The yeast S. cerevisiae is an invaluable in vivo test tube for examining human gene functions and disease including, including cell signaling, DNA metabolism and mitochondrial function. We have focused on two human genes with broad health significance: the Friedreich's ataxia gene frataxin and the tumor suppressor p53. FRATAXIN AND FRIEDREICHS ATAXIA--The mitochondrial protein frataxin helps maintain appropriate iron levels in the mitochondria. Friedreich?s ataxia (FRDA) is a progressive neuro-degenerative disease with early onset that results from a deficiency in frataxin, a protein localized to the mitochondria (mt). Several model systems have been developed in an effort to understand the disease. None had been developed to investigate the relationship between mitochondrial damage and nuclear integrity. Deletion of the frataxin homolog YFH1 in yeast results in a 10-fold increase in iron within the mitochondria and this leads to loss of mitochondrial function and the appearance of a petite phenotype in nearly all strains that have been examined. We anticipated that a study of the consequences of frataxin loss could provide an understanding of the relationship between the mitochondria and the nucleus in terms of genome stability. In particular, we were interested in whether defects in the mitochondrial frataxin could lead to mitochondrial damage as well as lesions in mitochondrial and nuclear DNA. Furthermore, we wanted to develop a system that better represents the reduced levels of frataxin in FRDA. Using a highly regulatable system, we have shown that excess iron due to loss of frataxin within the mitochondria can generate ROS that in turn can cause nuclear genome instability, as measured by increases in mutation and recombination rates. However it is important to note that in recapitulating FRDA in model systems, the consequences of reduced activity and/or levels of proteins needs to be considered since the disease is associated with a deficiency of the protein frataxin rather than a complete absence. The highly regulatable, GAL1 promoter based system enabled the expression of variable levels of frataxin. Using this system we have been able to identify several consequences of reduced levels of frataxin including iron accumulation, mt protein damage, lesions in mtDNA, loss of mtDNA, the appearance of petites that lack mitochondrial DNA and the appearance of nuclear DNA damage in a sensitized rad52 mutant background. Our findings have implications for how mitochondrial 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 mitochondria. 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. 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. Although several methods have been attempted to classify p53 mutants, based on physical/chemical, or immunological/structural parameters, it is not presently possible to predict a priori the behavior of a mutant protein. p53 responds to a variety of stress signals by controlling, as a homotetramer, the expression of over 50 genes. 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. We have utilized yeast as an in vivo test tube to address the transactivation capacity of p53 and various mutants, as well as p53 family members (p63 and p73). These are expressed with a tightly controlled ?rheostatable promoter? so that the level of expression is proportional to level of inducer (galactose) in the medium. We have also systems with constitutive high expression (i.e., the ADH1 promoter). 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. The REs can be easily changed so that it is possible to determine transactivation capacity from many REs. Therefore, by changing levels of expressed p53 as well as RE?s, many issues can be addressed including rules of binding and consequences of mutations on activating various REs. Because of the ease of targeted mutagenesis in yeast, it is now relatively easy to address rapidly the consequences of tumor associated p53 mutations. On a broader scale, the approach has enabled us to investigate the evolution of transcription networks. In addition the system allows us to address other types of sequence specific transcription factors, such as NF kappa beta and NKX2.5 that act on many genes. 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. For example, mutants might affect specific pathways through altering the transcription network controlled by p53. Along this line, we have demonstrated that a p53 hotspot mutant in UV-induced skin tumors in mice, does in fact result in an altered spectrum of target gene tranasctivation by the mutant p53. Gene expression studies using both real time PCR and microarray technologies are being used to probe and better understand the global changes in gene expression underlying the complex selection of p53 downstream pathways.
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