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 FRIEDREICHS ATAXIA--The mitochondrial protein frataxin helps maintain iron levels in the mitochondria (mt). Friedreichs ataxia is a progressive neuro-degenerative disease with early onset that results from a reduced level 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 frataxin loss leads to nuclear genome stability leading us to develop a highly regulatable expression system that better represents reduced levels of frataxin characteristic of FRDA. The lowered levels led to changes normally associated with complete loss of frataxin including 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 conclude 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. 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 as well as 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 reports. Therefore, by changing levels of expressed p53 as well as REs, many issues can be 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 syndrome mutation R337C and the R337H mutation that give rise to adrenal cortical carcinoma and previously thought to be a silent mutation (R337H). In the yeast system they have very different impacts on transactivation kinetics. 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 address 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. 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. The evolution of the p53 transcriptional network between rodents and primates is being examined. We are comparing the transactivation capacity of murine and human p53 proteins and assessing the level of conservation (both sequence and predicted function) of the REs located in the promoter of p53 target genes in different species. SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) IN P53 TARGET REs-- Genetic variation in promoter REs of individual p53 target genes could alter biological responses to stress and influence risk for diseases. Examination of p53-mediated transactivation in our yeast system revealed vast variation in transactivation potential among 44 RE sequences, despite close relatedness to p53 consensus. Thus substantial diversity in transcription might result from polymorphisms in p53 REs, possibly leading to variability in cellular responses to stress. We developed custom bioinformatics approaches to search the dbSNP database for SNPs that might alter p53-mediated responses by interfering with potential p53 binding at RE sequences. Once candidates were identified, we utilized our functional yeast and mammalian cell assays to demonstrate the capacity of selected SNPs to differentially affect p53 transactivation. SNP IN FLT1 AND REGULATION BY P53--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. 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.
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