Correction: Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

Following the publication of this article the authors noted that two images were duplicated in Figure 2B. The corrected figure 2B is below. The authors wish to apologize for any inconvenience caused.

p53 responsive elements in human retrotransposons.

Long interspersed nuclear elements-1 (L1s) are highly repetitive DNA elements that are capable of altering the human genome through retrotransposition. To protect against L1 retroposition, the cell downregulates the expression of L1 proteins by various mechanisms, including high-density cytosine methylation of L1 promoters and DICER-dependent destruction of L1 mRNAs. In this report, a large number of p53 responsive elements, or p53 DNA binding sites, were detected in L1 elements within the human genome. At least some of these p53 responsive elements are functional and can act to increase the levels of L1 mRNA expression. The p53 protein can directly bind to a short 15-nucleotide sequence within the L1 promoter. This p53 responsive element within L1 is a recent addition to evolution, appearing approximately 20 million years ago. This suggests an interplay between L1 elements, which have a rich history of causing changes in the genome, and the p53 protein, the function of which is to protect against genomic changes. To understand these observations, a model is proposed in which the increased expression of L1 mRNAs by p53 actually increases, rather than decreases, the genomic stability through amplification of p53-dependent processes for genomic protection.

Pro-proliferative FoxM1 is a target of p53-mediated repression.

The p53 tumor suppressor protein acts as a transcription factor to modulate cellular responses to a wide variety of stresses. In this study we show that p53 is required for the downregulation of FoxM1, an essential transcription factor that regulates many G2/M-specific genes and is overexpressed in a multitude of solid tumors. After DNA damage, p53 facilitates the repression of FoxM1 mRNA, which is accompanied by a decrease in FoxM1 protein levels. In cells with reduced p53 expression, FoxM1 is upregulated after DNA damage. Nutlin, a small-molecule activator of p53, suppresses FoxM1 levels in two cell lines in which DNA damage facilitates only mild repression. Mechanistically, p53-mediated inhibition of FoxM1 is partially p21 and retinoblastoma (Rb) family dependent, although in some cases p21-independent repression of FoxM1 was also observed. The importance of FoxM1 to cell fate was indicated by the observation that G2/M arrest follows FoxM1 ablation. Finally, our results indicate a potential contribution of p53-mediated repression of FoxM1 for maintenance of a stable G2 arrest.

Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function?

Although still controversial, the presence of mutant p53 in cancer cells may result in more aggressive tumors and correspondingly worse outcomes. The means by which mutant p53 exerts such pro-oncogenic activity are currently under extensive investigation and different models have been proposed. We focus here on a proposed mechanism by which a subset of tumor-derived p53 mutants physically interact with p53 family members, p63 and p73, and negatively regulate their proapoptotic function. Both cell-based assays and knock-in mice expressing mutant forms of p53 support this model. As more than half of human tumors harbor mutant forms of p53 protein, approaches aimed at disrupting the pathological interactions among p53 family members might be of clinical value.

Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

The p53 tumor suppressor is a nucleocytoplasmic shuttling protein that is found predominantly in the nucleus of cells. In addition to mutation, abnormal p53 cellular localization is one of the mechanisms that inactivate p53 function. To further understand features of p53 that contribute to the regulation of its trafficking within the cell, we analysed the subnuclear localization of wild-type and mutant p53 in human cells that were either permeabilized with detergent or treated with the proteasome inhibitor MG132. We, here, show that either endogenously expressed or exogenously added p53 protein localizes to the nucleolus in detergent-permeabilized cells in a concentration- and ATP hydrolysis-dependent manner. Two discrete regions within the carboxyl terminus of p53 are essential for nucleolar localization in permeabilized cells. Similarly, localization of p53 to the nucleolus after proteasome inhibition in unpermeabilized cells requires sequences within the carboxyl terminus of p53. Interestingly, genotoxic stress markedly decreases the association of p53 with the nucleolus, and phosphorylation of p53 at S392, a site that is modified by such stress, partially impairs its nucleolar localization. The possible significance of these findings is discussed.

Transcriptional regulation by p53: one protein, many possibilities.

The p53 tumor suppressor protein is a DNA sequence-specific transcriptional regulator that, in response to various forms of cellular stress, controls the expression of numerous genes involved in cellular outcomes including among others, cell cycle arrest and cell death. Two key features of the p53 protein are required for its transcriptional activities: its ability to recognize and bind specific DNA sequences and to recruit both general and specialized transcriptional co-regulators. In fact, multiple interactions with co-activators and co-repressors as well as with the components of the general transcriptional machinery allow p53 to either promote or inhibit transcription of different target genes. This review focuses on some of the salient features of the interactions of p53 with DNA and with factors that regulate transcription. We discuss as well the complexities of the functional domains of p53 with respect to these interactions.

P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation.

p21(Cip1/WAF1) is a known inhibitor of the short-gap filling activity of proliferating cell nuclear antigen (PCNA) during DNA repair. In agreement, p21 degradation after UV irradiation promotes PCNA-dependent repair. Recent reports have identified ubiquitination of PCNA as a relevant feature for PCNA-dependent DNA repair. Here, we show that PCNA ubiquitination in human cells is notably augmented after UV irradiation and other genotoxic treatments such as hydroxyurea, aphidicolin and methylmethane sulfonate. Intriguingly, those DNA damaging agents also promoted downregulation of p21. While ubiquitination of PCNA was not affected by deficient nucleotide excision repair (NER) and was observed in both proliferating and arrested cells, stable p21 expression caused a significant reduction in UV-induced ubiquitinated PCNA. Surprisingly, the negative regulation of PCNA ubiquitination by p21 does not depend on the direct interaction with PCNA but requires the cyclin dependent kinase binding domain of p21. Taken together, our data suggest that p21 downregulation plays a role in efficient PCNA ubiquitination after UV irradiation.

p53 tumor suppressor protein regulates the levels of huntingtin gene expression.

The p53 protein is a transcription factor that integrates various cellular stress signals. The accumulation of the mutant huntingtin protein with an expanded polyglutamine tract plays a central role in the pathology of human Huntington's disease. We found that the huntingtin gene contains multiple putative p53-responsive elements and p53 binds to these elements both in vivo and in vitro. p53 activation in cultured human cells, either by a temperature-sensitive mutant p53 protein or by gamma-irradiation (gamma-irradiation), increases huntingtin mRNA and protein expression. Similarly, murine huntingtin also contains multiple putative p53-responsive elements and its expression is induced by p53 activation in cultured cells. Moreover, gamma-irradiation, which activates p53, increases huntingtin gene expression in the striatum and cortex of mouse brain, the major pathological sites for Huntington's disease, in p53+/+ but not the isogenic p53-/- mice. These results demonstrate that p53 protein can regulate huntingtin expression at transcriptional level, and suggest that a p53 stress response could be a modulator of the process of Huntington's disease.

Transcriptional regulation by p53 and p73.

The tumor suppressor p53 exerts its effect through transactivation of a wide variety of genes leading to outcomes such as cell cycle arrest or apoptosis. Both p53 protein levels and modification status are thought to play a role in its ability to discriminate between different target genes and, thereby, cell fate. Here, we have determined the contribution of p53 levels to promoter selectivity when ectopically expressed in H1299 cells. Interestingly, p53AIP1, a pro-apoptotic p53 target gene, requires a significantly higher threshold level of p53 for its activation than p21WAF1, a cell cycle arrest gene. We also found that whereas exogenous p73 exhibits similar transcriptional activity to p53 in H1299 cells, the endogenous p73 that accumulates upon DNA damage in HCT116 cells is unable to compensate for p53 function. Quantification of protein expression levels revealed that the basal expression of TAp73 in HCT116 cells is very low and, even after induction by DNA damage, it accumulates to levels that are lower than basal uninduced levels of p53. These results might partially explain why, unlike p53, p73 does not function as a major tumor suppressor.

Cyclin a-CDK phosphorylation regulates MDM2 protein interactions.

The product of the MDM2 gene interacts with and regulates a number of proteins, in particular the tumor suppressor p53. The MDM2 protein is likely to be extensively modified in vivo, and such modification may regulate its functions in cells. We identified a potential cyclin-dependent kinase (CDK) site in murine MDM2, and found the protein to be efficiently phosphorylated in vitro by cyclin A-containing complexes (cyclin A-CDK2 and cyclin A-CDK1), but MDM2 was either weakly or not phosphorylated by other cyclin-containing complexes. Moreover, a peptide containing a putative MDM2 cyclin recognition motif specifically inhibited phosphorylation by cyclin A-CDK2. The site of cyclin A-CDK2 phosphorylation was identified as Thr-216 by two-dimensional phosphopeptide mapping and mutational analysis. Phosphorylation of MDM2 at Thr-216 both weakens its interaction with p53 and modestly augments its binding to p19(ARF). Interestingly, an MDM2-specific monoclonal antibody, SMP14, cannot recognize MDM2 phosphorylated at Thr-216. Changes in SMP14 reactivity of MDM2 in staged cell extracts indicate that phosphorylation of MDM2 at Thr-216 in vivo is most prevalent at the onset of S phase when cyclin A first becomes detectable.

A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain.

The p53 protein is related by sequence homology and function to the products of two other genes, p63 and p73, that each encode several isoforms. We and others have discovered previously that certain tumor-derived mutants of p53 can associate and inhibit transcriptional activation by the alpha and beta isoforms of p73. In this study we have extended these observations to show that in transfected cells a number of mutant p53 proteins could bind and down-regulate several isoforms not only of p73 (p73 alpha, -beta, -gamma, and -delta) but also of p63 (p63 alpha and -gamma; Delta Np63 alpha and -gamma). Moreover, a correlation existed between the efficiency of p53 binding and the inhibition of p63 or p73 function. We also found that wild-type p63 and p73 interact efficiently with each other when coexpressed in mammalian cells. The interaction between p53 mutants and p63 or p73 was confirmed in a physiological setting by examining tumor cell lines that endogenously express these proteins. We also demonstrated that purified p53 and p73 proteins interact directly and that the p53 core domain, but not the tetramerization domain, mediates this interaction. Using a monoclonal antibody (PAb240) that recognizes an epitope within the core domain of a subset of p53 mutants, we found a correlation between the ability of p53 proteins to be immunoprecipitated by this antibody and their ability to interact with p73 or p63 in vitro and in transfected cells. Based on these results and those of others, we propose that interactions between the members of the p53 family are likely to be widespread and may account in some cases for the ability of tumor-derived p53 mutants to promote tumorigenesis.

A role for p53 in base excision repair.

Wild-type p53 protein can markedly stimulate base excision repair (BER) in vitro, either reconstituted with purified components or in extracts of cells. In contrast, p53 with missense mutations either at hot-spots in the core domain or within the N-terminal transactivation domain is defective in this function. Stimulation of BER by p53 is correlated with its ability to interact directly both with the AP endonuclease (APE) and with DNA polymerase beta (pol beta). Furthermore, p53 stabilizes the interaction between DNA pol beta and abasic DNA. Evidence that this function of p53 is physiologically relevant is supported by the facts that BER activity in human and murine cell extracts closely parallels their levels of endogenous p53, and that BER activity is much reduced in cell extracts immunodepleted of p53. These data suggest a novel role for p53 in DNA repair, which could contribute to its function as a key tumor suppressor.

p53 down-regulates CHK1 through p21 and the retinoblastoma protein.

Both fission yeast and mammalian cells require the function of the checkpoint kinase CHK1 for G2 arrest after DNA damage. The tumor suppressor p53, a well-studied stress response factor, has also been shown to play a role in DNA damage G2 arrest, although in a manner that is probably independent of CHK1. p53, however, can be phosphorylated and regulated by both CHK1 as well as another checkpoint kinase, hCds1 (also called CHK2). It was therefore of interest to determine whether reciprocally, p53 affects either CHK1 or CHK2. We found that induction of p53 either by diverse stress signals or ectopically using a tetracycline-regulated promoter causes a marked reduction in CHK1 protein levels. CHK1 downregulation by p53 occurs as a result of reduced CHK1 RNA accumulation, indicating that repression occurs at the level of transcription. Repression of CHK1 by p53 requires p21, since p21 alone is sufficient for this to occur and cells lacking p21 cannot downregulate CHK1. Interestingly, pRB is also required for CHK1 downregulation, suggesting the possible involvement of E2F-dependent transcription in the regulation of CHK1. Our results identify a new repression target of p53 and suggest that p53 and CHK1 play interdependent and complementary roles in regulating both the arrest and resumption of G2 after DNA damage.

p53 accumulates but is functionally impaired when DNA synthesis is blocked.

p53 is required for the induction of a G(1) and/or G(2) irreversible arrest after gamma irradiation (IR), whereas blocked DNA replication causes a p53-independent S-phase arrest. We have examined the response to p53 when DNA synthesis is blocked by hydroxyurea (HU) or aphidicolin or when DNA is damaged by gamma IR. Similarly to gamma IR, blocked DNA synthesis induces high levels of phosphorylated nuclear p53. Surprisingly, several (but not all) p53 transcriptional targets that are rapidly induced by gamma IR are weakly or not induced when DNA replication is blocked. Moreover, the p53 response to gamma IR is inhibited by pretreatment of cells with HU or aphidicolin, suggesting that blocked DNA replication prevents p53 from being fully active as a transcription factor. HU-induced stabilization of p53 neither requires functional ATM (ataxia telangiectasia mutated), nor interferes with the gamma IR-dependent activation of the ATM kinase. Thus, stalled replication forks activate kinases that modify and stabilize p53, yet act downstream of ATM to impair p53 transcriptional activity. The ramifications of this novel regulation of p53 are discussed.

Why is p53 acetylated?

Recent studies suggest that acetylation of the p53 tumor suppressor protein is not important for its DNA binding activity, as was previously thought. We discuss here a number of theories as to how this modification may serve to regulate the protein's functions.

The N terminus of p53 regulates its dissociation from DNA.

It is important to gain insight into p53 DNA binding and how it is regulated. By using electrophoretic mobility shift assays and DNase I footprinting, we show that a region within the N terminus of the protein controls the dissociation of p53 from a p53-binding site. When p53 is bound by a number of N-terminal-specific monoclonal antibodies, its rate of dissociation from DNA is reduced, and its ability to protect a cognate site from DNase I digestion is increased. Moreover, greatly reduced dissociation is observed with p53 protein lacking the N-terminal 96 amino acids. By contrast, deletion of the C terminus does not affect p53 dissociation from DNA or DNase I protection. p53 protein expressed in and purified from bacterial cells displays markedly more instability on its consensus DNA-binding site than does p53 produced in insect cells, suggesting that post-translational modifications may affect the stability of the protein. Our results provide evidence that the N terminus of p53 possesses an auto-inhibitory function that is mechanistically different from the inhibitory region at the C terminus.