Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants. 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. The tumor suppressor protein p53 Valifenalate responds to DNA damage to slow cell growth and promote programmed cell death (21, 31, 35). p53 achieves its antiproliferative properties through its action as a DNA-binding transcriptional activator, to induce expression of downstream target genes. These include (16), (30), (46), Valifenalate (40), (11), and (6, 60), whose gene products are involved in cell cycle arrest, apoptosis, and regulation of p53 function in cells exposed to DNA-damaging agents. Three major functional domains have been identified in p53: an amino (N)-terminal transactivation domain (residues 1 to 80) (12, 17, 20, 49), a central sequence-specific DNA-binding domain (residues 94 to 293) (7, 24, 57), and a carboxyl (C)-terminal oligomerization domain (residues 325 to 355) (14, 28, 34, 50, 55). In addition to the oligomerization domain, the C terminus of p53 contains two regions (residues 290 to 325 [58] and residues 356 to 393 [26]) that negatively regulate its DNA-binding activity. Multiple posttranslational modifications to these regulatory domains, such as phosphorylation, affect p53 function through modulation of DNA binding (26, 56). In addition, the highly positively charged C-terminal regulatory region may interact with the core DNA-binding domain and lock p53 in an inactive conformation (42). Evidence that supports this idea is the activation of DNA binding by (i) deletion either of the C-terminal region or of the polyproline region at the N-terminal border of the core DNA-binding domain, (ii) binding of 14-3-3 proteins or the monoclonal antibody PAb421 to the C-terminal regulatory domain, and (iii) phosphorylation within the regulatory regions (24C26, 29, 42, 56, 58). CREB binding protein (CBP) and p300 are structurally related transcriptional factors, involved in cell cycle control and differentiation, which coactivate numerous transactivators, including p53 (3, 23, 36, 52). CBP/p300 have extensive structural and functional similarity, including the capacity to bind both to the adenovirus oncoprotein E1A (1a) and to transactivators, such as CREB (1a, 33, 38), c-Jun/c-Fos (2, 5), c-Myb/v-Myb (15, 43), MyoD (62), and Stat1 Valifenalate (63) and Stat2 (9), and to p53. p300 and CBP associate with PCAF (p300 and CBP associated factor), which has been implicated as an important factor for cell cycle progression (61) and differentiation (48, 61). The complex formed between CBP and PCAF is disrupted by E1A (61), leading to suppression of p53 transactivation (36, 48, 52). These observations suggest that interaction of CBP and PCAF with p53 is critical for p53 function. Supportive evidence is provided by the finding Vax2 that CBP/p300 and PCAF function as transcriptional coactivators for p53 to fully activate endogenous gene expression (52). An important feature common to coactivators CBP/p300 and PCAF is their intrinsic histone acetyltransferase (HAT) activity (45, 61). Acetylation of lysine residues in the N-terminal tails of histones facilitates gene activation, perhaps by reducing histone tail affinity for DNA and thereby promoting transcription factor binding to nucleosomal DNA (10, 37, 41, 54). The finding that coactivators are HATs has led to an appealing model that transactivators recruit these enzymes to provide Valifenalate promoter-specific chromatin remodeling. Although both are HATs, CBP/p300 and PCAF have little sequence similarity within their HAT domains (44, 45) and, accordingly, exhibit differences in substrate specificities: recombinant CBP/p300 equally acetylate all four histones (H3, H2A, H2B, and H4), even when incorporated into nucleosomes, while PCAF preferentially acetylates H3 and primarily while in a free, nonnucleosomal state. Other proteins, including components of the transcriptional machinery, such as TFIIE and TFIIF, are acetylated in vitro by CBP/p300 as well as by other HATs (27). Recently, p300 was shown to acetylate p53 on its C terminus and to enhance p53s DNA-binding activity in vitro (22). This observation is consistent with the model discussed above, that p53s C terminus regulates DNA binding. The observation that HATs acetylate substrates other than histones has generated increased interest in the role of acetylation in regulation of gene expression. However, the physiological significance of acetylation of targets other than histones remains an open question. Given the ability of p300 to acetylate p53 in vitro, we investigated whether PCAF is also able to acetylate p53. In this study, we demonstrate that PCAF does indeed acetylate p53 and that it does so at a specific lysine.