There is growing evidence that the p53 tumor suppressor protein not only can function to activate gene transcription but also to repress the expression of specific genes. Although recent studies have implicated the transcriptional repression function of p53 in the pathway of apoptosis, the molecular basis of this activity remains poorly understood. This study takes a first step toward elucidating this mechanism. We report that trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs), abrogates the ability of p53 to repress the transcription of two genes that it negatively regulates, Map4 and stathmin. Consistent with this finding, we report that p53 physically associates in vivo with HDACs. This interaction is not direct but, rather, is mediated by the corepressor mSin3a. Both wild-type p53 and mSin3a, but not mutant p53, can be found bound to the Map4 promoter at times when this promoter preferentially associates with deacetylated histones in vivo. Significantly, inhibition of p53-mediated transcriptional repression with TSA markedly inhibits apoptosis induction by p53. These data offer the first mechanistic insights for p53-mediated transcriptional repression and underscore the importance of this activity for apoptosis induction by this protein.

Activation of the p53 tumor suppressor protein has been demonstrated to block cell growth by inducing either a transient cell cycle arrest or programmed cell death (apoptosis). Although evidence exists linking p53's function as an activator of transcription to its ability to effect cell cycle arrest, the role of this activity in the induction of apoptosis remains unclear. To gain insight into the molecular mechanisms underlying p53-mediated antiproliferative pathways, a study was initiated to explore the functions of a putative p53 signaling domain. This region of the human p53 protein is localized between amino acids 61 and 94 (out of 393) and is noteworthy in that it contains five repeats of the sequence PXXP (where P represents proline and X any amino acid). This motif has been shown to play a role in signal transduction via its SH3 domain binding activity. A p53 cDNA deletion mutant (⌬proAE), which lacks this entire proline-rich domain (deleted for amino acids 62-91), was created and characterized for a variety of p53 functions. The entire domain has been shown to be completely dispensable for transcriptional activation. On the other hand, this deletion of the p53 proline-rich domain impairs p53's ability to suppress tumor cell growth in culture. Amino acid substitution mutations at residues 22 and 23 of p53 (eliminates transcriptional activity) also impair p53-mediated inhibition of cell growth in culture. Unlike wild-type p53, the ⌬proAE mutant cDNA can be stably expressed in tumor derived cell lines with few immediate detrimental effects. These cells express physiologic levels of p53 protein that are induced normally in response to DNA damage, indicating that removal of the proline-rich domain does not disrupt p53's upstream regulation by DNA damage. These data indicate that, in addition to the transcriptional activation domain, the p53 proline-rich domain plays a critical role in the transmission of antiproliferative signals downstream of the p53 protein and may link p53 to a direct signal transduction pathway.The p53 tumor suppressor protein plays a pivotal role in the prevention of cellular transformation by curtailing the proliferation of cells harboring potentially oncogenic lesions. The unusually high frequency of p53 mutations observed in human cancers (1) indicates the complexity of the antiproliferative pathways under p53 regulation. Indeed, evidence now suggests that p53 can respond to multiple signals of cellular alarm, including DNA damage (2-4) and perturbations of cell cycle regulation (5-7), by inducing either a transient growth arrest (8-10) or programmed cell death (apoptosis; refs. 11-13). At the molecular level, p53 has been demonstrated to function as a transcriptional activator (14, 15). Transcriptional activation is dependent upon three independent structural domains that mediate: (i) sequence-specific DNA binding (amino acid residues 100-290 out of 393) (16, 17); (ii) interactions with the basal transcription factor TFIID (residues 1-40; refs. 18 and 19); and (iii) homooligo...

Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear. Our efforts to clarify these relationships have revealed two novel properties of nuclear receptors. First, we demonstrate that the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and that increased expression of PCAF leads to enhanced retinoid-responsive transcription. Second, we demonstrate that, in vitro, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, therefore defining a novel cofactor interaction surface. Furthermore, our results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors. This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself. On the basis of these observations, we suggest that PCAF may play a more central role in nuclear receptor function than previously anticipated.

It has been reported recently that the wild-type p53 gene product can positively regulate the expression of a test gene adjacent to the enhancer-promoter elements of the murine muscle-specific creatine kinase (MCK) gene. This discussion reports the identification of a wild-type p53 protein-specific DNA-binding element located within the p53-responsive region of the MCK enhancer-promoter element. This p53 protein/DNA-binding element has been defined by DNase I footprint analysis, which identified a 50-bp region. This 50-bp sequence was sufficient to confer wild-type p53 responsiveness on a heterologous minimal promoter. The mutant forms of p53 protein are much less capable of stimulating this DNA element. This study has identified the first example of a naturally occurring wild-type p53-specific DNA-binding element that is able to mediate positive regulation of a test gene. The results suggest a biological function in gene regulation for the wild-type p53 protein that is lost or altered in the mutant p53 proteins.[Key Words: Wild-type p53 protein; DNA-binding element; enhancer]