Extended Data Fig. 7: Early dysregulation of chromatin regulatory features of advanced PDAC.
From: A gene–environment-induced epigenetic program initiates tumorigenesis
a, Heat map representation of RNA-seq data showing the relative expression of gene sets associated with the ATAC-seq clusters identified in Fig. 1 across mKate2+ pancreatic epithelial cells isolated from normal, injury, Kras*, Kras* + injury and PDAC tissue states (as defined in Fig. 1a). Heat map colour represents median expression of all genes associated with each cluster, z-scored for comparison across conditions. Each column represents an independent mouse. b, Chromatin dynamics at ATAC peaks at promoter, distal or intron regions associated with differentially expressed genes (DEGs; RNA-seq fold change > 2, adjusted P < 0.05) between mKate2+ pancreatic epithelial cells isolated from the indicated tissue states versus normal pancreas (n = independent mice per condition as in a). DEGs were classified depending on whether they exhibit significant chromatin accessibility change (chromatin-dynamic DEGs) or no accessibility change (chromatin-stable DEGs, in grey) at associated peaks in the respective experimental condition versus normal pancreas. UP-DEGs, upregulated genes; DN-DEGs, downregulated genes. c, Heat map of RNA-seq data showing top upregulated pathways in the Kras* + injury condition, separated depending on whether they exhibit ATAC gains at associated peaks (promoter or distal). Upregulated genes associated with ATAC gain (left) are linked to distinct biological traits commonly acquired in PDAC (for example, differentiation, inflammation, fibrosis, signalling), whereas those with no ATAC change (that is, ‘primed’ in normal pancreas) are linked to general cellular processes (for example, cell proliferation, translation) (right). See Supplementary Table 6 for additional tissue states and pathways. d, Relative enrichment of the indicated gene sets in shBrd4.1448 versus shRen.713-expressing pancreatic epithelial cells (mKate2+GFP+) isolated from KCsh mice (n = 3 per shRNA genotype, Kras* + injury) as determined by GSEA. UP-DEGs (left bars) and DN-DEGs (right bars) between the Kras* + injury and normal conditions were classified depending on whether they exhibit or not significant accessibility changes (ATAC gain or ATAC loss) at associated ATAC peaks. Negative normalized enrichment scores (NES) indicate downregulation of gene sets in shBrd4 cells as compared to shRen counterparts. e, Heat map representation of ATAC–RNA combined scores for the indicated TFs and tissue states. The ATAC–RNA combined score infers the probability of differential binding of a specific TF to a motif significantly enriched in the ATAC gain or loss regions of each condition versus normal, based on a consistent gene expression change in the same comparison (see Methods for details). Top TFs scoring for the Kras* + injury or PDAC conditions versus normal are shown. DN-TFs, downregulated TFs (versus normal); UP-TFs, upregulated TFs (versus normal). f, Heat map of RNA-seq data from lineage-traced (mKate2+) pancreatic epithelial cells isolated from the indicated tissue states, showing the relative expression of TFs whose binding motifs are enriched in ATAC gain or loss loci by effects of tissue damage, mutant Kras or the combination of both (see Extended Data Fig. 2a, ATAC clusters) or in the transition to full-blown adenocarcinoma PDAC (PDAC versus Kras* + injury). Each column represents an individual mouse. The boxes highlight modules of TFs that are: (i) similarly expressed in normal regeneration and cancer context (green, black); (ii) selectively induced in early neoplasia and PDAC (red); (iii) selectively overexpressed in late disease (dark blue); (iv) that become increasingly suppressed by effects of injury, mutant Kras (light blue) or both (orange); or (v) selectively induced in early-stage but not late-stage disease (purple), with names of TF examples to the right. Injury and mutant Kras differentially induce diverse members of the same TF families, including several AP-1 JUN–FOS complex members (marked with arrows) and other TFs known known to also bind AP-1 motifs. In addition, note that the Kras* and injury combination suppresses the expression of master regulators of acinar differentiation (marked with asterisks) more potently than either insult alone. g, Representative IHC of two AP-1 family members in Kras-mutant or wild-type pancreatic tissues in the presence and absence of tissue damage (48 h after caerulein treatment) and compared to advanced PDAC (n = 4 mice per group). Whereas the AP-1 family member FOSL1 is induced in non-injured Kras-mutant pancreases, JUNB protein levels increase only after injury, with a potent co-activation occurring in the presence of both stimuli, suggesting that cooperative gene–environment interactions modulate the expression of AP-1 TF complex members. Scale bars, 100 μm.
