3.

Effects of H₂O₂ and TSA on the promoter status of p21^WAF1 in HCT116 p53⁺/⁺ cells. (A) Schematic drawing of the relative positions of PCR primers for the amplification of Sp1 site or p53 responsible element (RE) of the p21^WAF1 promoter. (B–H) ChIP experiments using immunoprecipitation with a p53 (B, C), Ac-H4 (D, E), Ac-H3 (F, G) and HDAC1 antibody (H) in control cells (C), 6 hrs and 24 hrs after treatment with TSA for 6 hrs (200 ng/ml, T), and with H₂O₂ (30 mM, 3 min, H), and with TSA and H₂O₂ (T + H), respectively. (B, C) There is an increase in p53 binding only at the p21^WAF1-p53 RE promoter region after H₂O₂. TSA pre-treatment enables p53 to bind also at the Sp1 site of the p21^WAF1 promoter in the early response, while this binding was diminished in the late response. (D–G) Pre-treatment with TSA increases H₂O₂-induced accumulation of acH4 (D, E), but not acH3 (F, G) in the chromatin associated with both p21^WAF1 promoter regions. (H) Combination of TSA and H₂O₂ treatment displaces HDAC1 from the Sp1 binding site.