Figure 5
Figure 5. Co-occurrence of RUNX1 and AP-1 at de novo RUNX1 bound sites and RUNX1-mediated increase of AP-1 in response to TPA. (A) RUNX1 and AP1 motifs were inferred directly from de novo RUNX1 bound sites. Distributions of differential (after vs before TPA treatment) RUNX1 ChIP-seq readouts are plotted for groups of regions with increasing binding energy of RUNX (left) or AP-1 (right) motifs. Unlike RUNX motifs, AP-1 motifs are significantly correlated with differential (± TPA) RUNX1 occupancy. (B) Correlation between differential occupancy of RUNX1 and AP-1 occupancy. Shown are the distributions of increased RUNX-1 occupancy (y-axis; after vs before TPA treatment) in groups of loci with range of AP-1 occupancy level after TPA treatment (x-axis). AP-1 occupancy is a good predictor for TPA-induced RUNX1 recruitment. (C) Co-occurrence of AP-1 and RUNX1 ChIP-seq peaks at de novo RUNX1-occupied sites. Shown are the distributions of distances between RUNX1 peaks and most proximal AP-1 peaks at constitutively (light green) or de novo (+TPA blue) RUNX1 sites. For a reference, the distribution of distances for the nearest AP-1 sites from random H3K4me1 enhancers lacking RUNX1 binding is presented (gray). (D) Combinatorial analysis of RUNX1 and AP-1 sites and motifs. The relative fold enrichments of RUNX (orange) and AP-1 (green) motifs relative to the genomic background are shown for groups I to IX of loci with various combinatorial ChIP-seq readouts of the 2 factors. Constitutive sites are those observed in K562 cells both before and after TPA treatment. De novo sites are those observed only after TPA treatment. The pattern represented in groups VI and VIII suggest that RUNX1 occupancy enables AP-1 recruitment to regions lacking AP-1 motif; and vice versa, AP-1 binding enables recruitment of RUNX1 to sites lacking RUNX motifs. (E) TPA-dependent binding of RUNX1 to 3 AP-1 genes. RUNX1 ChIP-seq tracks at loci encompassing FOS, FOSB, and JUN before (light green) and after (blue) TPA, in K562 cells. De novo RUNX1 bound sites in remote enhancers are noted. (F) Quantitative RT-PCR analysis of FOS, FOS-B, and JUN expression in K562 cells. Data are mean ± SD of 3 independent experiments performed in triplicates. The increased expression of FOS, FOS-B, and JUN in K562-TPA relative to K562 and in K562-TPA with RUNX1 knockdown control (TPA neg) relative to K562 KD was significant (K562 ± TPA, P = .01, .005, and .005; and K562 ± KD, P = .01, .001, and .01).

Co-occurrence of RUNX1 and AP-1 at de novo RUNX1 bound sites and RUNX1-mediated increase of AP-1 in response to TPA. (A) RUNX1 and AP1 motifs were inferred directly from de novo RUNX1 bound sites. Distributions of differential (after vs before TPA treatment) RUNX1 ChIP-seq readouts are plotted for groups of regions with increasing binding energy of RUNX (left) or AP-1 (right) motifs. Unlike RUNX motifs, AP-1 motifs are significantly correlated with differential (± TPA) RUNX1 occupancy. (B) Correlation between differential occupancy of RUNX1 and AP-1 occupancy. Shown are the distributions of increased RUNX-1 occupancy (y-axis; after vs before TPA treatment) in groups of loci with range of AP-1 occupancy level after TPA treatment (x-axis). AP-1 occupancy is a good predictor for TPA-induced RUNX1 recruitment. (C) Co-occurrence of AP-1 and RUNX1 ChIP-seq peaks at de novo RUNX1-occupied sites. Shown are the distributions of distances between RUNX1 peaks and most proximal AP-1 peaks at constitutively (light green) or de novo (+TPA blue) RUNX1 sites. For a reference, the distribution of distances for the nearest AP-1 sites from random H3K4me1 enhancers lacking RUNX1 binding is presented (gray). (D) Combinatorial analysis of RUNX1 and AP-1 sites and motifs. The relative fold enrichments of RUNX (orange) and AP-1 (green) motifs relative to the genomic background are shown for groups I to IX of loci with various combinatorial ChIP-seq readouts of the 2 factors. Constitutive sites are those observed in K562 cells both before and after TPA treatment. De novo sites are those observed only after TPA treatment. The pattern represented in groups VI and VIII suggest that RUNX1 occupancy enables AP-1 recruitment to regions lacking AP-1 motif; and vice versa, AP-1 binding enables recruitment of RUNX1 to sites lacking RUNX motifs. (E) TPA-dependent binding of RUNX1 to 3 AP-1 genes. RUNX1 ChIP-seq tracks at loci encompassing FOS, FOSB, and JUN before (light green) and after (blue) TPA, in K562 cells. De novo RUNX1 bound sites in remote enhancers are noted. (F) Quantitative RT-PCR analysis of FOS, FOS-B, and JUN expression in K562 cells. Data are mean ± SD of 3 independent experiments performed in triplicates. The increased expression of FOS, FOS-B, and JUN in K562-TPA relative to K562 and in K562-TPA with RUNX1 knockdown control (TPA neg) relative to K562 KD was significant (K562 ± TPA, P = .01, .005, and .005; and K562 ± KD, P = .01, .001, and .01).

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