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 Fig. 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. Drawings in b–e were created with BioRender.com. 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.