Figure 2.

iASPP enhances the association of p300 and TAp73 with the promoter regions of p73 target genes. (a) Levels of chromatin-bound p300 and TAp73 decrease in iASPP-depleted cells upon cisplatin treatment. HCT116 cells were treated for 8 h with 20 μM cisplatin before chromatin preparation and immunoblot analysis. (b) p300 binds poorly to p300/p73 target sites in iASPP-depleted cells. P300- or IgG-ChIP as control was performed in cisplatin-treated HCT116 cells (8 h, 20 μM cisplatin), followed by quantification of the percentage of precipitated DNA from specific genomic loci. The relative binding describes the fold enrichment of p300 over IgG control. Results from the control knockdown were set to 100% and the relative loss of p300 binding in iASPP knockdown cells was calculated. The average binding from three independent experiments is shown. Significance was determined by the Student's t-test and the P-value is shown by asterisks (*P-value<0.05, **P-value<0.01, ***P-value<0.001). A detailed ChIP analysis of one biological with all controls is shown in Supplementary File S2. (c) iASPP depletion causes a decrease of TAp73-binding to its specific target gene promoters. P73- or IgG ChIP as well as the subsequent analysis was performed as in (b). (d) iASPP knockdown results in a shift of TAp73 from the chromatin bound to the soluble fraction upon cisplatin treatment. HCT116 cells were treated with cisplatin (16 h, 20 μM cisplatin) before separation of cell lysates into triton-soluble (soluble fraction) and triton-insoluble (chromatin-bound) fractions