Figure 2
Figure 2. TPA-dependent recruitment of RUNX1 to preprogrammed remote enhancer regions. (A) Validation of anti-RUNX1 antibody efficacy and specificity in immunoprecipitation (IP). IP of RUNX1 from CMK, K562, or K562-TPA whole cell lysates by the anti-RUNX1 antibodies used in the ChIP-seq experiments. Lane 1 indicates protein-A agarose beads (supplemental data) were incubated without cell lysate. Lane 2, Western blot using 50 μg of protein of cell lysate and anti-RUNX1 antibodies (α RUNX1). Lane 2*, Longer exposure of lane 2. Lanes 3 to 5, Western blot analysis of proteins immunoprecipitated from CMK (2), K562 (3), and K562-TPA cell lysates. Lane 6, Western blot analysis of CMK cell lysate using anti-GAPDH antibodies (α GAPDH). Lane 7, Western blot analysis, using anti-GAPDH antibodies, of proteins immunoprecipitated from CMK by anti-RUNX1 antibodies. (B) Venn diagram showing genome-wide RUNX1 occupancy in K562 and K562-TPA cells. RUNX1-binding peaks from ChIP-seq analysis (covered beyond a threshold and lacking significant coverage in control experiment) before and 24 hours after TPA treatment were compared. (C) Distribution of RUNX1 ChIP-seq peaks relative to the TSS. Shown are RUNX1 peak frequencies relative to the distance from the nearest annotated TSS. Although regions proximal to TSSs show increased RUNX1 binding, the majority of RUNX1 peaks (inset) were localized more than 10 kb from the nearest TSS. (D) Enrichment of RUNX1 binding in regions with open chromatin. Distributions of H3K4me1 ChIP-seq readouts at RUNX1 bound sites (light green) and at background regions (gray) demonstrate that RUNX1 binding was largely restricted to H3K4me1-marked remote enhancers. (E) Change in H3K4me1-marked regions after TPA induction. Histograms represent the numbers of ChIP-seq H3K4me1-marked genomic regions that were identified in both pre– and post–TPA-treated cells (constitutive, gray), in pre–TPA-treated cells (green), and in TPA-induced cells alone (blue). (F) De novo occupied RUNX1 sites are marked by H3K4me1 before TPA induction. Histograms represent the distributions of H3K4me1 ChIP-seq readouts before (light green) and after (blue) TPA in de novo occupied RUNX1 sites. The distribution of H3K4me1 levels at background regions (gray) served as a control. The data demonstrate that de novo RUNX1-occupied regions were marked open by H3K4me1 before TPA induction and before RUNX1 binding. (G-H) RUNX1 binding is negatively correlated with H3K27me3 ChIP-seq occupancy. A RUNX1 enrichment value is computed by dividing the frequency of RUNX1 peaks over the defined part of the genome by the RUNX1 peak frequency over the entire genome. Plotted are RUNX1 enrichment values as a function of H3K27me3 occupancy, before (G) and after (H) TPA induction.

TPA-dependent recruitment of RUNX1 to preprogrammed remote enhancer regions. (A) Validation of anti-RUNX1 antibody efficacy and specificity in immunoprecipitation (IP). IP of RUNX1 from CMK, K562, or K562-TPA whole cell lysates by the anti-RUNX1 antibodies used in the ChIP-seq experiments. Lane 1 indicates protein-A agarose beads (supplemental data) were incubated without cell lysate. Lane 2, Western blot using 50 μg of protein of cell lysate and anti-RUNX1 antibodies (α RUNX1). Lane 2*, Longer exposure of lane 2. Lanes 3 to 5, Western blot analysis of proteins immunoprecipitated from CMK (2), K562 (3), and K562-TPA cell lysates. Lane 6, Western blot analysis of CMK cell lysate using anti-GAPDH antibodies (α GAPDH). Lane 7, Western blot analysis, using anti-GAPDH antibodies, of proteins immunoprecipitated from CMK by anti-RUNX1 antibodies. (B) Venn diagram showing genome-wide RUNX1 occupancy in K562 and K562-TPA cells. RUNX1-binding peaks from ChIP-seq analysis (covered beyond a threshold and lacking significant coverage in control experiment) before and 24 hours after TPA treatment were compared. (C) Distribution of RUNX1 ChIP-seq peaks relative to the TSS. Shown are RUNX1 peak frequencies relative to the distance from the nearest annotated TSS. Although regions proximal to TSSs show increased RUNX1 binding, the majority of RUNX1 peaks (inset) were localized more than 10 kb from the nearest TSS. (D) Enrichment of RUNX1 binding in regions with open chromatin. Distributions of H3K4me1 ChIP-seq readouts at RUNX1 bound sites (light green) and at background regions (gray) demonstrate that RUNX1 binding was largely restricted to H3K4me1-marked remote enhancers. (E) Change in H3K4me1-marked regions after TPA induction. Histograms represent the numbers of ChIP-seq H3K4me1-marked genomic regions that were identified in both pre– and post–TPA-treated cells (constitutive, gray), in pre–TPA-treated cells (green), and in TPA-induced cells alone (blue). (F) De novo occupied RUNX1 sites are marked by H3K4me1 before TPA induction. Histograms represent the distributions of H3K4me1 ChIP-seq readouts before (light green) and after (blue) TPA in de novo occupied RUNX1 sites. The distribution of H3K4me1 levels at background regions (gray) served as a control. The data demonstrate that de novo RUNX1-occupied regions were marked open by H3K4me1 before TPA induction and before RUNX1 binding. (G-H) RUNX1 binding is negatively correlated with H3K27me3 ChIP-seq occupancy. A RUNX1 enrichment value is computed by dividing the frequency of RUNX1 peaks over the defined part of the genome by the RUNX1 peak frequency over the entire genome. Plotted are RUNX1 enrichment values as a function of H3K27me3 occupancy, before (G) and after (H) TPA induction.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal