Figure 2

HIPK2 stimulates co-recruitment of p53Lys382 and p300 on apoptotic promoters. (a) MCF7/indsi/HIPK2 cells were treated with Dox (+Dox) for 5 days (for HIPK2 knockdown) and then cultured without Dox (-Dox) for additional 5 days (for HIPK2 recover) before analysing HIPK2 expression by RT-PCR. GAPDH was used as a control of efficiency for RNA extraction and transcription. (b) ChIP analysis with antibodies specific for p53 and p53 acetylated on Lys382 was performed with MCF7indsi/HIPK2 cells treated with Dox for 5 days and then cultured without Dox for additional 5 days, with or without ADR (2 μg/ml) treatment for 16 h. PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for p53 target promoters, as indicated. (c) ChIP analysis with antibodies specific for p53, p53 acetylated on Lys373/382 and PAN-acetylated Histone H4 (ac-H4) was performed with RKO cells, transfected with siRNA control, left untreated or treated with ADR (2 μg/ml) for 16 h. Re-ChIP analysis was performed by immunoprecipitating the p53-containing complexes with an antibody against p300. PCR analysis was performed on the immunoprecipitated DNA samples using specific primers for p53 target promoters. A sample representing linear amplification of the total input chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunoglobulins (No Ab). (d) ChIP analysis with antibody specific for p53 and PAN-acetylated Histone H4 (ac-H4) was performed with RKO cells stable depleted of HIPK2 function by pSuper-HIPK2 transfection (siHIPK2) and treated with ADR (2 μg/ml) and zinc (150 μM) for 16 h. PCR analysis was performed on the immunoprecipitated DNA samples using specific primers for p53 target promoters. A sample representing linear amplification of the total input chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunoglobulins (No Ab). (e) MCF7indsi/HIPK2 cells were treated as in (b) and induction of p53 target genes p53AIP1 and Puma was evaluated by semiquantitative RT-PCR, with GAPDH serving as control.