a, Competitive proliferation assays of MLL–AF9;NrasG12D leukaemia cells transduced with pLMN constructs expressing the indicated shRNAs. Shown is the fraction of GFP+/shRNA+ cells (relative to the initial ratio) under treatment with JQ1 (50 nM) or DMSO over time (continuation from Fig. 1d). JQ1 treatment was initiated at the indicated time points (red arrow). b, Competitive proliferation assays evaluating one validated shRNA targeting Eed as an additional core component of the PRC2 complex (as in a). c, Competitive proliferation assays of Tet-on competent MLL–AF9;NrasG12D leukaemia cells expressing the indicated shRNAs from a Tet‐inducible vector (pRT3GEN-miR30). Transduced cells were selected with G418 and subsequently mixed with wild-type (wt) cells in a ratio of 95% to 5%. shRNA-expression was induced using dox treatment (1 µg ml−1; from day 0), and the fraction of GFP+ cells was measured over time and plotted relative to day 2. JQ1 treatment (50 nM) was initiated in one of the duplicate samples at the indicated time point (red arrow). Once the percentage of viable cells was below 10%, measurements were discontinued (indicated in the graph by the discontinuation of the respective sample). The negative effect induced by Suz12 suppression is reverted upon treatment with JQ1, which is not the case when Myc is suppressed. d, Bar chart showing colony-forming cell frequencies of MLL–AF9;NrasG12D leukaemia cells expressing Ren.713 or Suz12.1676 shRNAs in the presence of DMSO or 200 nM JQ1; type 1, myeloblasts; type 2, maturing myeloblasts; type 3, terminally differentiated myeloid cells (n = 3, mean ± s.e.m.). e, Pie charts depicting the fraction of JQ1-response genes which are re-expressed to the indicated extent in mouse resistant AML cells expressing shRNAs targeting Dnmt3a and Psip1 (continuation of Fig. 2b). JQ1-response genes (Fig. 2a) were grouped into four categories based on the divergence of their expression in resistant AML compared to AML expressing Ren.713 (not changed compared to expression after 24 h JQ1, less than 1.5-fold; restoration relative to DMSO control: full restoration, less than 1.5-fold; enhanced, above 1.5-fold; partial restoration, restored but less than 1.5 fold) f, Heat map showing the distribution of H3K27me3, H3K36me3 and H3K4me3 ChIP-seq peaks (fold enrichment >5 over input, FDR <1%) relative to Brd4 enhancer and promoter binding sites in MLL–AF9;NrasG12D AML cells with and without JQ1 treatment. g, ChIP-seq occupancy profiles of Brd4 and H3K27me3 and H3K36me3 chromatin marks at enhancer regions downstream of Myc and upstream of Tifab following 3 days of treatment with vehicle or JQ1 (25 nM). y axis reflects the number of normalized cumulative tag counts in each region.