The gene encoding the p53 tumor suppressor is the most commonly mutated locus in human cancer (64
). p53 functions as a sequence-specific transcription factor to regulate genes controlling growth arrest and apoptosis (15
). Until recently, little was known of the biochemical mechanism utilized by p53 to modulate transcription. Several studies have recently provided indications that the role of p53 in target gene expression may involve altering levels of histone acetylation. For example, Murphy et al. demonstrated that p53-mediated repression of the Map4 and stathmin genes relies on recruitment of HDAC complexes (49
). Furthermore, a recent in vitro study showed that activation of at least one p53 target gene, that encoding p21, may result from recruitment of the HAT p300 to the p21 promoter (16
). The present study was undertaken to define the biochemical mechanism utilized by p53 to activate transcription in vivo by examining well-characterized p53 target gene mdm2
. We report here that p53-dependent expression of mdm2
involves recruitment of HAT complexes containing the ATM-related TRRAP cofactor. Specifically, we demonstrated that p53 and TRRAP act synergistically to activate mdm2
transcription and that both proteins are necessary for optimal mdm2
expression. Two potential models for the mechanism by which p53 and TRRAP cooperate were initially considered. In the first model, TRRAP simply functions as a transcriptional cofactor for p53 by mediating the recruitment of STAGA, TFTC, hSAGA/PCAF, or NuA4/Tip60 acetyltransferase complexes. These complexes in turn would catalyze the modification of histones associated with the mdm2
promoter and thereby facilitate transcription. The second model is one in which TRRAP overexpression results in the stabilization of the p53 protein via its acetylation or through some other posttranscriptional mechanism. The data presented here support the first model, as an acetylation-negative p53 mutant still cooperates with TRRAP, no TRRAP-induced acetylation of p53 was observed, and direct measurement of p53 protein levels revealed no stabilization following TRRAP overexpression (Fig. ). It is worth noting that the acetylation-negative p53 mutant utilized here lacks the carboxy-terminal lysines which have been shown to be modified by the TRRAP-associated HAT PCAF and by p300/CBP (20
). Most importantly, we show that TRRAP and p53 interact directly in vitro and in vivo, that blocking TRRAP function with antisense treatment blocks p53-mediated transcription of mdm2
, that TRRAP is directly recruited to the endogenous mdm2
promoter by p53, and that this recruitment correlates with increased histone acetylation.
In an elegant study of p53 acetylation, Barlev et al. recently reported that the transcriptional cofactors TRRAP and CBP are bound more tightly to acetylated than nonacetylated p53 (4
). Our studies of the physical interaction between p53 and TRRAP were conducted using only nonacetylated p53, but the Barlev study suggests that the binding we observed would be increased severalfold by p53 acetylation. As in our study, Barlev et al. found no correlation between p53 DNA binding affinity and its acetylation status. Like us, they also found that TRRAP (or CBP) recruitment to a p53 target gene promoter correlated with increased levels of histone acetylation. Our failure to find a defect in mdm2
transcription when p53 acetylation sites were mutated differs from what Barlev et al. found at the p21 promoter. However, our study is consistent with several previous reports that found no transcriptional defect for acetylation-deficient p53 on the p21 promoter or on synthetic reporter genes (16
). The p21 promoter contains a single, upstream p53 binding site, while there are two intronic binding sites for p53 in the mdm2
gene. The presence of only a single binding site in the p21 gene may make it more sensitive to the effects of increased cofactor recruitment following p53 acetylation, while the dual binding sites in mdm2
may make increased cofactor recruitment unnecessary for maximum transcription. Clearly the effect of p53 acetylation on the transcription of individual target genes requires further study.
We have reported here a direct interaction between p53 and TRRAP. Another recently published study found a direct interaction between p53 and ADA3 (65
). Like TRRAP, ADA3 is a component of the PCAF/SAGA, STAGA, and TFTC acetyltransferase complexes. Considered together with our results, these data suggest that multiple subunits of these complexes (e.g., TRRAP and ADA3) can make independent, direct contact with p53. This scenario is similar to what has recently been described for the SWI/SNF chromatin-remodeling complex. There, a single sequence-specific activator was shown to bind several SWI/SNF subunits independently (51
). The ability to interact with multiple subunits in a single cofactor complex may serve to stabilize recruitment by sequence-specific activators such as p53.
TRRAP is the only subunit shared by mammalian HAT complexes PCAF/SAGA, NuA4/Tip60, STAGA, and TFTC. Among these, NuA4/Tip60, STAGA, and TFTC have been firmly implicated not just in transcription but also in the repair of DNA damage (7
). This dual role in transcription and DNA repair is common to p53 and the TFTC and NuA4/Tip60 complexes. The remaining TRRAP complex, PCAF/SAGA, has been postulated to function with p53 (58
), perhaps in a signaling capacity, to enhance its tumor suppressor effects. This hypothesis was based in part on the homology between TRRAP and the ATM family of proteins, all of which can transmit signals to p53 in the form of increased phosphorylation. Whether there is a physiological link between p53 and the DNA damage repair and apoptosis functions of the TRRAP acetyltransferase complexes is worthy of further investigation, particularly in light of the transcriptional link reported here.
Several issues related to p53 function are raised by this studies. Among the most important issues that remain to be addressed is how p53 mediates recruitment of the correct type of chromatin-modifying complex to each target gene. For example, how can p53 recruit HDACs to the Map4 and stathmin genes to repress transcription while recruiting p300 to the p21 gene and recruiting TRRAP complexes to the mdm2 gene to activate transcription? A second issue raised by these studies is whether p53 preferentially recruits any of the distinct TRRAP/HAT complexes to the mdm2 promoter. Our histone H3 and H4 acetylation data suggest that at least two distinct TRRAP complexes are recruited to the endogenous mdm2 promoter. Current efforts are aimed at identifying these complexes.
It is also of interest to determine the extent to which TRRAP plays a role in the regulation of other p53 target genes. Evidence for Gadd45 (Fig. ) suggests that the synergistic transactivation of the mdm2
promoter by p53 and TRRAP is not common to all p53 targets. This may be due to different chromatin contexts in which distinct p53 target gene promoters reside. For example, nucleosomal histones at the mdm2
promoter may be in such a configuration that their repositioning is necessary before transcription can occur, and recruitment of the TRRAP-associated HATs may be critical for this process. Conversely, the positioning of nucleosomes on the promoters of other p53 target genes may not be inhibitory to transcription or may require modifications distinct from those catalyzed by the TRRAP/HAT complexes. Finally, the robust TRRAP responsiveness of the mdm2
promoter may result from the unique juxtaposition of two p53 binding sites in this gene (2
) or from their intronic location.
In general, human cancers accumulate mutations that impair the p53 pathway (64
). These mutations can be within the p53 gene itself or in genes encoding other components of the p53 pathway. In tumors carrying wild-type p53, overexpression of the p53-degrading mdm2 protein is a common mechanism utilized to block p53 function (reviewed in reference 48
). Our demonstration that the TRRAP/HAT complexes play a role in mdm2
expression may have important implications in this subset of human tumors.
This study documents a novel biochemical mechanism utilized by p53 to activate transcription. In addition, p53 (along with c-Myc and E2F1) represents the third transcriptional regulator critical in human cancer that utilizes the TRRAP acetyltransferase complexes to activate gene expression. Finally, the direct recruitment of TRRAP acetyltransferase complexes to the mdm2 promoter by p53 to activate transcription provides a mechanistic symmetry to previous studies showing that recruitment of HDAC complexes by p53 mediates transcriptional repression.