P53 Sequence-specific DNA Binding Research Articles

Inhibition of p53 DNA binding by a small molecule protects mice from radiation toxicity.

Transcription factors are attractive therapeutic targets that are considered non-druggable because they do not have binding sites for small drug-like ligands. We established a cell-free high-throughput screening assay to search for small molecule inhibitors of DNA binding by transcription factors. A screen was performed using p53 as a target, resulting in the identification of NSC194598 that inhibits p53 sequence-specific DNA binding in vitro (IC50 = 180 nM) and in vivo. NSC194598 selectively inhibited DNA binding by p53 and homologs p63/p73, but did not affect E2F1, TCF1, and c-Myc. Treatment of cells with NSC194598 alone paradoxically led to p53 accumulation and modest increase of transcriptional output owing to disruption of the MDM2-negative feedback loop. When p53 was stabilized and activated by irradiation or chemotherapy drug treatment, NSC194598 inhibited p53 DNA binding and induction of target genes. A single dose of NSC194598 increased the survival of mice after irradiation. The results suggest DNA binding by p53 can be targeted using small molecules to reduce acute toxicity to normal tissues by radiation and chemotherapy.

Resveratrol Rescues SIRT1-Dependent Adult Stem Cell Decline and Alleviates Progeroid Features in Laminopathy-Based Progeria

Abnormal splicing of LMNA gene or aberrant processing of prelamin A results in progeroid syndrome. Here we show that lamin A interacts with and activates SIRT1. SIRT1 exhibits reduced association with nuclear matrix (NM) and decreased deacetylase activity in the presence of progerin or prelamin A,leading to rapid depletion of adult stem cells (ASCs) in Zmpste24(-/-) mice. Resveratrol enhances the binding between SIRT1 and A-type lamins to increases its deacetylase activity. Resveratrol treatment rescues ASC decline, slows down body weight loss, improves trabecular bone structure and mineral density, and significantly extends the life span in Zmpste24(-/-) mice. Our data demonstrate lamin A as an activator of SIRT1 and provide a mechanistic explanation for the activation of SIRT1 by resveratrol. The link between conserved SIRT1 longevity pathway and progeria suggests a stem cell-based and SIRT1 pathway-dependent therapeutic strategy for progeria.

Quaternary structure of the specific p53–DNA complex reveals the mechanism of p53 mutant dominance

The p53 tumour suppressor is a transcriptional activator that controls cell fate in response to various stresses. p53 can initiate cell cycle arrest, senescence and/or apoptosis via transactivation of p53 target genes, thus preventing cancer onset. Mutations that impair p53 usually occur in the core domain and negate the p53 sequence-specific DNA binding. Moreover, these mutations exhibit a dominant negative effect on the remaining wild-type p53. Here, we report the cryo electron microscopy structure of the full-length p53 tetramer bound to a DNA-encoding transcription factor response element (RE) at a resolution of 21 Å. While two core domains from both dimers of the p53 tetramer interact with DNA within the complex, the other two core domains remain available for binding another DNA site. This finding helps to explain the dominant negative effect of p53 mutants based on the fact that p53 dimers are formed co-translationally before the whole tetramer assembles; therefore, a single mutant dimer would prevent the p53 tetramer from binding DNA. The structure indicates that the Achilles’ heel of p53 is in its dimer-of-dimers organization, thus the tetramer activity can be negated by mutation in only one allele followed by tumourigenesis.

RNA content in the nucleolus alters p53 acetylation via MYBBP1A

A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53-p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity.

The prolyl cis/trans isomerase cyclophilin 18 interacts with the tumor suppressor p53 and modifies its functions in cell cycle regulation and apoptosis

The functional diversity of the tumor suppressor protein p53 is mainly regulated by protein interactions. In this study, we describe a new interaction with the peptidyl-prolyl cis/trans isomerase cyclophilin 18 (Cyp18). The interaction reduced the sequence-specific DNA binding of p53 in vitro, whereas the inhibition of the interaction increased p53-reporter gene activity in vivo. The active site of the folding helper enzyme Cyp18 was directly involved in binding. The proline-rich region (amino acids 64-91) of p53 was most likely responsible for the observed binding because a synthetic peptide comprising amino acids 68-81 of p53 inhibited this interaction, and a p53 variant containing a proline residue at position 72 (p53(P72)) interacted with Cyp18 more effectively than the corresponding p53(R72) variant. Impairment of the Cyp18-p53 interaction induced an accumulation of cells in the G2/M phase of the cell cycle, which was more pronounced when p53(P72) was expressed compared with p53(R72) in an otherwise isogenic cellular background. Moreover, p53-dependent apoptosis was elevated in Cyp18 knockout cells, suggesting an antiapoptotic potential of Cyp18-p53 complexes. Functional in vivo data hint to a possible clinical relevance of the p53-Cyp18 interaction observed.

Redox-Dependent Translocation of p53 to Mitochondria or Nucleus in Human Melanocytes after UVA- and UVB-Induced Apoptosis

The p53 protein is an important transcription factor and tumor suppressor that is induced in response to many forms of cellular stress. UVA irradiation of human melanocytes caused generation of reactive oxygen species, which altered the intracellular redox balance and was accompanied by translocation of p53 to mitochondria. In contrast, UVB did not affect the redox status and p53 was translocated to the nucleus. Although different intracellular location of p53, UVA/B induced apoptosis through the intrinsic pathway detected as translocation of Bax to mitochondria, release of cytochrome c, and activation of caspases. These events were all prevented by inhibition of p53 with pifithrin-alpha. Furthermore, inhibition of p53 prevented lysosomal membrane permeabilization, detected as translocation of cathepsins to the cytosol, after UVB exposure, whereas UVA-induced lysosomal release was unaffected by inhibition of p53. In control cells, p53 coimmunoprecipitated with the antiapoptotic proteins Bcl-2 and Bcl-x(L) and upon UVA exposure the interaction was replaced by binding to the proapoptotic proteins Bax, Noxa, and Puma. Our findings suggest that UVA-induced apoptosis is caused by extensive oxidative damage leading to p53-regulated mitochondrial release, whereas UVB induces DNA damage and apoptosis signaling upstream of lysosomal membrane permeabilization.

Reversible Dysfunction of Wild-Type p53 following Homeodomain-Interacting Protein Kinase-2 Knockdown

About half of cancers sustain mutations in the TP53 gene, whereas the other half maintain a wild-type p53 (wtp53) but may compromise the p53 response because of other alterations. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of p53 oncosuppressor function. Here, we show, by microarray analysis, that wtp53 lost the target gene activation following stable knockdown of HIPK2 (HIPK2i) in colon cancer cell line. Our data show that the stable knockdown of HIPK2 led to wtp53 misfolding, as detected by p53 immunoprecipitation with conformation-specific antibodies, and that p53 protein misfolding impaired p53 DNA binding and transcription of target genes. We present evidence that zinc supplementation to HIPK2i cells increased p53 reactivity to conformation-sensitive PAb1620 (wild-type conformation) antibody and restored p53 sequence-specific DNA binding in vivo and transcription of target genes in response to Adriamycin treatment. Finally, combination of zinc and Adriamycin suppressed tumor growth in vivo and activated misfolded p53 that induced its target genes in nude mice tumor xenografts derived from HIPK2i cells. Bioinformatics analysis of microarray data from colon cancer patients showed significant association of poor survival with low HIPK2 expression only in tumors expressing wtp53. These results show a critical role of HIPK2 in maintaining the transactivation activity of wtp53 and further suggest that low expression of HIPK2 may impair the p53 function in tumors harboring wtp53.

DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites

The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.

Quantitative Proteomics Analysis of the Effects of Ionizing Radiation in Wild Type and p53K317R Knock-in Mouse Thymocytes

The tumor suppressor protein p53 is a sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. Following exposure to a variety of stresses, p53 becomes post-translationally modified with concomitant increases in activity and stability. To better understand the role of acetylation of Lys-317 in mouse p53, the effect of ionizing radiation (IR) on the thymocytes of p53(K317R) knock-in mice was studied at the global level. Using cleavable ICAT quantitative mass spectrometry, the effect of IR on protein levels in either the wild type or p53(K317R) thymocytes was determined. We found 102 proteins to be significantly affected by IR in the wild type thymocytes, including several whose expression has been shown to be directly regulated by p53. When the effects of IR in the wild type and p53(K317R) samples were compared, 46 proteins were found to be differently affected (p < 0.05). The p53(K317R) mutation has widespread effects on specific protein levels following IR, including the levels of proteins involved in apoptosis, transcription, and translation. Pathway analysis of the differently regulated proteins suggests an increase in p53 activity in the p53(K317R) thymocytes as well as a decrease in tumor necrosis factor alpha signaling. These results suggest that acetylation of Lys-317 modulates the functions of p53 and influences the cross-talk between the DNA damage response and other signaling pathways.

An ATM- and Rad3-related (ATR) Signaling Pathway and a Phosphorylation-Acetylation Cascade Are Involved in Activation of p53/p21Waf1/Cip1 in Response to 5-Aza-2′-deoxycytidine Treatment

Most agents that damage DNA act through posttranslational modifications of p53 and activate its downstream targets. However, whether cellular responses to nucleoside analogue-induced DNA damage also operate through p53 posttranslational modification has not been reported. In this study, the relationship between p53 activation and its posttranslational modifications was investigated in the human cancer cell lines A549 and HCT116 in response to 5-aza-2'-deoxycytidine (5-aza-CdR) or cytarabine treatment. 5-Aza-CdR induces p53 posttranslational modifications through activation of an ATM- and Rad3-related (ATR) signaling pathway, and 5-aza-CdR-induced association of replication protein A with chromatin is required for the binding of ATR to chromatin. Upon treatment with 5-aza-CdR, ATR activation is clearly associated with p53 phosphorylation at Ser(15), but not at Thr(18), Ser(20), or Ser(37). This specific p53 phosphorylation at Ser(15) in turn results in acetylation of p53 at Lys(320) and Lys(373)/Lys(382) through transcriptional cofactors p300/CBP-associated factor and p300, respectively. These p53 posttranslational modifications are directly responsible for 5-aza-CdR induced p21(Waf1/Cip1) expression because the binding activity of acetylated p53 at Lys(320)/Lys(373)/Lys(382) to the p21(Waf1/Cip1) promoter, as well as p21(Waf1/Cip1) expression itself are significantly increased after 5-aza-CdR treatment. It is of interest that p53 phosphorylation at Ser(15) and acetylations at Lys(320)/Lys(373)/Lys(382) mutually interact in the 5-aza-CdR induced p21(Waf1/Cip1) expression shown by transfection of artificially mutated p53 expression vectors including S15A, K320R, and K373R/K382R into p53-null H1299 cells. These data taken together show for the first time that 5-aza-CdR activates the ATR signaling pathway, which elicits a specific p53 phosphorylation-acetylation cascade to induce p21(Waf1/Cip1) expression.

DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner

Proteins p53 and p73 act as transcription factors in cell cycle control, regulation of cell development and/or in apoptotic pathways. Both proteins bind to response elements (p53 DNA-binding sites), typically consisting of two copies of a motif RRRCWWGYYY. It has been demonstrated previously that DNA modification with the antitumor drug cisplatin inhibits p53 binding to a synthetic p53 DNA-binding site. Here we demonstrate that the effects of global DNA modification with cisplatin on binding of the p53 or p73 proteins to various p53 DNA-binding sites differed significantly, depending on the nucleotide sequence of the given target site. The relative sensitivities of protein-DNA binding to cisplatin DNA treatment correlated with the occurrence of sequence motifs forming stable bifunctional adducts with the drug (namely, GG and AG doublets) within the target sites. Binding of both proteins to mutated p53 DNA-binding sites from which these motifs had been eliminated was only negligibly affected by cisplatin treatment, suggesting that formation of the cisplatin adducts within the target sites was primarily responsible for inhibition of the p53 or p73 sequence-specific DNA binding. Distinct effects of cisplatin DNA modification on the recognition of different response elements by the p53 family proteins may have impacts on regulation pathways in cisplatin-treated cells.

C-Abl acetylation by histone acetyltransferases regulates its nuclear-cytoplasmic localization.

c-Abl function is strictly dependent on its subcellular localization. Using an in vitro approach, we identify c-Abl as a new substrate for p300, CBP (CREB-binding protein) and PCAF (p300/CBP-associated factor) histone acetyltransferases. Remarkably, acetylation markedly alters its subcellular localization. Point mutagenesis indicated that Lys 730, located in the second nuclear localization signal, is the main target of p300 activity. It has previously been reported that c-Abl accumulates in the cytoplasm during myogenic differentiation. Here, we show that c-Abl protein is acetylated at early stages of myogenic differentiation. Indeed, acetylation on Lys 730 drives c-Abl accumulation in the cytoplasm and promotes differentiation. Thus, Lys 730 acetylation is a novel post-translational modification of c-Abl and a novel mechanism for modulating its subcellular localization that contributes to myogenic differentiation.

The versatile interactions of p53 with DNA: when flexibility serves specificity

The tumor suppressor p53 exerts versatile interactions withDNA. Of importance is the binding of wild-type p53 to p53-responsive elements in promoters of p53 target genes. Thisinteraction is complex and determined by DNA sequence andstructure, and involves the p53 core DNA binding and theC-terminal domain. In addition, wild-type p53 binds linearDNA with high affinity in a sequence-independent manner,andnon-BDNAinaDNAstrictlystructure-selectivemodethatis not dependent on the presence of a p53-responsiveelement. Mutant p53 has lost sequence-specific DNA bindingand high-affinity binding to linear DNA, but has retained theability to interact with DNA in a structure-selective manner.We discuss the interactions of p53 with DNA, which arecharacterized by a high flexibility, both on the side of p53 andDNA, thereby providing p53 with the specificity required for itsfunctions.The major molecular property of p53 is that of a DNAbinding protein. Consequently, its interaction with DNA iscentral to various p53-mediated activities. Best known andanalyzed is the sequence-specific DNA binding of p53 (p53-SSDB), followed by transcriptional activation of genesinvolved in regulation of the cell cycle, DNA repair orapoptosis,uponactivationofthep53pathwaybyendogenousor exogenous stress factors.

Binding of Rad51 and Other Peptide Sequences to a Promiscuous, Highly Electrostatic Binding Site in p53

Homologous recombination is repressed by the binding of p53 to Rad51. We identified by fluorescence and NMR spectroscopy that peptides corresponding to residues 179-190 of Rad51 bind to the core domain of p53 in a site that overlaps with its specific DNA binding site. The p53 site is quite promiscuous, since it also binds peptides derived from 53BP1, 53BP2, Hif-1alpha, and BCL-X(L) in overlapping regions. Binding is mediated mainly by a strong, nonspecific, electrostatic component and is fine tuned by specific interactions. Competition of the different proteins with each other and with specific DNA for a single site in p53 could be a factor in regulation of its activity.

Activation of the DNA-binding ability of latent p53 protein by protein kinase C is abolished by protein kinase CK2.

p53 is one of the most important regulators of cell proliferation and differentiation and of programmed cell death, triggering growth arrest and/or apoptosis in response to different cellular stress signals. The sequence-specific DNA-binding function of p53 protein can be activated by several different stimuli that modulate the C-terminal domain of this protein. The predominant mechanism of activation of p53 sequence-specific DNA binding is phosphorylation at specific sites. For example, phosphorylation of p53 by PKC (protein kinase C) occurs in undamaged cells, resulting in masking of the epitope recognized by monoclonal antibody PAb421, and presumably promotes steady-state levels of p53 activity in cycling cells. In contrast, phosphorylation by cdk2 (cyclin-dependent kinase 2)/cyclin A and by the protein kinase CK2 are both enhanced in DNA-damaged cells. We determined whether one mechanism to account for this mutually exclusive phosphorylation may be that each phosphorylation event prevents modification by the other kinase. We used non-radioactive electrophoretic mobility shift assays to show that C-terminal phosphorylation of p53 protein by cdk2/cyclin A on Ser315 or by PKC on Ser378 can efficiently stimulate p53 binding to DNA in vitro, as well as binding of the monoclonal antibody Bp53-10, which recognizes residues 371-380 in the C-terminus of p53. Phosphorylation of p53 by CK2 on Ser392 induces its DNA-binding activity to a much lower extent than phosphorylation by cdk2/cyclin A or PKC. In addition, phosphorylation by CK2 strongly inhibits PKC-induced activation of p53 DNA binding, while the activation of p53 by cdk2/cyclin A is not affected by CK2. The presence of CK2-mediated phosphorylation promotes PKC binding to its docking site within the p53 oligomerization domain, but decreases phosphorylation by PKC, suggesting that competition between CK2 and PKC does not rely on the inhibition of PKC-p53 complex formation. These results indicate the crucial role of p53 C-terminal phosphorylation in the regulation of its DNA-binding activity, but also suggest that antagonistic relationships exist between different stress signalling pathways.

General transcriptional coactivator PC4 activates p53 function.

The function of p53 is modulated by several transcriptional coactivators that regulate its tumor suppressor activity. Here we report that human transcriptional coactivator PC4 enhances the DNA binding of p53 to its cognate site in vitro and directly interacts with p53 in vivo. In vitro interaction studies demonstrated that the C-terminal 30 amino acids (364 to 393) of p53 strongly interact with PC4. Surprisingly, PC4 also stimulates the sequence-specific DNA binding of p53 with the C-terminal 30 amino acids deleted (p53Delta30), suggesting that PC4 mediates enhancement of p53 DNA binding by a unique mechanism. We also demonstrated that PC4 can stimulate p53- and p53Delta30-mediated transactivation from a p53-responsive promoter. Furthermore, PC4 enhances p53- and p53Delta30-dependent apoptosis by inducing bax (a p53-targeted proapoptotic gene) gene expression. These results establish the first physiological role of PC4 as a transcriptional coactivator.

Specific Interaction of p53 with Target Binding Sites Is Determined by DNA Conformation and Is Regulated by the C-terminal Domain

Transcriptional activation of p53-regulated genes is initiated by sequence-specific DNA binding of p53 to target binding sites. Regulation of sequence-specific DNA binding is complex and occurs at various levels. We demonstrate that DNA topology is an important parameter for regulating the selective and highly specific interaction of p53 with its target binding sites. Specific binding of wild-type p53 is greatly enhanced when cognate binding sites are present in a non-linear stem-loop conformation. The C-terminal domain plays a key role in regulating the specific interactions of p53 with target binding sites in a DNA conformation-dependent manner. The C-terminal domain is required for binding to target sites in a non-linear DNA conformation in contrast to the strong inhibitory effects of the C terminus on p53 interaction with linear DNA. We propose that selective binding of p53 to various promoters may be determined by the DNA conformation within p53 cognate sites.

Efficient specific DNA binding by p53 requires both its central and C-terminal domains as revealed by studies with high-mobility group 1 protein.

The nonhistone chromosomal protein high-mobility group 1 protein (HMG-1/HMGB1) can serve as an activator of p53 sequence-specific DNA binding (L. Jayaraman, N. C. Moorthy, K. G. Murthy, J. L. Manley, M. Bustin, and C. Prives, Genes Dev. 12:462-472, 1998). HMGB1 is capable of interacting with DNA in a non-sequence-specific manner and causes a significant bend in the DNA helix. Since p53 requires a significant bend in the target site, we examined whether DNA bending by HMGB1 may be involved in its enhancement of p53 sequence-specific binding. Accordingly, a 66-bp oligonucleonucleotide containing a p53 binding site was locked in a bent conformation by ligating its ends to form a microcircle. Indeed, p53 had a dramatically greater affinity for the microcircle than for the linear 66-bp DNA. Moreover, HMGB1 augmented binding to the linear DNA but not to the microcircle, suggesting that HMGB1 works by providing prebent DNA to p53. p53 contains a central core sequence-specific DNA binding region and a C-terminal region that recognizes various forms of DNA non-sequence specifically. The p53 C terminus has also been shown to serve as an autoinhibitor of core-DNA interactions. Remarkably, although the p53 C terminus inhibited p53 binding to the linear DNA, it was required for the increased affinity of p53 for the microcircle. Thus, depending on the DNA structure, the p53 C terminus can serve as a negative or a positive regulator of p53 binding to the same sequence and length of DNA. We propose that both DNA binding domains of p53 cooperate to recognize sequence and structure in genomic DNA and that HMGB1 can help to provide the optimal DNA structure for p53.

Latent and active p53 are identical in conformation.

p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.

The Gene Encoding p202, an Interferon-inducible Negative Regulator of the p53 Tumor Suppressor, Is a Target of p53-mediated Transcriptional Repression

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We reported previously that overexpression of p202, an interferon-inducible negative regulator of cell growth, negatively regulates the transcriptional activity of p53. Now we identify the gene encoding p202 as one whose mRNA and protein expression decrease in cells following the expression of wild-type, but not mutant, p53. Furthermore, the levels of p202 also decrease after exposure of cells to ultra violet light, which correlate with increase in the levels of p53. We report that the sequence-specific DNA binding of p53 to the 5'-regulatory region of the 202 gene contributes to the transcriptional repression of the 202 gene. Interestingly, overexpression of p202 in cells induced to undergo p53-dependent apoptosis significantly delays this process, indicating that the negative regulation of the 202 gene by wild-type p53 is important to potentiate apoptosis.