The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53(S315A) knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53(S315A) ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.

Summary Coactivators CBP and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short α-helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p531-39 phosphorylated at Thr18 is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser15 and Thr18.

Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms Δ133p53 and p53β are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8 + T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28 -CD57 + ) with decreased Δ133p53 and increased p53β expression. Human lung tumor-associated CD8 + T lymphocytes also harbored senescent cells. Cultured CD8 + blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and Δ133p53 protein. In poorly proliferative, Δ133p53-low CD8 + CD28 -cells, reconstituted expression of either Δ133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, Δ133p53 knockdown or p53β overexpression in CD8 + CD28 + cells inhibited cell proliferation and induced senescence. This study establishes a role for Δ133p53 and p53β in regulation of cellular proliferation and senescence in vivo. Furthermore, Δ133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection.

Nanog is a transcription factor required for maintaining the pluripotency of embryonic stem cells, and is not expressed in most normal adult tissues. However, recent studies have indicated that Nanog is overexpressed in many types of human cancers, including breast cancer. To elucidate the physiological roles of Nanog in tumorigenesis, we developed an inducible Nanog transgenic mouse model, in which the expression of Nanog in adult tissues can be induced via LoxP/Cre-mediated deletion. Our findings indicate that overexpression of Nanog in the mammary gland is not sufficient to induce mammary tumor. However, when co-expressed with Wnt-1 in the mouse mammary gland, it promotes mammary tumorigenesis and metastasis. In this context, Nanog promotes the migration and invasion of breast cancer cells. Microarray analysis has shown that the ectopic expression of Nanog deregulates the expression of numerous genes associated with tumorigenesis and metastasis, such as the PDGFRα gene. Our findings demonstrate the involvement of Nanog in breast cancer metastasis and provide the basis for the reported correlation between Nanog expression and poor prognosis of human breast cancer patients. Since Nanog is not expressed in most adult tissues, these findings identify Nanog as a potential therapeutic target in the treatment of Nanog-expressing metastatic breast cancer.

Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERTnegative counterpart containing a transferred, normal chromosome 3 (RCC23ϩ3). This E-box element mediated repression of hTERT transcription in RCC23ϩ3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23ϩ3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box-binding protein complex in both RCC23 and RCC23ϩ3 and, importantly, detected an RCC23ϩ3-specific, E-box-binding factor that was distinct from the USF and Myc/Mad families. The E-box-mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.

The p53 tumor suppressor is a critical component of the cellular response to stress. As it can inhibit cell growth, p53 is mutated or functionally inactivated in most tumors. A multitude of protein-protein interactions with transcriptional cofactors are central to p53-dependent responses. In its activated state, p53 is extensively modified in both the N- and C-terminal regions of the protein. These modifications, especially phosphorylation of serine and threonine residues in the N-terminal transactivation domain, affect p53 stability and activity by modulating the affinity of protein-protein interactions. Here, we review recent findings from in vitro and in vivo studies on the role of p53 N-terminal phosphorylation. These modifications can either positively or negatively affect p53 and add a second layer of complex regulation to the divergent interactions of the p53 transactivation domain.

The initial stages in the repair of damaged DNA by the Escherichia coli uvr system involve the recognition of damage by UvrA. We have examined in detail the binding of UvrA to DNA randomly damaged by ultraviolet light, undamaged DNA, and single-stranded DNA using nitrocellulose filter binding and gel mobility shift assays to arrive at the following model: UvrA dimers bind specifically to damaged DNA both in the presence and in the absence of ATP. The dimerization of UvrA is promoted by UvrA concentrations greater than 1 nM, the presence of ATP, or physiological temperatures, and the dimerization step dominates the temperature dependence of UvrA binding to DNA damaged by ultraviolet light. The apparent association constant for specific binding is dependent on the concentration of UvrA due to coupled dimerization, aggregation, and nonspecific binding reactions. At 1 nM UvrA, either with or without ATP, Kuv approximately 10(9) M-1. The binding of UvrA to undamaged DNA is 10(3)-10(4)-fold weaker than the damage-specific binding. Both the strength of damage-specific binding and the discrimination between damaged and undamaged sites are affected by the salt concentration. The kinetics of association and dissociation reactions indicate that the primary effects of ATP are on the extent of UvrA dimerization rather than on the properties of the UvrA-uvDNA complex. The complexity of the interaction of UvrA, ATP, and DNA is indicated by the opposing effects of ATP binding and hydrolysis on UvrA dimerization.

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser 376 or Ser 378 completely abolishes this inhibition. Following blockage of DNA replication, Ser 15 phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases. Bloom and Werner syndromes (BS and WS)1 are autosomal recessive disorders characterized by immune deficiency, cancer predisposition, and chromosomal instability (1). The products of the genes responsible for these disorders, BLM and WRN, are ATP-dependent DNA helicases that exhibit 3Ј to 5Ј polarity. Mutations in the BLM or WRN genes disrupt their helicase activity, which may be important for the phenotypic traits associated with these hereditary diseases (2).Homologous recombination (HR) is required for genetic exchange during meiosis, repair of complex lesions in DNA, and the segregation of chromosomes at cell division. Expression of the BLM or WRN helicases in Saccharomyces cerevisiae containing a mutation in sgs1, a BLM and WRN homolog, suppresses their increased rates of illegitimate recombination and HR (3). BLM and its yeast homologue, Sgs1, functionally interact with topoisomerase III (4), whereas the WRN interaction with DNA polymerase ␦ is apparently required for some aspect of DNA replication and/or repair (5, 6). Recent reports indicate that both of these helicases recognize and disrupt alternative DNA structures, including G-quadruplex DNA and Holliday junctions (HJ) (7-11). HJ arise as intermediates during HR and can occur spontaneously, or during DNA replication and the repair of DNA damage (12). BLM and WRN may promote ATP-dependent translocation of HJ to eliminate DNA recombination intermediates, thereby reducing inappropriate DNA recombination in vivo (10, 11). p53 suppresses genomic instability, particularly in response to DNA damage (13,14). p53 also has been implicated in HR. Evidence of p53 modulation of HR includes the following: (a) overexpression of wild-type p53 (WT p53) can down-regulate the rate of HR between SV40 molecules (15); (b) the rate of HR is increased in p53 mutant cell lines (16 -18); (c) p53 has 3Ј to 5Ј exonuclease and DNA strand transfer activities (19); and...