Extended Data Fig. 2: Cooperative recruitment of EZH2 and cMyc to common targets leads to gene activation in leukaemia.
(a-d) Immunoblotting for EZH2 (a) or cMyc (c) and growth of MV4;11 cells following the doxycycline (Dox)-induced EZH2 knockdown (KD; shEZH2, b) or cMyc knockout (KO; by either sgcMyc-1 or sgcMyc-5, d), relative to respective empty vector (EV) controls. Y-axis shows growth after normalization to controls (n = 3; mean ± SD; unpaired two-tailed Student’s t-test). iCas9, Dox-inducible Cas9. (e) Venn diagram using downregulated DEGs, identified by RNA-seq with (blue) or without (red) the spike-in control normalization, in MV4;11 cells following EZH2 KD or cMyc KO. (f) Venn diagram using downregulated DEGs, identified by RNA-seq with spike-in control normalization, in MV4;11 cells post-depletion of EZH2 or cMyc. (g) Venn diagram using the EZH2/cMyc co-upregulated genes, identified by RNA-seq with (blue) or without (red) spike-in control normalization, in MV4;11 cells. (h) Averaged signals of the indicated protein bound at the EZH2/cMyc co-upregulated genes (n = 129; shown in f). TSS, transcriptional start site; TES, transcriptional end site. (i) Gene Ontology (GO) analysis (top) and enrichment of the DisGeNet category (bottom) using the EZH2/cMyc co-upregulated genes in f. (j-k) Box plot showing overall expression of all genes (left) and those associated with EZH2-‘solo’ (middle) or -‘ensemble’ (right) peaks in EOL-1 (j) or MV4;11 (k) cells. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers (dashed) indicate 1.5× the interquartile range. Paired two-sided t-test. (l) Box plots showing log2-converted ratios for the indicated sample comparisons by using the 204 genes identified in main Fig. 2a. Box plot was defined the same as above. (m) IGV views of the indicated factor at GADD45B, CD55 and ADAM9 in MV4;11 cells. (n-p) Immunoblotting of the indicated protein in MV4;11 cells post-treatment with C24 (n), UNC6852 (o) or A-485 (p) for 24 hours. (q) EZH2 immunoblot following EZH2 KO. *, **, and *** denote P < 0.05, 0.01 and 0.005, respectively. NS denotes not significant. Numerical source data, statistics, exact P values and unprocessed blots are available as source data.
