Proximity of RUNX1-occupied sites to ETS motifs in CMK cells and schematic illustration of regulatory interplay between RUNX1 and cooperating TFs. (A) Venn diagram showing the relationships btween RUNX1 ChIP-seq occupancy profiles in K562, K562-TPA, and CMK cells. A substantial number (∼ 7000) of RUNX1 bound sites are CMK specific. (B) RUNX1 occupancy profile at several CMK specific loci. Shown are RUNX1 occupancy profiles in K562 cells before (light green) and after (blue) TPA, and in CMK cells (orange), at loci spanning the ETS1, FLI1, PIK3R6, and RAB27b genes. (C) A multimodal RUNX1 occupancy landscape of distinct TF motif combinations characterizing the megakaryopoietic gene expression program. RUNX1 bound sites in K562, K562-TPA, and CMK were grouped according to their co-occurrence with motifs of RUNX, GATA, AP-1, and ETS. Histograms show percentage of occurrence of motifs with binding energies in the top 5% of background. ETS motifs are highly prevalent in CMK cells, whereas the AP-1 motif is clearly biased to de novo occupied TPA-induced sites. (D) A schematic model summarizing our hypothesis about stage-specific RUNX1-mediated regulation. RUNX1 (orange crescent) is preferentially bound to remote enhancers and cooperates with GATA1 (blue cluster) to regulate early myeloid genes (orange rectangles). On induction, RUNX1 recruits coactivators (purple crescent) to activate the AP1 genes. Thereafter, AP1 TFs (green clusters) facilitate the binding of RUNX1 to early megakaryocytic genes, thereby launching and driving the differentiation program. At subsequent differentiation stage (CMK), RUNX1 cooperates with ETS family TFs (yellow ellipses) to activate a different set of megakaryocytic genes. This scenario underscores the notion that RUNX1 functions in a context-dependent manner to regulate the transcriptional program in differentiating megakaryocytic cell lines.