The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53's ability to bind to its cognate DNA site. We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

Chemicals that target microtubules induce mitotic stress by affecting several processes that occur during mitosis. These processes include separation of the centrosomes in prophase, alignment of the chromosomes on the spindle in metaphase and sister-chromatid separation in anaphase. Many human cancers are sensitive to mitotic stress. This sensitivity is being exploited for therapy and implies checkpoint defects. The known mitotic checkpoint genes, which prevent entry into anaphase when the chromosomes are not properly aligned on the mitotic spindle, are, however, rarely inactivated in human cancer. Here we describe the chfr gene, which is inactivated owing to lack of expression or by mutation in four out of eight human cancer cell lines examined. Normal primary cells and tumour cell lines that express wild-type chfr exhibited delayed entry into metaphase when centrosome separation was inhibited by mitotic stress. In contrast, the tumour cell lines that had lost chfr function entered metaphase without delay. Ectopic expression of wild-type chfr restored the cell cycle delay and increased the ability of the cells to survive mitotic stress. Thus, chfr defines a checkpoint that delays entry into metaphase in response to mitotic stress.

The ability of p53 to function as a tumor suppressor is linked to its function as a transcriptional activator, since p53 mutants that do not transactivate are unable to suppress tumor cell growth. Previous studies identi®ed an activation domain in the amino terminal 40 residues of the protein, a region that binds to several general transcription factors and to some oncogene products. For example, mdm-2, a cellular oncoprotein, binds to this region and represses p53 transactivation. Here we describe a new activation domain within the amino terminus of p53 that maps between amino acids 40 ± 83, and whose residues trp-53 and phe-54 are critical for function both in yeast and in mammalian cells. In vivo studies in yeast show that the new activation subdomain, unlike the previously described, is mdm-2 independent. Both p53 activation subdomains (1 ± 40 and 40 ± 83) require the yeast adaptor complex ADA2/ADA3/GCN5 for transcriptional activation. Moreover, since activation by p53 requires GCN5's enzymatic histone acetyltransferase domain, p53 may regulate gene expression by in¯uencing chromatin modi®cation.Keywords: adaptor; genetics; mdm-2; p53; transcription IntroductionThe tumor suppressor protein p53 (for a review, see Donehower and Bradley, 1993;Ko and Prives, 1996;Levine, 1993 and references therein) is inactivated in more than half of all human tumors . p53 is a sequence-speci®c transcription factor that suppresses oncogenic transformation (Eliyahu et al., 1989;Finlay et al., 1989) and induces cell cycle arrest (Leonardo et al., 1994) or programmed cell death (Clarke et al., 1993; Lowe et al., 1993a,b) in response to DNA damage.Three distinct domains have been identi®ed within p53: the acidic aminoterminus is an activation domain (Fields and Jang, 1990;Funk et al., 1992;Raycroft et al, 1990), the hydrophobic central region contains a sequence-speci®c DNA-binding domain (Bargonetti et al., 1993;Wang et al., 1993), and the basic carboxyterminus comprises an oligomerization domain (Clore et al., 1994;Jerey et al., 1995;Lee et al., 1994;Sakamoto et al., 1994;Sturzbecher et al., 1992) and a region that regulates DNA binding anity Hupp et al., 1992;Waterman et al., 1995). Hot-spots for p53 mutations in tumors (Hollstein et al., 1991;Levine et al., 1991) are found predominantly in the central DNA-binding domain.In addition, p53 can act as a transcriptional repressor, to down-regulate promoters lacking wild type p53 binding sites (Ginsberg et al., 1991;Santhanam et al., 1991;Subler et al., 1992). The amino terminal transactivation domain of p53 also is required for its inhibitory eects on transcription (Sang et al., 1994;Subler et al., 1994).Tumor suppression and transcriptional activation are strongly correlated functions of p53 (Farmer et al., 1992;Fields and Jang, 1990;Funk et al., 1992). Mutations or deletions in the aminoterminus of p53 coordinately abolish transactivation and tumor suppressor functions. Tumor-derived p53 mutants fail to activate transcription of wild type p53 responsive genes. Furthermore, mdm-2,...

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