The yeast Saccharomyces cerevisiae is invaluable as an in vivo test tube for human gene functions that extend to cell signaling, DNA metabolism and mitochondrial activities. We are focusing on several genes that have broad health significance: the Friedreich's ataxia (FRDA) gene frataxin, the tumor suppressor p53, and the homeodomain transcription factor NKX2.5 associated with heart disease.? FRATAXIN AND FRDA--The mitochondrial protein frataxin helps maintain iron levels in mitochondria (mt). FRDA is a progressive neuro-degenerative disease with early onset that results from a 70% to 90% reduction in levels of frataxin, a protein localized to the mt. Deletion of the frataxin gene YFH1 in yeast results in a 10-fold increase in iron within the mt and this leads to loss of mt function. ? We found that decreases in frataxin leads to nuclear genome stability, iron accumulation within the mt and the induction of mt petite mutants. There was considerable oxidative damage to mt proteins and accumulation of mt DNA lesions. Chronically-reduced frataxin levels resulted in similar response patterns. Furthermore nuclear DNA damage was detected in a rad52 mutant, deficient in double-strand break repair. We concluded that reduced frataxin levels result in oxidative damage in both mt and nuclear DNA.? In order to further understand these effects as well as the biological impacts of FRDA disease in humans, we are developing RNA interference systems in human cells that can merge potential damage from frataxin deficiency to cell signaling systems mediated by p53. We have established an efficient system for reducing levels of frataxin to those found in many patients. The reduced levels greatly increase sensitivity to hydrogen peroxided generated radical damage. ? TUMOR SUPPRESSOR p53--The p53 gene is central to many stress responses and genome stability in human cells. Nearly 50% of all cancers have an associated p53 mutation and most of these are missense mutants. We are addressing the sequence-specific transactivation function of p53 to better understand the consequences of tumor mutations and to use human p53 to approach the general issue of how in vivo transactivation specificity and selectivity are achieved.? Different biological responses can be elicited by p53-induced transcriptional networks, including cell cycle arrest, programmed cell death, cellular senescence and differentiation, and stimulation of DNA repair. The extent and kinetics of transcriptional modulation of individual genes likely dictates which biological response will be elicited but the mechanisms regulating such specificity remain to be clarified. p53 target genes contain in their promoters p53 response elements (REs) whose sequences are related to a degenerate 20 bp consensus and deviations from the consensus sequence in individual REs are common. Given the broad spectrum of p53 functions as a transcription factor and the many different p53 alleles with single amino acid changes that are aberrantly expressed in cancer cells, a detailed knowledge of the functional status of p53 mutants could have clinical value, especially for therapies tailored to specific tumors. ? Using yeast we address the transactivation capacity of p53 and various mutants. These are expressed with a tightly regulated (rheostatable) promoter so that the level of expression is proportional to level of inducer (galactose) in the medium. The ability of p53 and various mutants to act as sequence specific transactivation factors is determined by its ability to activate REs at promoters placed upstream of various reporters. By changing levels of p53 as well as changing REs, many issues are addressed including rules of binding and consequences of mutations on activating various REs. The system has also been useful for examining mutants outside the binding domain, in particular the Li-Fraumeni syndrom mutation R337C and the R337H mutation that give rise to adrenal cortical carcinoma and previously thought to be a silent mutation (R337H). ? We are also addressing the biological and functional impact of ectopic expression of the p53 mutants with altered transactivation capacity in human cell lines, including transformed and non-transformed cells with different p53 status and evaluating the effects on cell cycle progression, apoptosis, DNA repair, and activation of p53 targets. The differential consequences of the functional p53 mutants with altered transactivation capacity may result in changes in the transactivation patterns that would be advantageous during tumorigenesis and could be selected in particular cellular or genetic environments. In order to examine the consequences of functional p53 mutants, we also generated stable cell lines expressing p53 from a inducible responsive promoter. p53 null SasOS-2 cells were stably transfected with constructs expressing p53 from a tetracycline-regulated promoter. The levels of expression correspond to levels found after DNA damage stress. We are combining the luciferase reporter assays, real time PCR and microarray technologies to probe and better understand the global changes in gene expression underlying the complex selection of p53 downstream pathways. Remarkably, different p53 functional mutants can have dramatically different biological consequences including sensitivity to DNA damaging agents. ? The evolution of the p53 transcriptional network between rodents and primates is being examined. We are assessing the level of conservation (both sequence and predicted function) of the REs located in the promoter of p53 target genes in different species. Surprisingly, there are many genes associated with DNA metabolism that are under p53 control in human cells but not in mouse cells. Since rodents are often a model for human systems, these results suggest that for many affecting DNA metabolic processes, comparisons across species must be done with caution. ? SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) IN FLT1 PROMOTER AND REGULATION BY P53 --Genetic variation in promoter REs of p53 target genes could alter biological responses to stress and influence risk for diseases. The vascular endothelial growth and angiogenic factor (VEGF) exerts its biological effects, such as cell proliferation, differentiation, and apoptosis through the two high affinity receptors? Fms-like tyrosine kinase 1 (Flt-1)/vascular endothelial growth factor (VEGF) receptor 1 (VEGFR-1) and VEGF receptor-2 (=FLK-1/KDR or VEGFR-2). There is variation in the expression of the FLT-1 between individuals which appears to be due to a C/T SNP in its promoter sequence. This change was predicted to result in a p53 half site target RE (T allele) with limited responsiveness to p53 based on rules that we have developed. The C-allele would not be expected to exhibit p53 mediated transactivation. We have investigated FLT-1 induction and the consequences of the SNP and have established allele discrimination by p53. Importantly, we now establish that the regulation is dependent on an estrogen receptor half-site and binding by ligand associated estrogen receptor. ? NKX2.5--The transcription factor NKX2-5, is essential in early cardiac development. Sporadic NKX2-5 mutations can lead to septal heart defects and are usually present as multiple mutations. We developed a transcription system in yeast to functionally dissect the complex mutations that combines tight regulation of NKX2-5 and gene reporter assays under control of different naturally occurring variants of the NKX2-5 response element. Individual mutations were shown to contribute to NKX2-5 loss of function. The mutations do not exhibit dominance over the wild type, but instead show gene dosage effects. The system has been expanded to other heart development transcription factors. ? Public Health or Environmental Health Significance: FRATAXIN AND FRIEDREICHS ATAXIA?Results with our model frataxin gene system demonstrate that the mt compartment can be source of damage to nuclear material. This concept is novel in the sense that nuclear DNA lesions are generally thought to occur as a result of errors in nuclear metabolism, including replication and repair, or due to exposure to extraneous sources such as ionizing radiation or other genotoxicants. Oxidative damage to the mt DNA is an established fact accounting for the greater mutation rates in that organelle. Our results demonstrating that this organelle can be an internal source of nuclear DNA damage under certain conditions may impact on therapeutic strategies for mt-linked diseases including FRDA. Overall, our findings in yeast and emerging findings in human cells have implications for how mt associated syndromes could have impacts on nuclear genome stability. For the case of FRDA, our system is expected to prove helpful in the development of therapeutic strategies for FRDA and other neurodegenerative diseases that cause oxidative damage in mt. P53--Classification of p53 mutations based on p53 transactivation ability towards many target genes can help predict tumor aggressiveness and responsivenes. We predict that the number of mutations retaining partial function is likely to be greater than 20% of all p53 tumor mutations. The tools we developed make feasible the development of a functional matrix for each p53 mutant in terms of transactivation from specific REs. These data can be organized to develop a p53 mutant functionality database to be linked with the database of p53 mutations observed in tumor samples and cell lines that is updated yearly at the International Agency for Cancer Research (IARC), France. The functional profiles are likely to provide directions for effective patient management in the clinical setting. Our assays also provide sensitive p53 mutation screening tools of human tissues and may be useful for understanding the etiologies and the clinical manifestation of several cancers. Furthermore they allow for categorizing biochemical pathways induced by p53 and developing structure function/analyses of p53 mutant alleles. In a more general sense the system of tight regulation of protein expression, rapid construction of responsive promoters and phenotypic/quantitative assessment of transcription levels offers opportunities to investigate the regulation by other families of transcription factors that recognize several sequence-related response elements and/or have tightly regulated expression. SNPs IN P53 RESPONSE ELEMENTS--Our results suggest that there may be considerable variation in stress responses between individuals carrying RE polymorphisms and this has strong implications for cancer risk. Establishing a relationship between RE alleles and disease risk will require epidemiological investigation. Our study also supports and extends the concept of master genes of diversity which we previously proposed. Diversity can arise through functional mutations in a master regulator that change both the spectrum and intensity of downstream gene expression responses. The present observations demonstrate that diversity can also result from various combinations of REs and even half-REs that are contained in the genome of each individual. This diversity could be an important factor in governing environmental responses and the potential for disease. We suggest that the impact of identical cancer-associated functional p53 mutations could differ between individuals because of polymorphisms in response elements present in their genomes. NKX2.5 and FLT1--Our approaches with regulatable transcriptions, SNPs, and expression from different REs are enabling us to examine a wide variety of transcrition factors and the consequences of mutations. The recent studies with NKX2.5 and FLT1 are expected to prove important in the addressing their functions in heart disease and various cancers.. ?
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