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. Author manuscript; available in PMC: 2009 Nov 20.
Published in final edited form as: Cell. 2007 Aug 24;130(4):624–637. doi: 10.1016/j.cell.2007.06.013

Figure 3. p53 bound to Hzf is preferentially recruited to p21 and 14-3-3σ promoter.

Figure 3

(A, B) Loss of Hzf enhances expression of proapoptotic targets of p53, but it suppresses the expression of p21 and 14-3-3σ. Western blotting of p53 targets in Hzf+/+ and Hzf−/−MEFs. Hzf+/+ and Hzf−/− MEFs were infected with Ad-GFP or Ad-p53 (A) or treated with ETO (40 μM) (B). 24 hours later, Western blot analysis was carried out for the indicated proteins.

(C) Re-expression of Hzf restores preferential induction of p21 and 14-3-3σ and represses proapoptotic targets in response to p53. Hzf−/− MEFs were infected with Ad-GFP or Ad-Hzf 6 hours prior to infection with Ad-GFP or Ad-p53. 24 hours post infection, Western blots were carried out for the indicated proteins.

(D) Multiple ChIP analysis on the promoters of p53 targets. Wt and Hzf−/− MEFs were treated with ETO (40 μM). 24 hours post drug treatment ChIP was carried out using control mouse IgG or anti-p53 antibody and PCR was done for the indicate promoters.

(E) Part of the chromatin immunoprecipitated with p53 antibody was again subjected to ChIP using control rabbit IgG or Hzf antibody. Input represents 2% of total chromatin from untreated Hzf+/+ MEFs used for immunoprecipitation.

(F, G) EMSA showing the DNA binding activity of p53 to oligonucleotides containing the p53 binding site in p21 promoter, Bax first intron, and Mdm2 promoter in wt-MEF (F) and Hzf-null MEF (G). Wt and Hzf−/− MEFs were treated with ETO (40 μM). 24 hours post drug treatment, nuclear extracts were prepared, and EMSA carried out as described in supplementary methods. The arrows indicate the position of the p53-DNA complex (lower) and supershifted complex containing anti-p53 antibody (pAb421).