Fig 2: Promoters of INK4a/ARF interact with multiple, but not all, enhancers in the cluster irrespective of their functional marks(A) Ranking of TADs based on number of H3K27ac peaks.(B) Plot showing INK4a/ARF enhancer cluster contains a super-enhancer overlaid by enhancer annotations (purple boxes E1–E24) with the super-enhancer marked (E5–E21).(C) 4C plot showing the interactions from CDKN2A viewpoint; red dots represent the significant interactions. The 4C plot is overlaid with ChIP-seq and GRO-seq tracks in HeLa.(D) The UCSC browser shot shows the zoomed region from E1–E21. Tracks are overlaid with STARR-seq track in HeLa..(E) ChromHMM profiles at the region from E1–E21 across different cell types and distances among E8, E12, E17, and E21 are shown.

Fig 3: Each interacting enhancer is essential and fully regulates INK4a/ARF genes(A–C) mRNA levels of (A) INK4a, (B) ARF, and (C) INK4b genes upon CRISPRi on E8, E12, E17, and E21 (n = 4). Plots are overlaid with a schematic of the INK4a/ ARF locus marked with enhancer annotation, H3K4me1, and H3K27ac tracks, and highlighted enhancers were blocked (orange and green solid circles).(D–I) H3K27ac and PolII enrichment on promoters (D) INK4a, (E) ARF, and (F) INK4b upon CRISPRi on E8, E12, and E17 individually or E21 (G–I) (n = 3).(J–L) mRNA levels of (J) INK4a, (K) ARF, and (L) INK4b genes upon deletion of E8, E12, E17, and E21 enhancers (n = 4). Plots are overlaid with histone tracks, and highlighted enhancers were deleted. Statistical significance is determined by unpaired t test (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001; ns p > 0.05), and error bars denote SEM.

Fig 4: Loss of a single enhancer results in EZH2 enrichment at the INK4a/ARF promoters(A) UCSC genome browser snapshot at the INK4a/ARF locus shows ChIP-seq tracks for EZH2 and H3K27me3, H3K27ac, and PolII in hESCs and HeLa.(B) EZH2 enrichment on INK4a, ARF, and INK4b promoters upon deletion of E8, E12, and E17 enhancers (n = 3).(C) H3K27me3 enrichment on INK4a, ARF, and INK4b promoters upon deletion of E8, E12, and E17 enhancers (n = 3).(D–F) EZH2 and H3K27me3 enrichment on (D) E12 and E17 upon E8 deletion (n = 3), (E) E8 and E17 upon E12 deletion (EZH2 n = 3 and H3K27me3 n = 2), and (F) E8 and E12 upon E17 deletion (n = 3).(G) Expression of different isoforms of ANRIL upon deletion of E8, E12, and E17 enhancer (n = 3).(H and I) Pearson correlation plots depicting positive correlation between (H) CDKN2A and CDKN2BAS, and (I) CDKN2B and CDKN2BAS in cervical cancer tumors from TCGA cohort; Pearson correlation value and p value, respectively, are shown at top of each plot. Statistical significance is determined by unpaired t test (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns p > 0.05), and error bars denote SEM.

Fig 5: MDM2’s binding to the E2F1 mRNA is distinct from the binding to the p53 mRNA and blocked by the tumour suppressor p14Arf. (A) mRNA translation stress-mediated induction of E2F1 stimulates growth-promoting genes (cyclin E and c-myc) and ribosome biogenesis (45S pre-rRNA) in cells expressing MDM2. (B) WBs show the levels of p53 and E2F1 in cells expressing indicated MDM2 constructs under DNA damage (doxorubicin (1 µM for 4 h)) or mRNA translation stress conditions (GAr). (C) The relative levels of p53 or E2F1 mRNAs bound to indicated recombinant MDM2 proteins. The left graph shows recombinant MDM2-166D protein binding to indicated mRNAs isolated from cells expressing, or not, the GAr. The right graph shows the binding of indicated mRNAs derived from non-treated cells to a recombinant MDM2 carrying the ATM kinase MDM2-S395D phosphomimetic mutant. No MDM2 protein was used as reference value. (D) WBs show p53 and E2F1 levels in p14Arf null U2OS cells expressing p14Arf or MDM2. (E) Autoradiograph shows the rate of E2F1 synthesis in the presence of MDM2 and the GAr with, or without, expression of p14Arf. (F) PLA shows the interactions between MDM2 and the E2F1 mRNA in cells expressing p14Arf, or an p14Arf that lacks the MDM2 binding domain (amino acids 2–14) (p14Arf-MUT). The graph below shows the quantification of interactions from three independent experiments. (G) The relative amount of E2F1 mRNA bound to recombinant MDM2. The RNA was isolated from cells expressing indicated constructs. The p14Arf WT protein, but not the p14Arf-MUT, prevented MDM2 from interacting with the E2F1 mRNA. WBs represents one of three independent experiments and actin was used as a loading control. Statistical significance was calculated using t tests (***P < 0.001, **P < 0.01 and *P < 0.05).