FIG. 3.

p53 inhibits Hbo1 HAT activity in vitro and in vivo. (A) Addition of p53 inhibits Hbo1 HAT activity in vitro. The results of HAT assays using [³H]acetyl-CoA and chicken-free core histones are shown. Control incubations with histones only (lanes 1 and 9) or with histones plus GST-p53 only (lane 7, 12.5 pmol GST-p53; lane 8, 50 pmol GST-p53) did not give histone acetylation. However, the addition of His-Hbo1 (14 pmol) to the reaction mixtures gave strong histone acetylation (lanes 2 and 10). This activity was inhibited by the addition of GST-p53 (lane 3, 12.5 pmol; lane 4, 50 pmol) but not GST alone (lane 5, 12.5 pmol; lane 6, 50 pmol). Inhibition activity was retained in only the C-terminal regulatory domain represented on GST-p53(328-393) (lane 11, 12.5 pmol; lane 12, 50 pmol). (B) The inhibition of Hbo1 by p53 in vitro is specific. Hbo1 HAT activity is not inhibited by GST-p53(286-330), a fragment that does not bind well to the enzyme (Fig. 2B). HAT assays with and without the enzyme served as positive and negative controls (lanes 1 and 2). His-Hbo1 (14 pmol) was incubated with the GST-p53(286-330) protein (lanes 3 and 4) or GST alone (lanes 5 and 6) at 12.5 pmol (lanes 3 and 5) and 50 pmol (lanes 4 and 6). A different histone H4 acetyltransferase, Hat1, is not inhibited by p53 in vitro (lanes 7 to 12). Purified human Hat1 (0.7 pmol) was incubated with free chicken core histones and [³H]acetyl-CoA in the absence (lane 8) or presence (lanes 9 and 10) of GST-p53 or GST alone (lanes 11 and 12) at 12.5 pmol (lanes 9 and 11) or 50 pmol (lanes 10 and 11). Reactions were separated by SDS-PAGE and assayed by measuring the incorporation of [³H]acetate into histones (“Fluorogram”). The amounts of histone substrate were measured by Coomassie staining of the gels (“Stain”). Histone acetylation activity in these reaction mixtures required the addition of the Hat1 enzyme as expected (lane 7). (C) Expression of p53 in cells inhibits Hbo1 acetylation activity. Saos-2 cells (p53^−/−) were transiently transfected with either empty pcDNA3.1Zeo(+) plasmid (lane 1) or a pcDNA3.1Zeo(+) clone expressing p53 (lane 2). Whole-cell extracts were separated by PAGE and assayed by Western blotting with antibodies against p53, Hbo1, Mdm2, p21, and α-tubulin. Immunoprecipitates pulled down from the extracts with anti-Hbo1 antibody were assayed for HAT activity using [³H]acetyl-CoA incorporation (“Fluorogram”) with chicken core histones as a substrate (“Stain”). (D) Expression of the C-terminal domain of p53 is sufficient to inhibit the HAT activity of Hbo1 in cells. Saos-2 cells were transfected with either adenovirus expressing GFP (lane 1) or adenovirus expressing only the C-terminal regulatory domain of p53, residues 328 to 393 (lane 2). Whole-cell extracts were probed by Western blotting as described above (B), with the exception that p53C was detected by slot blot Western analysis of the chromatin-enriched fractions using an antibody (PAb 122) specific for the C-terminal region of p53. Immunoprecipitation (IP) and HAT assays were carried out as described above (B).