Sharlyn J. Mazur

p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression

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 p53S315A knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53S315A 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.

Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 Apoptotic Activities after DNA Damage

ABSTRACT Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53K317R mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53K317R MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53K317R thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors.

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.

Structural Basis for p300 Taz2/p53 TAD1 Binding and Modulation by Phosphorylation

Coactivators CREB-binding protein 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 alpha 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 p53(1-39) phosphorylated at Thr(18) 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 Ser(15) and Thr(18).

INHIBITION OF RNA POLYMERASE II TRANSCRIPTION IN HUMAN CELL EXTRACTS BY CISPLATIN DNA DAMAGE

The anticancer drug cisplatin induces a spectrum of lesions in DNA. The effect of such DNA damage on transcription by RNA polymerase II (RNA pol II) in human cell extracts was investigated at the level of initiation and elongation. RNA pol II transcription directed from the adenovirus major late promoter was inhibited following treatment of the promoter-containing template with increasing concentrations of cisplatin. Furthermore, transcription from an undamaged promoter fragment was depleted in the presence of increasing amounts of cisplatin DNA damage on an exogenous plasmid, suggesting such damage may hijack an essential factor for transcription initiation. The effect of cisplatin damage on RNA pol II elongation was investigated using site-specifically-placed cisplatin adducts. The GTG adduct was an effective block to RNA pol II elongation, inhibiting the polymerase by 80%. In contrast, RNA pol II completely bypassed the cisplatin GG intrastrand adduct. These studies suggest that the inhibition of RNA pol II transcription observed following the treatment of cells with cisplatin is likely to reflect the combined effects of DNA damage at the level of both transcription initiation and elongation.

p53 N-terminal phosphorylation: a defining layer of complex regulation

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.

Oxidative DNA damage processing and changes with aging.

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.

Regulation of the Wip1 phosphatase and its effects on the stress response.

Wip1 (PPM1D) is a stress responsive PP2C phosphatase that plays a key role in stress signaling. Although originally identified as a gene induced by p53 after genotoxic stress, we now know that Wip1 expression is additionally regulated by other mechanisms. Wip1 is not only a target of p53, but is also a target of other transcription factors, including Estrogen Receptor-alpha and NF-kappaB. Additionally, Wip1 expression is regulated by post-transcriptional mechanisms such as mRNA stabilization and alternative splicing. Upon induction, Wip1 dampens the stress response by dephosphorylating and inactivating proteins such as p53, p38 MAPK, and ATM, usually as part of a negative feedback loop. As a result, Wip1 functions to abrogate cell cycle checkpoints and inhibit senescence, apoptosis, DNA repair, and the production of inflammatory cytokines. Furthermore, Wip1 is overexpressed in several types of human cancers and has oncogenic functions. The regulation of Wip1, the role of Wip1 in stress signaling, and the cooperation of Wip1 with oncogenes in promoting tumorigenesis will be discussed in this review.

The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis.

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 coexpressed 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. As 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.

Two Distinct Motifs within the p53 Transactivation Domain Bind to the Taz2 Domain of p300 and Are Differentially Affected by Phosphorylation

The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive posttranslational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogues CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser(15) or Thr(18) increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser(15) and Ser(37) or Thr(18) and Ser(20) further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 posttranslational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation, the change in heat capacity at constant pressure, DeltaC(p), was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of DeltaC(p) for Taz2 binding to the first (-330 cal/(mol.K)) compared to the second site (-234 cal/(mol.K)) suggests that the importance of nonpolar and polar interactions differs between the two sites.