Fig 2: Sustained Notch activation is required to maintain the enhanceosome.a, b Sequential ChIP-qPCR analysis of binding co-occupancy of RBPJ with P300, CBP, and SETD1A within the Nrg1 enhancer region in Met1 cancer cells treated with LG or HG. c–j ChIP-qPCR analysis of binding occupancy of RBPJ, NOTCH intracellular domain (NICD), P300, CBP, SETD1A, HDAC and enrichment of H3K4me1 and H3K27ac within the Nrg1 enhancer region in Met1 cancer cells treated with vehicle or DAPT under HG. Data represented mean ± SEM of technical replicates in a–j, and n = 3 independent experiments were performed. Statistical significance was evaluated using two-way ANOVA followed by Bonferroni’s post hoc test a–j. *P < 0.05; **P < 0.01; ***P < 0.001, a (all ***P < 0.0001), b (***P < 0.0001), c (from left to right, *P = 0.0133, **P = 0.0013, ***P < 0.0001), d (from left to right, *P = 0.0234,***P < 0.0001, *P = 0.0378, ***P = 0.0006), e (from left to right, ***P < 0.0001, *P = 0.0403, **P = 0.0049, ***P < 0.0001), f (from left to right, **P = 0.0019, **P = 0.0016, ***P < 0.0001), g (from left to right, ***P = 0.0003, *P = 0.0323), h (from left to right, **P = 0.0066, *P = 0.0349, ***P < 0.0001), i (from left to right, *P = 0.033, ***P = 0.0005, **P = 0.0047), j (from left to right, *P = 0.0279, ***P < 0.0001, *P = 0.0126). Source data are provided in Source Data file.

Fig 3: Global chromatin state and deposition of KMT2 components in mouse urothelial cells.a, Representative IGV tracks of H3K36me3 Cut&Run indicating the successful depletions of Exon 3 in Kmt2c and Exons 50-51 in Kmt2d. b, Western blot of KMT2 components in WT and dKO urothelial cells (n = 2 independent experiments). c, Genotyping of Kmt2c and Kmt2d floxed alleles in WT and dKO urothelial cells (n = 3 independent experiments). Primers amplifying single LoxP were used to only detect intact allele. Primers amplifying double LoxP were used to identify both intact (long) and deleted (short) allele. d, Overview of ChromHMM in WT urothelial cells. Left, a darker blue color corresponds to a higher probability of observing specific modifications in each chromatin state. Middle and right, a darker blue color corresponds to a higher fold enrichment of chromatin state at the given coordinates. State annotations were shown on the right side of the heatmap. e, Genomic distributions of KMT2 components. KMT2A+Menin and SET1A + CXXC1 groups show the features of combined peaks. f, Overlap of KMT2A and Menin, SET1A and CXXC1 on promoter-distal and promoter-proximal peaks. g, Comparison of tag-counts (log2) of KMT2A and Menin, SET1A and CXXC1 at promoter-proximal and promoter-distal peaks. Source data

Fig 4: Kmt2c/d loss suppresses activities of KMT2D-bound TSS and redistributes KMT2A–menin to CpG-high promoters.a, The overlap of KMT2D, KMT2A–menin and SET1A–CXXC1 peaks on promoter-proximal regions. b,c, The fraction of CpG dinucleotide (b) and log2 fold changes (c) of PRO-cap, H3K4me1, H3K4me3 and H3K27me3 signal in three subgroups of active TSS. The center line represents the median, the box limits represent the upper and lower quartiles and the minimum and maximum whiskers represent the 10th and 90th percentiles, respectively. Data were analyzed with a two-tailed t-test. d, Fold enrichment over the genome of chromatin states at all TSS and the top 500 PRO-cap up- (Up) or downregulated (Dn) TSS. e, A dot plot showing H3K4me3 and H3K27me3 modifications at all TSS in WT urothelial cells. The signal of H3K4me3 and H3K27me3 were normalized with RPGC. The red points indicate TSS with upregulated PRO-cap signal with Kmt2c/d KO (top 500). The gray points indicate all remaining TSS. Here we defined TSS with H3K4me3 ≥4 and H3K27me3 <3 as H3K4me3 only, while TSS with H3K4me3 ≥4 and H3K27me3 ≥3 as bivalent. f, Left: the log2 fold change of PRO-cap signal at H3K4me3 only (n = 8,152) and bivalent (n = 1,756) TSS. Middle: the fraction of CpG. Right: the log2 fold change of KMT2A enrichment. The center line represents the median, the box limits represent the upper and lower quartiles and the minimum and maximum whiskers represent the 10th and 90th percentiles, respectively. Data were analyzed with a two-tailed t-test. g, A heat map showing the changes of KMT2A, SET1A, H3K4me1, H3K4me2, H3K4me3 and H3K27ac between the WT and dKO groups. Data are shown as the average of replicate samples. The purple color indicates signal upregulated in dKO cells, while cyan indicates signal downregulated in dKO cells. h, The log2 fold change of KMT2A, menin, SET1A, CXXC1 and PRO-cap with Kmt2c/d KO. The center line represents the median, the box limits represent the upper and lower quartiles and the minimum and maximum whiskers represent the 10th and 90th percentiles, respectively. Data were analyzed with a two-tailed t-test. i, A dot plot of each TSS with linear correlation of KMT2A and PRO-cap alterations (log2 fold change, dKO/WT) at active TSS. Linear regression coefficient, R2 = 0.4807.Source data.

Fig 5: Kmt2c/d deletion suppresses enhancer activity and decreases KMT2A/SET1A deposition at active enhancers.a, Mass spectrometry comparing the fractions of H3K4 modifications in WT and dKO cells (n = 3 independent experiments). Data are shown as mean ± s.d. and were analyzed with a two-tailed t-test. b, Comparison of 12 chromatin states in WT and dKO groups using ChromHMM annotation. A darker blue color corresponds to a higher probability of observing specific modifications in each state. Annotations of each state are shown on the right side of the heat map. c, The genomic faction of each chromatin state in WT and dKO urothelial cells. d, The genomic distributions of KMT2D, KMT2A–menin and SET1A–CXXC1 peaks in WT or dKO urothelial cells. Note that peaks within −1.0 kb/+0.5 kb to the TSS were annotated as promoter proximal and the remaining peaks were annotated as promoter distal. e, The overlap of KMT2D, KMT2A–menin and SET1A–CXXC1 peaks on promoter-distal regions. f, KMT2A, menin, SET1A, CXXC1, ATAC-seq and PRO-cap signal at KMT2D-positive (pos) and KMT2D-negative (neg) enhancers in WT and dKO urothelial cells. The center line represents the median, the box limits represent the upper and lower quartiles and the minimum and maximum whiskers represent the 10th and 90th percentiles, respectively. Data were analyzed with a two-tailed t-test. g, A heat map showing the enrichment of KMT2D, H3K4me1, H3K4me2, H3K27ac, H3K27me3, KMT2A, SET1A and ATAC signal at two subgroups of KMT2D-bound enhancers. Data are shown as the average of replicate samples. h, The log2 fold change of KMT2A, menin, SET1A, CXXC1, H3K4me1, H3K27ac and PRO-cap signal (signal diff) at two subgroups of KMT2D-bound enhancers. The center line represents the median, the box limits represent the upper and lower quartiles and the minimum and maximum whiskers represent the 10th and 90th percentiles, respectively. Data were analyzed with a two-tailed t-test. i, The log2 fold change of PRO-cap signal (PRO-cap diff) at active TSS containing a nearest enhancer within or outside ±10 kb. For our analysis, duplicated enhancers were removed as we kept only the closest enhancer to each TSS. The center line represents the median, the box limits represent the upper and lower quartiles and the minimum and maximum whiskers represent the 10th and 90th percentiles, respectively. Data were analyzed with a two-tailed t-test.Source data.