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. Author manuscript; available in PMC: 2012 Jan 21.
Published in final edited form as: Oncogene. 2011 Mar 21;30(29):3207–3221. doi: 10.1038/onc.2011.35

Figure 5. p53 is recruited to a binding site in the second intron of the FoxO3 gene.

Figure 5

(A) Location and sequence of the p53 binding sites (p53-1, p53-2, p53-3, and p53-4) in the promoter and second intron of the mouse FoxO3 gene. R: G or A; W: T or A; Y: C or T; E: exon; I: intron. Also depicted are the consensus for p53 binding sites, the p53 binding site in p21Cip1 promoter, and the mutant of critical bases in p53-4 (p53-4m). (B) ChIP on MEFs treated with Doxorubicin (Dox, 0.2 μg/ml) for 16–20 hours, using antibodies to p53 (colored bars) or control IgG (white bars). The chromatin bound to p53 or to the control IgG was analyzed by quantitative-PCR with primers surrounding a region that did not contain p53 binding sites (−), the distal p53 binding site in FoxO3 promoter (p53-1), the p53-4 binding site in FoxO3 intron 2 (p53-4), and the p53 binding site in the p21Cip1 promoter (p53 p21Cip1). The fold enrichment over the IgG control is represented. Mean +/− SEM of three independent experiments. **: p<0.01, one-way ANOVA. (C) ChIP on p53+/+ and p53−/− MEFs in the absence or presence of Doxorubicin (Dox, 0.2 μg/ml) for 6 hours, using antibodies to p53 or control IgG. The chromatin bound to p53 or to the control IgG was analyzed by quantitative-PCR with primers surrounding a region that did not contain p53 binding sites (−), the distal p53 binding site in FoxO3 promoter (p53-1), the p53-4 binding site in FoxO3 intron 2 (p53-4) and the p53 binding site in the p21Cip1 promoter (p53 p21Cip1). The fold enrichment over the IgG control is represented. Mean +/− SD from triplicates of one experiment. (D) Normalized activity of luciferase reporter constructs driven by 500bp surrounding the p53 binding sites p53-1 or p53-4 in p53+/+ (black) and p53−/− (white) MEFs. Mean +/− SEM of four independent experiments conducted in triplicate. *: p<0.05 between p53-4 and control in p53+/+ MEFs, **: p<0.01 between p53+/+ and p53−/− MEFs for p53-4, one-way ANOVA. (E) Normalized activity of luciferase reporter constructs driven by the region surrounding the p53-4 binding site or by the region surrounding the p53-4 binding site in which the p53 binding site was mutated (p53-4m) in p53+/+ (black) and p53−/− (white) MEFs. Mean +/− SEM of three independent experiments conducted in triplicate. **: p<0.01 between p53-4m and p53-4 in p53+/+ MEFs, one-way ANOVA.