT) or Apc^Fl/Fl ileal organoids. d, Abundant Axin2 in a representative ISC-Cre adenoma (in situ hybridization, N=3 mice); WT crypt levels are lower. Dashed line demarcates crypt bottoms; scale bar 50 μm. e, Five days PCI, Sec-Cre showed predominantly solitary Apc^−/− clones, whereas ISC-Cre labelling was clustered, as expected from patchy Lgr5^Cre variegation. TdTom quantified in 3 cross-sections per ISC-Cre (N=6 mice) or Sec-Cre (N=15 mice) Apc^Fl/Fl ileum. ISC-Cre vs. Sec-Cre, p <0.0001, Kolmogorov-Smirnov (K-S) test, two-tailed. f, Cumulative frequencies of clustered crypt distributions. Quantitation in ileal whole mounts confirmed Apc^Fl/Fl crypt clustering in ISC-Cre 5 days PCI (N=3 mice), in contrast to predominance of solitary crypts in Sec-Cre 98 days PCI (N=4 mice). ISC-Cre vs. Sec-Cre, p <0.0001 in 2D tissue sections and 3D whole mounts (note different x-axis ranges for cluster size), two-tailed K-S test. g, Fractions of clustered (≥2 consecutive Tom⁺) crypts in ISC-Cre and Sec-Cre ileal whole mounts (circled dots) relative to simulations (violin plots). Note different y-axis ranges. h, Hypothesis: adenomas arise from sizable patches of mutant crypts in ISC-Cre, not from isolated Apc^−/− crypts or small clusters in Sec-Cre or ISC-Cre.

Fig. 3 |. Adenomas arise mainly from Apc-null crypt aggregates.

a, MKI67⁺ cell numbers per Apc-null (Tom⁺) crypt are increased in large ISC-Cre clusters (≥5 adjacent crypts in 2D sections) compared to small clusters (≤3 contiguous crypts) or Apc^+/+ (WT) crypts. N=3 mice per group, 40 Tom⁺ crypts per mouse, unpaired t test with Welch’s correction, one-tailed. Right: representative immunostains, scale bars 50 µm. b, Increased rates of clonal crypt marking in Sec-Cre;Apc^Fl/Fl mice with 12 injections of Tam or by ablating Lgr5^DTR-Gfp ISCs with Diphtheria toxin (DT, Extended Data Fig. 5b) elicited small increases in cluster size. K-S test, two-tailed. c, These small increases in crypt clustering did not increase the incidence of adenomas. N, mouse numbers; Mann-Whitney test, one-tailed. d, Sec-Cre labels 4.91% of Apc^+/+ (WT) colonic, compared to 0.88% of WT ileal, crypts (N=6 per intestinal region). Unpaired t test with Welch’s correction, one-tailed. e, Tumour incidence in Sec-Cre;Apc^Fl/Fl ileum and colon after 2X Tam (standard), ISC ablation, or 12X Tam. N, mouse numbers; Fisher Exact test, two-tailed. f, Clonal labelling in ISC-Cre ilea treated with 0.1 mg Tam (N=8 mice) is reduced by 67.9% from mice treated with 1 mg Tam (N=6 mice), to approximately the same rate as Sec-Cre (N=13 mice, both compared to Fig. 1d). Unpaired t test with Welch’s correction, one-tailed. g, Reduced labelling decreased crypt clustering, as measured in ISC-Cre ileal 2D tissue sections and whole mounts (note different x-axis scales). N, mouse numbers; P <0.0001 for 1 mg vs 0.1 mg Tam in both measures (K-S test, two-tailed). h, Lack of adenomas 3 weeks and 8 weeks PCI after 0.1 mg Tam in ISC-Cre mice. N, mouse numbers; Fisher Exact test, two-tailed. Representative micrograph (scale bar 100 μm) shows predominance of clonal ribbons 8 weeks PCI. Bars in all graphs: mean ±SD.

Fig. 4 |. Distinct complement of accessible enhancers in tumour-resident Apc-null ISCs.

a, Two weeks post-Tam, all ISC-Cre;Apc^Fl/Fl;R26R^Tom intestines carried adenomas (N=8 mice). Images: representative outcropping of fused, dysmorphic crypts, scale bar 100 μm, and effaced Tom⁺ crypt architecture, scale bar 200 μm. b, Compared to WT Lgr5⁺ ISCs, Apc-null ISCs 14 days PCI showed increased chromatin access (called ATAC-seq peaks ≥log₂ 2.5-fold over WT, FDR <0.05, N=2 duodeni each) at 5,704 enhancers (>−1 kb and >2 kb from transcription start sites). WT duodenal ISCs lack H3K27ac or H3K4me2 at these sites (ChIP-seq) and CpG dinucleotides are methylated (whole-genome bisulfite sequencing, WGBS); public data sources listed in the Online Methods; WGBS data deposited in GEO series GSE241384. Enhancers accessible in both conditions are shown for comparison. c, Significant correlation (p = 4.69*10⁻⁴, one-tailed K-S test) between newly accessible enhancers and Apc^−/− ISC expression of genes within 100 kb; genes reduced in expression are not correlated. Below, representative PyGenome tracks (Pi3kgc) illustrate newly accessible enhancers (asterisks) and increased RNA expression in Apc^−/− ISCs. d, Only 419 of 5,704 enhancers (7.4%) identified in duodenal Apc^−/− ISCs from ISC-Cre are accessible in Apc^−/− ISCs from Sec-Cre (N=4 mice) or from adenoma-free ISC-Cre;Apc^−/− (N=2 mice) ilea after 0.1 mg Tam (≥log₂ 2.5-fold compared to WT ileal ISCs, N=2 mice); the other 92.6% are enriched only in duodenal and ileal ISCs from adenoma-bearing mice. Columns in grey repeated from panel B for reference. Signals at 5,000 Control regions (accessible in all conditions) are shown as background. e, Illustrative PyGenome tracks (ATAC-seq) from samples represented in panel d. Enhancers selectively accessible in adenoma-resident ISCs are highlighted. f, Study conclusion: ISCs in clustered Apc^−/− crypts generate adenomas with new chromatin accessibility at >5,000 enhancers. ISCs in dispersed Apc^−/− crypts neither form adenomas nor access those enhancers.