Journal: Nature cell biology

Article Title: Lineage-determining transcription factors constrain cohesin to drive multi-enhancer oncogene regulation

doi: 10.1038/s41556-025-01827-2

Figure Lengend Snippet: a , Box-and-whisker plots depicting higher expression (left) and essentiality (right) of EBF1 in Non-Hodgkin B lymphomas compared to T cell leukaemia/lymphoma from DepMap. The number of cell lines are indicated in parentheses. Box-and-whisker plots: centre line, median; box limits, upper (75th) and lower (25th) percentiles; whiskers, 1.5 interquartile range. b , Western blotting of EBF1 in Cas9-expressing JVM-2, Granta519 and PGA-1 MCL showing efficient depletion of EBF1 three days after transfection of sgRNA targeting EBF1. β-actin is loading control. c , d , Flow cytometry plots ( c ) and quantification ( d ) of cell apoptosis and death measured by Annexin V and ToPro-3 staining. Ctrl and EBF1-KO Cas9-expressing MCL cells were sorted three days post lentiviral transduction and cultured for three days. See Supplementary Fig. 1 for gating strategy. Data represent mean ± S.D. of 3 replicates per condition. P value: two-sided t-test. e , Cumulative distribution plot (CDF) of distances between each reproducible EBF1 peak to the closest expressed gene TSS in Granta519 showing > 75% EBF1 peaks are located 10 kb away from TSS. f , Schematics of knocking in FKBP F36V domain at the stop codon of endogenous EBF1 gene in Granta519-Cas9 cells. Single cell clones were selected with blasticidin and successful insertion of the FKBP F36V cassette was validated with genomic DNA PCR. Clones with efficient EBF1 degradation after dTAG V -1 treatment (referred to as dTAG) are used. g , Hierarchical clustering of normalized reads (RPKM) from Ctrl and EBF1-KO Granta519-Cas9, and EBF1-FKBP-KI clones 26, 27, 29, 97 with 0, 6, 24-h 125 nM dTAG treatment showing higher similarity of clones 27 and 97 with Granta519-Cas9 transcriptome. h , Volcano plot showing ATAC-seq signal fold enrichment (x axis) versus false discovery rate (FDR) (y axis) in Granta519-EBF1-FKBP-KI clone 27 treated with 125 nM dTAG for 6, 24, 48, 72 h compared with untreated cells. Each point depicts an accessible element, colour coded by blue, red, and black based on significantly decreased, increased, or unchanged accessibility in treated cells, respectively. Significance cutoff: FDR < 1E-5 and Log2(fold change) => 1. i , Heatmaps displaying overall unchanged normalized accessibility levels at dynamic EBF1-binding sites. Each column of ATAC-seq signals is centred on bona fide EBF1-bound elements per Fig. 1d ± 2 Kb flanking sequences with 50 bp resolution. j , Scatterplot of PC1 values of Hi-C contact matrices in Ctrl and EBF1-KO Granta519-Cas9 cells showing largely invariable A/B compartments. k , Box-and-whisker plots of EBF1 (left), YY1 (middle) and H3K27ac (right) loading at the promoters (P) and enhancers (E) interacting with significant long-range DNA loops of SMC1 HiChIP in Granta519. EBF1 preferentially binds enhancers, while YY1 preferentially binds promoters. H3K27ac is negative control. EE: n = 11,818; PE: n = 10,739; PP: n = 3,806. Box-and-whisker plots: see panel a . FWER-adjusted p values: two-side Wilcoxon rank-sum test. l , Venn diagram comparing overlapping of the genomic coordinates of enhancer–promoter hubs in Granta519 SMC1 and EBF1 (RG and MP) HiChIP showing high concordance of hubs identified by three assays. m , Cumulative distribution plots showing expressed genes count in each enhancer–promoter hub defined by EBF1 (RG and MP) and SMC1 HiChIP. n , Box-and-whisker plots showing higher number of genes per megabase in gene-dense compared to gene-sparse hubs defined by EBF1 HiChIP using RG (left) and MP (right) antibodies. The number of hubs in each group is listed in parentheses. Box-and-whisker plots: see panel a . P values: two-sided Wilcoxon rank-sum test. Source numerical data and unprocessed blots are available in Source data .

Article Snippet: DND41, JVM-2 and PGA-1 were purchased from the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Lines (DSMZ, cat. nos.

Techniques: Whisker Assay, Expressing, Western Blot, Transfection, Control, Flow Cytometry, Staining, Transduction, Cell Culture, Clone Assay, Binding Assay, Hi-C, HiChIP, Negative Control