[show abstract][hide abstract] ABSTRACT: Transcriptional activation by the tumor suppressor p53 is considered to depend on cellular level, although there are few systems where this dependence on cellular level of p53 has been directly addressed. Previously, we reported that transactivation from p53 targets was sensitive to both p53 amount and DNA sequence, with some sequences being responsive to much lower p53 levels than others when examined in yeast model systems or human cells. Because p53 is normally present at low levels and perturbations might lead to small increases, we examined transactivation under limiting p53. Unlike the positive relationship between transactivation and binding affinity from target sequences at high cellular levels of human p53 in yeast, no such relationship was found at low levels. However, transactivation in the yeast system and the torsional flexibility of target sequences were highly correlated, revealing a unique structural relationship between transcriptional function and sequence. Surprisingly, a few sequences supported high transactivation at low p53 levels in yeast or when transfected into human cells. On the basis of kinetic and flexibility analyses the "supertransactivation" property was due to low binding off rates of flexible target sites. Interestingly, a supertransactivation response element can differentiate transcriptional capacities of many breast cancer-associated p53 mutants. Overall, these studies, which are relevant to other transcription factors, address the extent to which transactivation properties of p53 target sequences are determined by their intrinsic physical properties and reveal unique rules of engagement of target sequences at low p53 levels.
Proceedings of the National Academy of Sciences 08/2012; 109(36):14387-92. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4.
Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i) variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.
We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.
PLoS ONE 01/2011; 6(6):e20643. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mutations of the sequence-specific master regulator p53 that alter transactivation function from promoter response elements (RE) could result in changes in the strength of gene activation or spectra of genes regulated. Such mutations in this tumor suppressor might lead to dramatic phenotypic changes and diversification of cell responses to stress. We have determined "functional fingerprints" of sporadic breast cancer-related p53 mutants, many of which are also associated with familial cancer proneness such as the Li-Fraumeni syndrome and germline BRCA1/2 mutant-associated cancers. The ability of p53, wild-type and mutants, to transactivate from 11 human target REs has been assessed at variable expression levels using a cellular, isogenomic yeast model system that allows for the rapid analysis of p53 function using a qualitative and a quantitative reporter. Among 50 missense mutants, 29 were classified as loss of function. The remaining 21 retained transactivation toward at least one RE. At high levels of galactose-induced p53 expression, 12 of 21 mutants that retain transactivation seemed similar to wild-type. When the level of galactose was reduced, transactivation defects could be revealed, suggesting that some breast cancer-related mutants can have subtle changes in transcription. These findings have been compared with clinical data from an ongoing neoadjuvant chemotherapy treatment trial for locally advanced breast tumors. The functional and nonfunctional missense mutations may distinguish tumors in terms of demographics, appearance, and relapse, implying that heterogeneity in the functionality of specific p53 mutations could affect clinical behavior and outcome.
Molecular Cancer Research 05/2010; 8(5):701-16. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Germline CDKN2A mutations are observed in 20-50% of melanoma-prone families. We identified melanoma patients that were heterozygous for non-coding germline variants in the 5'-UTR of CDKN2A (c.-21C > T; c.-25C > T&c.-180G > A; c.-56G > T; c.-67G > C) and examined their impact on the p16(INK4a) 5'-UTR activity using two luciferase-based reporter vectors that differ in basal transcription level and that were transfected into the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells. The wild-type 5'-UTR sequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G > T), was included as controls. Results revealed that the variants at -21 and -34 severely reduced the reporter activity. The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C were dependent on the plasmid type. Quantification of the luciferase mRNA indicated that the effects of the variants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmed that c.-21C > T and c.-34G > T had a severe negative impact in both cell lines. We also applied a polysomal profiling technique to samples heterozygous for the 5'-UTR variants, including patient-derived lymphoblasts. Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > C variants also reduced mRNA translation efficiency. Overall, our results suggest that the c.-21C > T sequence variant is a melanoma-predisposing mutation. The c.-25C > T&c.-180G > A and particularly the c.-56G > T variants showed a range of intermediate functional defects in the different assays, and were not observed in the control population. We propose that these variants should be considered as potential mutations.
Human Molecular Genetics 04/2010; 19(8):1479-91. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0-13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0-13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi-in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical (1/2)-(a single decamer) and (3/4)-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of (1/2)- and (3/4)-site REs greatly expands the p53 master regulatory network.
[show abstract][hide abstract] ABSTRACT: The TP53 tumor suppressor gene encodes a sequence-specific transcription factor that is able to transactivate several sets of genes, the promoters of which include appropriate response elements. Although human cancers frequently contain mutated p53, the alleles as well as the clinical expression are often heterogeneous. Germ line mutations of TP53 result in cancer proneness syndromes known as Li-Fraumeni, Li-Fraumeni--like, and nonsyndromic predisposition with or without family history. p53 mutants can be classified as partial deficiency alleles or severe deficiency alleles depending on their ability to transactivate a set of human target sequences, as measured using a standardized yeast-based assay (see http://www.umd.be:2072/index.html). We have investigated the extent to which the functional features of p53 mutant alleles determine clinical features in patients who have inherited these alleles and have developed cancer.
We retrieved clinical data from the IARC database (see http://www.p53.iarc.fr/Germline.html) for all cancer patients with germ line p53 mutations and applied stringent statistical evaluations to compare the functional classification of p53 alleles with clinical phenotypes.
Our analyses reveal that partial deficiency alleles are associated with a milder family history (P = 0.007), a lower numbers of tumors (P = 0.007), and a delayed disease onset (median, 31 versus 15 years; P = 0.007) which could be related to distinct tumor spectra.
These findings establish for the first time significant correlations between the residual transactivation function of individual TP53 alleles and clinical variables in patients with inherited p53 mutations who develop cancer.
Clinical Cancer Research 08/2007; 13(13):3789-95. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The p53 master regulatory network provides for the stress-responsive direct control of a vast number of genes in humans that can be grouped into several biological categories including cell-cycle control, apoptosis and DNA repair. Similar to other sequence-specific master regulators, there is a matrix of key components, which provide for variation within the p53 master regulatory network that include p53 itself, target response element sequences (REs) that provide for p53 regulation of target genes, chromatin, accessory proteins and transcription machinery. Changes in any of these can impact the expression of individual genes, groups of genes and the eventual biological responses. The many REs represent the core of the master regulatory network. Since defects or altered expression of p53 are associated with over 50% of all cancers and greater than 90% of p53 mutations are in the sequence-specific DNA-binding domain, it is important to understand the relationship between wild-type or mutant p53 proteins and the target response elements. In the words of the legendary detective Sherlock Holmes, it is 'Elementary, my dear Mr. Watson'.
[show abstract][hide abstract] ABSTRACT: Inducible promoter fusions are commonly employed to study the biological functions of genes as well as to investigate mechanisms of transcription regulation. A concern for many studies of heterologous gene expression is that steady state transcription may be too high under non-inducing conditions, producing undesired phenotypes prior to induction. Fusions containing the galactose-inducible GAL1 promoter joined to PvuII, a bacterial DNA endonuclease gene, are toxic to yeast cells even under non-inducing conditions, i.e., in glucose media. This toxicity was utilized in conjunction with PCR-based mutagenesis of the GAL1 regulatory region to isolate mutant promoters that retained high inducibility but exhibited reduced basal level expression. The Mig1 repressor binding and putative TATA box regions were unchanged among four mutant promoters examined in detail. However, each promoter contained one or more mutations within previously identified binding sites for the Gal4 activator protein. Genetic assays developed to monitor GAL1p::I-SceI endonuclease-induced recombination demonstrated that basal expression from two of the new promoters (designated GAL1-V4 and GAL1-V10) was strongly reduced. These experiments and additional quantitative luciferase reporter gene assays demonstrate the utility of the approach for identifying promoters that permit more tightly controlled gene expression.