Phosphorylation of p53 by TAF1 inactivates p53-dependent transcription in the DNA damage response.

While p53 activation has long been studied, the mechanisms by which its targets genes are restored to their preactivation state are less clear. We report here that TAF1 phosphorylates p53 at Thr55, leading to dissociation of p53 from the p21 promoter and inactivation of transcription late in the DNA damage response. We further show that cellular ATP level might act as a molecular switch for Thr55 phosphorylation on the p21 promoter, indicating that TAF1 is a cellular ATP sensor. Upon DNA damage, cells undergo PARP-1-dependent ATP depletion, which is correlated with reduced TAF1 kinase activity and Thr55 phosphorylation, resulting in p21 activation. As cellular ATP levels recover, TAF1 is able to phosphorylate p53 on Thr55, which leads to dissociation of p53 from the p21 promoter. ChIP-sequencing analysis reveals p53 dissociates from promoters genome wide as cells recover from DNA damage, suggesting the general nature of this mechanism.

p53 acetylation is crucial for its transcription-independent proapoptotic functions.

Acetylation of p53 at carboxyl-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here we demonstrate that p53 acetylation at Lys-320/Lys-373/Lys-382 is also required for its transcription-independent functions in BAX activation, reactive oxygen species production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) suberoylanilide hydroxamic acid and LAQ824. Knock-out of p53 markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cells to HDACi-mediated BAX-dependent apoptosis, whereas knockdown of endogenous mutant p53 in cancer cells reduced HDACi-mediated cytotoxicity. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70. The association between these two proteins was acetylation-independent, but acetylation of p53 could prevent and disrupt the Ku70-BAX complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

An acetylation switch in p53 mediates holo-TFIID recruitment.

Posttranslational modifications mediate important regulatory functions in biology. The acetylation of the p53 transcription factor, for example, promotes transcriptional activation of target genes including p21. Here we show that the acetylation of two lysine residues in p53 promotes recruitment of the TFIID subunit TAF1 to the p21 promoter through its bromodomains. UV irradiation of cells diacetylates p53 at lysines 373 and 382, which in turn recruits TAF1 to a distal p53-binding site on the p21 promoter prior to looping to the core promoter. Disruption of acetyl-p53/bromodomain interaction inhibits TAF1 recruitment to both the distal p53-binding site and the core promoter. Further, the TFIID subunits TAF4, TAF5, and TBP are detected on the core promoter prior to TAF1, suggesting that, upon DNA damage, distinct subunits of TFIID may be recruited separately to the p21 promoter and that the transcriptional activation depends on posttranslational modification of the p53 transcription factor.

A specific PP2A regulatory subunit, B56gamma, mediates DNA damage-induced dephosphorylation of p53 at Thr55.

Protein phosphatase 2A (PP2A) has been implicated to exert its tumor suppressive function via a small subset of regulatory subunits. In this study, we reported that the specific B regulatory subunits of PP2A B56gamma1 and B56gamma3 mediate dephosphorylation of p53 at Thr55. Ablation of the B56gamma protein by RNAi, which abolishes the Thr55 dephosphorylation in response to DNA damage, reduces p53 stabilization, Bax expression and cell apoptosis. To investigate the molecular mechanisms, we have shown that the endogenous B56gamma protein level and association with p53 increase after DNA damage. Finally, we demonstrate that Thr55 dephosphorylation is required for B56gamma3-mediated inhibition of cell proliferation and cell transformation. These results suggest a molecular mechanism for B56gamma-mediated tumor suppression and provide a potential route for regulation of B56gamma-specific PP2A complex function.

Disinfection using ultraviolet radiation as an antimicrobial agent: a review and synthesis of mechanisms and concerns.

Control of microbes in sensitive health care settings cannot be maintained without effective disinfection methods. Recently, increasing microbial resistance to common chemical agents, including antibiotics, has required the implementation of new approaches to disinfection where such sanitary practices are critical. Pharmaceutical manufacturers and many interrelated industries also have a need for effective disinfection and sanitation strategies. As a result, standard practices that direct and maintain effective microbial controls are established. These practices exploit a thorough understanding of biological processes and manipulate them to preserve the quality and safety of a valuable product. Most biological processes are delicately balanced with their surroundings, or environment. When changes are made to the environment during disinfection, damaged microbes may respond by repairing the immediate damage or by adapting their biological processes through developing a resistance. Unless sound disinfection procedures are consistently applied, new strains of environmental microbes that can utilize multiple resistance markers may be artificially selected. Examples of related drug resistance are currently being studied and managed in many large hospitals, but non-chemical methods are also subject to similar concerns. Here, we review the use of ultraviolet-based disinfection practices, the biological basis for them, and some potential desensitization issues that may develop. Finally, we suggest some approaches to study and practically address these effects.

Mechanistic insights into maintenance of high p53 acetylation by PTEN.

Earlier studies have shown that PTEN regulated p53 protein stability both in a phosphatase-dependent manner through antagonizing Akt-Mdm2 pathway and in a phosphatase-independent manner through interacting with p53. In this study, we report that PTEN forms a complex with p300 in the nucleus and plays a role in maintenance of high p53 acetylation in response to DNA damage. Furthermore, p300 is required for nuclear PTEN-regulated cell cycle arrest. Interestingly, however, p53 acetylation was found to promote PTEN-p53 interaction. To investigate the molecular mechanisms, we show that acetylation promotes p53 tetramerization, which, in turn, is required for the PTEN-p53 interaction and subsequent maintenance of high p53 acetylation. Taken together, our results suggest a physiological role for the PTEN tumor suppressor in the nucleus and provide a molecular explanation for our previous observation that PTEN controls p53 protein levels independent of its phosphatase activity.

Purification of acetyl-p53 using p300 co-infection and the baculovirus expression system.

As cells persist in their environment, they are exposed to harmful agents that can damage their genomic DNA. When DNA becomes damaged, p53, a tumor suppressor, is stabilized and acts as a transcription factor to cause either cell cycle arrest or apoptosis. Strict p53 regulatory mechanisms have been well characterized relative to phosphorylation and dephosphorylation, but acetylation of p53 in response to DNA damage has also been shown to participate in p53 function. Proper investigation of the many roles that acetylated p53 plays in the cell requires accurate in vitro studies, which can only be easily conducted if highly pure acetyl-p53 is available. Purified p53 that is acetylated in vitro can routinely achieve 10-20%. Separating this acetylated fraction from the undesired unacetylated fraction can be technically challenging, inefficient, and time consuming. We have developed an in vivo strategy to rapidly produce microgram quantities of p53 preparations that are greater than 60% acetylated using co-infection of p53 and p300 baculoviruses in Sf21 insect cell culture. Immunoaffinity recovery followed by further depletion of unacetylated p53 results in a preparation that is greater than 70-75% in acetyl-p53 after a single round, and undetectable levels of unacetylated p53 after two rounds. This approach to preparing acetylated protein in vivo may also extend to other acetylated transcription factors and histones.