Fig 2: Different chromatin landscapes of IRX and HOXA subtypes of KMT2A-r infant ALL cell lines.ChIP-seq profiles of KMT2A, H3K4me3, H3K27ac, and RNA polymerase II (RNAP2) at the MEIS1, HOXA, and IRX1 loci. RNA sequencing profiles are also depicted.

Fig 3: Analysis of MECOM expression and downstream targets. a Box plot showing the RNA-Seq differential expression analysis of MECOM in the 293 FLP-In Empty, SETBP1-WT, and SETBP1-G870S cell models. The top and bottom of each box represent the first and third quartile, respectively; the internal line represents the median; the dot represents the mean. Experiments were performed in triplicate. b Q-PCR analysis of MECOM expression in the 293 FLP-In Empty, SETBP1-WT, and SETBP1-G870S cell models. The top and bottom of each box represent the first and third quartile, respectively; the internal line represents the median; the small square represents the mean. Experiments were performed in triplicate; statistical analysis was performed using t-test. c SETBP1 ChIP-Seq coverage track and peak alignment to the hg19 reference genome (blue tracks) are superimposed to the different histone methylation tracks and KMT2A (MLL) ChIP-Seq coverage track61 within the MECOM locus. The boxed histogram represents an independent ChIP experiment performed against the V5 flag in the FLP-In cells followed by a Q-PCR directed against the predicted SETBP1-G870S-binding locus on the MECOM promoter. ChIP was performed in triplicate; statistical analysis was performed using t-test. d Gene expression heatmap of MECOM target genes in three Empty/SETBP1-G870S FLP-In clones. e Differential MECOM expression as read counts per million of mapped reads (RPM) in 32 aCML patients carrying WT (21) or mutated (11) SETBP1. The top and bottom of the box represent the first and third quartile, respectively; the internal line represents the median. Statistical analysis was performed using t-test. f Linear correlation of MECOM expression as assessed by RNA-Seq (x axis) and Q-PCR (y axis). r represents the Pearson linear correlation coefficient. g Gene expression heatmap of MECOM target genes in 32 aCML patients carrying WT (21) or mutated SETBP1 (11). Error bars represent the standard error. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 4: SETBP1 interacts with the SET1/KMT2A COMPASS-like complex. a Co-immunoprecipitation was performed against the HCF1 protein (left) or the V5 flag (right) and blotted with an anti-V5 or HCF1 antibody. b, c FRET analysis showing physical interaction between SETBP1 and HCF1. Positive FRET signal was recorded in both couples of HCF1 and SETBP1 WT or G870S (b, green and blue lines), conversely no FRET signal was recorded for HCF1 and SETBP1ΔHBM or G870SΔHBM (b, red and orange lines). FRET between β-TrCP and SETBP1 variants was assayed to demonstrate that ΔHBM did not modify the known SETBP1–β-TrCP interaction (c). Acceptor photobleaching was performed after the third acquired frame and indicated with gray bars in both the graphs. Bars represent the standard error of three experiments. d Relative expression of SETBP1 target genes as assessed by Q-PCR in empty (black), SETBP1-G870S (orange), and SETBP1-G870S-ΔHBM (light orange) lines. e Co-immunoprecipitation was performed against the V5 flag and blotted with an anti-KMT2A antibody. f ChIP against H4K20me1 followed by Q-PCR on a set of SETBP1 target genes performed on Empty (black bars) and SETBP1-G870S cells (orange bars). g Co-immunoprecipitation was performed against the V5 flag and blotted with anti-PHF8 and anti-PHF6 antibodies. h Proposed model for SETBP1 epigenetic network. i Relative expression of SETBP1 target genes in cells transduced with empty vector, SETBP1-G870S, or SETBP1-G870S carrying deletion of the first (ΔATH1), second (ΔATH2), and both (ΔATH1,2) AT-hooks. j ChIP against SETBP1-G870S in cells transduced with empty vector, SETBP1-G870S, or SETBP1-G870S carrying deletion of the first (ΔATH1), second (ΔATH2), or both (ΔATH1,2) AT-hooks, followed by Q-PCR on a set of SETBP1 target genes. In panels d, f, i and j, statistical analysis was performed using t-test. Bars represent the standard error of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 5: Increased occupancy of MLL1 with chromatin in taspase1 knockout cells. (A–O) Coverage profiles, heat maps, and box plots of MLL1C (A–C), MLL1N (D–F), and MLL2C (G–I) in 293T cells and of MLL1C (J–L) and MLL1N (M–O) in MCF7 cells are shown. A region within 2.5 kb around the center of MLL1C, MLL1N, or MLL2C is displayed. For the heat maps, profiles are centered within 2.5 kb of MLL1- or MLL2-occupied peaks and sorted in descending order of MLL1C, MLL1N, or MLL2C occupancy in wild-type cells. Box plots show quantitation of the MLL1 occupancy in 293T cells in replicates. (P–R) Genome browser track examples of ZMYND11, NEK1, CLCN3, and CASC5 show the increased occupancy of noncleavable MLL1 in the taspase1 knockout 293T cells enriched in both the promoter region and the gene body.