a, Heat map showing distribution of MLLT3 peaks reflecting endogenous MLLT3 and MLLT3-OE ChIP–seq signal using MLLT3 and V5 antibodies. MLLT3 from uncultured FL-HSPCs and input control, MLLT3 ChIP signal from 4 week cultured FL-HSPCs with MLLT3-OE or empty control vector, and V5 ChIP from FL-HSPCs cultured for 4 weeks with MLLT3-OE or control vector. The region surrounding the peak summit (± 2 kb) is shown for the combined MLLT3 peaks from all conditions (FL-HSPC MLLT3 ChIP n = 3, MLLT3-OE V5 ChIP n = 2 and MLLT3-OE MLLT3 ChIP n = 1). IP, immunoprecipitate. b, Heat map showing hierarchical clustering for all MLLT3-bound genes as in a. Signal from all MLLT3-bound genes (significant MLLT3 peak at < 5 kb from TSS) is shown. c, UCSC genome browser tracks of representative MLLT3-bound HSC (yellow) and erythroid (red) genes. Tracks show ChIP–seq signal for input and MLLT3 in uncultured FL-HSPCs, MLLT3 ChIP–seq on FL-HSPCs cultured for 4 weeks with control and MLLT3-OE vectors; V5-tagged MLLT3 ChIP–seq from FL-HSPCs cultured for 4 weeks with control and MLLT3-OE vectors, and MLLT3 ChIP–seq in FL-erythroblasts. d, Probe values from microarray datasets4 for MLLT3 and MLLT3-bound and upregulated HSC genes (see Fig. 3) documenting the decline of gene expression over time in culture (day 0 (n = 3) 2 weeks (n = 3) and 5 weeks (n = 2) on OP9M2, mean and individual values). e–g, Knockdown of MLLT3 targets MECOM and HLF in MLLT3-OE cells. e, FACS plots at 15 and 30 days after MECOM-KD and HLF-KD, with or without MLLT3-OE (representative of three experiments). f, Quantification of CD34+CD38−/lo CD90+ HSPCs (percentage of live cells) in MECOM-KD and HLF-KD FL-HSPCs transduced with empty vector or MLLT3-OE after 5, 15, 30 and 42 days in culture (mean, n = 3). P values determined by two-sided t-test. g, Quantification of HLF downregulation in HepG2 cells by qRT–PCR. h, Quantification of MECOM downregulation in K562 cells by qRT–PCR.