The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. We report here that mGCN5/PCAF and TIP60 acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation.

The p53 tumor suppressor regulates the cellular response to genetic damage through its function as a sequence-specific transcription factor. Among the most well-characterized transcriptional targets of p53 is the mdm2 oncogene. Activation of mdm2 is critical in the p53 pathway because the mdm2 protein marks p53 for proteosome-mediated degradation, thereby providing a negative-feedback loop. Here we show that the ATMrelated TRRAP protein functionally cooperates with p53 to activate mdm2 transcription. TRRAP is a component of several multiprotein acetyltransferase complexes implicated in both transcriptional regulation and DNA repair. In support of a role for these complexes in mdm2 expression, we show that transactivation of the mdm2 gene is augmented by pharmacological inhibition of cellular deacetylases. In vitro analysis demonstrates that p53 directly binds to a TRRAP domain previously shown to be an activator docking site. Furthermore, transfection of cells with antisense TRRAP blocks p53-dependent transcription of mdm2. Finally, using chromatin immunoprecipitation, we demonstrate direct p53-dependent recruitment of TRRAP to the mdm2 promoter, followed by increased histone acetylation. These findings suggest a model in which p53 directly recruits a TRRAP/acetyltransferase complex to the mdm2 gene to activate transcription. In addition, this study defines a novel biochemical mechanism utilized by the p53 tumor suppressor to regulate gene expression.The gene encoding p53 is the most frequently mutated locus in human cancer (reviewed in reference 64). p53 is a tumor suppressor which induces either cell cycle arrest or apoptosis in response to DNA damage. These effects rely on the ability of p53 to function as a sequence-specific transcription factor. Following DNA damage, multiple signaling pathways result in the stabilization of the normally short-lived p53 protein. Stabilized p53 then activates or represses the transcription of a number of downstream target genes, many of which play critical roles in cell cycle arrest or apoptosis. The biochemical mechanism by which p53 represses certain target loci was recently described in studies showing that p53 recruits histone deacetylase (HDAC) complexes (49). In contrast, the precise biochemical mechanism by which p53 activates transcription is still being elucidated. Interactions between p53 and the TATA-binding protein (TBP) and between p53 and several of the TBP-associated factors have been reported (11,17,37,39,42,(59)(60)(61). These interactions are likely to play an important role in the loading of the RNA polymerase holoenzyme onto target gene promoters. They do not, however, explain how p53 resolves the inhibitory effects of the chromatin structure at target genes prior to polymerase loading. It has recently been learned that sequence-specific transcription factors such as p53 overcome the repressive effects of chromatin structure via the recruitment of multiprotein enzyme complexes (21, 54). In fact, DNA footprinting studies of p53 have demonstrated that the r...