Figure 2.
Repressive and activating functions of PML/RARα are attributed to distinct cobinding patterns with HDAC1 and P300. (A) Heatmaps of ChIP-seq data showing cobinding of HDAC1 and P300 at the PML/RARα binding sites, displayed separately for PML/RARα-repressed targets (the top half) and -activated targets (the bottom half). (B) Abundant enrichment of both HDAC1 and P300 on PML/RARα-activated targets vs abundant HDAC1 with moderate/minor P300 on PML/RARα-repressed targets, illustrated using aggregate plots of ChIP-seq signals of HDAC1 and P300 centered on the PML/RARα binding peak summit. (C) Genome browser tracks showing the binding patterns of PML/RARα, HDAC1, and P300 on the representative repressed gene (CEBPE) and activated gene (GFI1). (D) ChIP-qPCR validation of distinct HDAC1 and P300 cobinding patterns on PML/RARα-repressed and activated targets in blast cells from 2 primary APL patients. (E) The physical interaction of both HDAC1 and P300 with PML/RARα. Endogenous coimmunoprecipitation (co-IP) experiments were performed in NB4 cells using antibodies against HDAC1 and P300, respectively. The protein level of PML/RARα was detected by western blotting with anti-RARα antibody (C20). IgG, immunoglobulin G; WB, western blot. (F) ChIP-re-ChIP showing co-occupancy of PML/RARα, HDAC1, and P300 on chromatin. ChIP products immunoprecipitated by each of the 3 antibodies were subjected to re-ChIP using the other 2 antibodies or normal immunoglobulin G. Data represent the mean of 3 replicates ± SD. *P < .01. (G) Aggregate plots of H3K27ac ChIP-seq signals centered on the PML/RARα peak summit. Red, PML/RARα-activated targets; blue, PML/RARα-repressed targets. (H) Enrichment of RNA Pol II at genes activated and repressed by PML/RARα. The activity of Pol II was determined by the Pol II traveling ratio, which was calculated by the relative amount of Pol II binding at the gene body vs near the promoter. Red, PML/RARα-activated targets; blue, PML/RARα-repressed targets. The 2-sample Kolmogorov-Smirnov test was used to compare distributions (P < .001).

Repressive and activating functions of PML/RARα are attributed to distinct cobinding patterns with HDAC1 and P300. (A) Heatmaps of ChIP-seq data showing cobinding of HDAC1 and P300 at the PML/RARα binding sites, displayed separately for PML/RARα-repressed targets (the top half) and -activated targets (the bottom half). (B) Abundant enrichment of both HDAC1 and P300 on PML/RARα-activated targets vs abundant HDAC1 with moderate/minor P300 on PML/RARα-repressed targets, illustrated using aggregate plots of ChIP-seq signals of HDAC1 and P300 centered on the PML/RARα binding peak summit. (C) Genome browser tracks showing the binding patterns of PML/RARα, HDAC1, and P300 on the representative repressed gene (CEBPE) and activated gene (GFI1). (D) ChIP-qPCR validation of distinct HDAC1 and P300 cobinding patterns on PML/RARα-repressed and activated targets in blast cells from 2 primary APL patients. (E) The physical interaction of both HDAC1 and P300 with PML/RARα. Endogenous coimmunoprecipitation (co-IP) experiments were performed in NB4 cells using antibodies against HDAC1 and P300, respectively. The protein level of PML/RARα was detected by western blotting with anti-RARα antibody (C20). IgG, immunoglobulin G; WB, western blot. (F) ChIP-re-ChIP showing co-occupancy of PML/RARα, HDAC1, and P300 on chromatin. ChIP products immunoprecipitated by each of the 3 antibodies were subjected to re-ChIP using the other 2 antibodies or normal immunoglobulin G. Data represent the mean of 3 replicates ± SD. *P < .01. (G) Aggregate plots of H3K27ac ChIP-seq signals centered on the PML/RARα peak summit. Red, PML/RARα-activated targets; blue, PML/RARα-repressed targets. (H) Enrichment of RNA Pol II at genes activated and repressed by PML/RARα. The activity of Pol II was determined by the Pol II traveling ratio, which was calculated by the relative amount of Pol II binding at the gene body vs near the promoter. Red, PML/RARα-activated targets; blue, PML/RARα-repressed targets. The 2-sample Kolmogorov-Smirnov test was used to compare distributions (P < .001).

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