RUNX1-dependent transcriptional activation is linked to remote de novo RUNX1 occupancy sites. (A) Enrichment of de novo RUNX1 binding in proximity of activated genes. The density (peaks/20 kb, y-axis) of de novo RUNX1 bound sites is plotted relative to TSSs for 147 bona fide RUNX1-regulated genes after TPA induction (blue) and for genes not activated by TPA (gray). (B) De novo binding of RUNX1 at long-range enhancer regions significantly correlates with its de novo binding at gene promoters. Log of maximal difference in RUNX1 ChIP-seq coverage before and after TPA within 3 kb (promoter region) and 200 kb (enhancer region) around the TSS was computed. Boxplots represent the distribution of differential RUNX1 occupancy at enhancers (y-axis) for groups of promoters with similar differential RUNX1 occupancy (x-axis). The outliers represent values more than 90th percentile. (C) Remote constitutive and de novo RUNX1-occupied sites and H3K4me1/H3K27me3 profile at key TPA-responsive RUNX1-regulated megakaryocytic gene loci. RUNX1, H3K4me1, and H3K27me3 ChIP-seq readouts, before (green) and after (blue) TPA treatment, in several genomic loci encompassing megakaryocytic important genes that were activated after TPA in a RUNX1-dependent manner. (D) Quantitative evaluation of RUNX1 ChIP-seq results in K562 and K562-TPA cells and murine primary megakaryocytes. Quantitative PCR analysis of RUNX1 binding to regions spanning ITGB3, VEGFA, and CTNNB indicated in panel C. Data are mean ± SD of 2 independent ChIP-quantitative PCR experiments performed in quadruplicates using K562 cells (green), K562-TPA cells (blue), and murine primary megakaryocytes (gray). Red asterisks represent regions containing RUNX motifs. Primers used for quantitative PCR assays are listed in supplemental Table 2, and details are described in supplemental data.