Fig. 2: Distinct roles of YAP and AP-1 in OnF reprograming during CRC evolution.

a, Heatmap of ATAC–seq signal in WT, A and AKSP organoids at genomic DARs (top). Average ATAC–seq signal profile ±2 kb is around the peak center in clusters c1 and c2 (bottom). RPKM, reads per kilobase million. b–e, Elbow plots showing TF activity in A versus WT (b and d) and AKSP versus WT (c and e) using DARs from clusters c1 (b and c) and c2 (d and e). For a–e, n = 2 independent organoid cultures per group. CEBP, CCAAT-enhancer-binding protein; NRs, nuclear receptors; BATF, basic leucine zipper transcription factor, ATF-like; JUND, JunD proto-oncogene, AP-1 transcription factor subunit. f,g, Relative OnF signature enrichment during tumor initiation following knockdown (kd) of YAP or FOS (f) or the ectopic expression of a dominant-negative (DN) FOS (g). Each data point is a gene in the OnF signature (n = 4 independent experiments; ^***P < 0.001, two-sided Wilcoxon’s rank-sum test). h, Line plot of differential TF activity (combined score), from d and e, during CRC evolution. Two-sided, paired Student’s t-tests were used for significance. JUN, Jun proto-oncogene, AP-1 transcription factor subunit; MAFK, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog K. i–k, Motif activity of ASCL2 (i), FOS/AP-1 (j) and TEAD1 (k) in AKSP single cells across the SC–OnF phenotypic spectrum determined by chromVAR z-scores for the indicated TFs. The correlation coefficients between TF activity and the LGR5⁺ SC or OnF score are indicated in orange and green, respectively. Multiome data are from two independent organoid cultures (n = 11,030 cells). l–n, Relative enrichment of the OnF (l), esophagus (m) and small intestine (n) signatures in WT organoids following ectopic expression of YAP or YAP then FOS, sequentially. Each data point is a gene (n = 4 independent experiments; NS, not significant, ^***P < 0.001, two-sided Wilcoxon’s rank-sum test). EV, empty vector. o–q, GSEA of OnF (o), esophagus (p) and small intestine (q) gene signatures in YAP overexpression (OE) versus YAP + FOS sequential OE organoids, relative to l–n. Two-sided, permutation-based test was used for significance; P values were adjusted via the Benjamini–Hochberg method. r, Model of phenotypic spectrum evolution in neoplastic cells during CRC progression highlighting key TFs at play. Various gradients of ASCL2, YAP and FOS activity are pivotal to establishing a phenotypic continuum in neoplastic cells. In the box plots in f, g and l–n, the center line is the median, the box limits the IQR (25th to 75th percentiles) and the whiskers the highest and lowest values within ±1.5× the IQR. ^*P < 0.05; ^***P < 0.001; exact P values are given in Supplementary Table 8. NES, normalized enrichment score; TPM, transcripts per million.

Source data