Isolation And Characterization Of Human Genes Affecting Chromosome Metabolism

Environmental Health Sciences

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Abstract

P53 MASTER REGULATORY NETWORK[unreadable] TRANSACTIVATION [unreadable] The DNA binding activity of the master regulator p53 is critical to its tumor suppressor activity in response to cellular and environmental stresses.The role of binding to individual REs in transactivation specificity was investigated using a highly regulatable p53 assay in yeast and human cells. The consensus target RE consists (RRRCWWGYYY)N(RRRCWWGYYY) where R= purine, Y = pyrimidine, W = A or T, and N = spacer. Nearly all REs have at least 1 mismatch and spacers tend to consist of only a few bases at most. We are deconstructing the canonical p53 consensus sequence in order to understand the role of sequence, organization and level of p53 on transactivation in budding yeast and human cell systems. Contrary to early reports for in vitro binding, increases in spacer length of only a few bases greatly reduces p53 transactivation. This has been confirmed using extracts from human cells in a novel microsphere binding assay. While p53 lacked transactivation capacity from many full-sized RE canonical sequences, it functioned to different extents from several noncanonical sites that are frequent in the genome including 3/4 sites (i.e., a decamer and an adjacent pentamer). Surprisingly, there can be substantial transactivation even at half-sites depending on sequence and p53 expression level. We are also analyzing the effect of mutations in key structural domains. Efficient transactivation from canonical and noncanonical elements requires tetrameric p53, based on results with mutations that yield either dimeric or monomeric proteins. [unreadable] These findings demonstrate that sequence and organization of a RE can have a large impact on p53-mediated transactivation. Furthermore, the loosening of a p53 binding element to include noncanonical sequences greatly expands the p53 transcriptional network. [unreadable] [unreadable] FUNCTIONAL EVALUTION OF HUMAN p53 MUTANTS.[unreadable] p53 missense mutations in the DNA binding domain are often cancer-associated. As shown with yeast-based systems, p53 mutants can alter the spectrum and intensity of transactivation from individual REs. We addressed in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV, ionizing radiation or spontaneously. These patterns reflected changes in the spectra and activity of target genes, as demonstrated for P21, MDM2, BAX and MSH2. Thus, as we originally proposed for master genes of diversity, p53 mutations in human cells can differentially influence target gene transactivation resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.[unreadable] [unreadable] Recently we began to investigate some unexpected roles that p53 may play in cell biology including cell migration, centrosome assembly and mitochondrial function. [unreadable] [unreadable] EVOLUTION OF p53 TRANSCRIPTIONAL NETWORK. [unreadable] Networks can evolve through variation of master regulators and/or by changes in regulation of genes within networks. Using a combination of custom bioinformatics and multispecies alignment of promoter regions, we investigated the functional evolution of REs in terms of responsiveness to the sequence-specific transcription factor p53, a tumor suppressor and master regulator of stress responses. We identified REs orthologous to known p53 targets in human and rodent cells or alternatively REs related to the established p53 consensus. The orthologous REs were then assigned p53 transactivation capabilities based on rules determined from model systems, and a functional heat map was developed for p53 transactivation towards 38 genes in 14 species. This approach emphasizes not only conservation of functionality but also addresses conservation of level of responsiveness. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional binding sites during p53 network evolution. Functional differences were often not predicted from consensus sequence evaluations. Of the 43 established human p53 REs analyzed, 50% were nonfunctional in rodents. Surprisingly, there was almost no conservation of functional REs or compensatory REs for genes involved in DNA metabolism or repair suggesting important differences in p53 stress responses as well as cancer development between humans and rodents.[unreadable] [unreadable] VARIATION OF p53 NETWORK[unreadable] SNPs in p53 REs and Flt1--We had developed a system to identify SNPs in potential p53 REs that are predicted to modify p53 control of target genes and establish their functional transactivation consequences. Recently we characterized a SNP in the promoter of the angiogenic factor FLT-1 receptor that might result in p53 transactivation. We examined the potential for p53 induced transactivation at the two SNP alleles. The expected good allele was much more responsive to p53 than the bad allele. However, the isolated p53 RE, which was actually a half-site, did not function. Instead, transactivation required another upstream site that corresponded to an estrogen receptor half-site. These results suggest that the FLT1 gene could be a novel target for p53 in a significant part of the population and that its transcriptional activation requires the cooperation between p53 and ERs acting on two partial REs, one of which is a SNP, then the p53 master regulatory network can become integrated directly with the estrogen master regulatory network. Our results demonstrate that different agents can cause a synergistic transcriptional response at a biologically important gene.[unreadable] [unreadable] EXPANSION OF p53 TRANSCRIPTIONAL NETWORK In a more global sense, these findings dramatically increase the size of a master regulatory network because of half-sites contributing to regulation, and they demonstrate new opportunities for cross-talk between networks. Using the p53 transactivation rules in combination with bioinformatic approaches we are employing genome-wide approaches to determine other putative p53 target genes containing p53 half-site REs that could present similar regulation. [unreadable] [unreadable] FRATAXIN--Friedreichs ataxia is a quantitative disease in that the level of reduction of frataxin appears to correlate with disease severity. The defect in most FRDA individuals is due to a triplet repeat expansion in intron I of the FXN gene. Given that slight alterations in frataxin expression can alter disease severity, we addressed frataxin expression levels in forty-seven FRDA patients by real-time PCR as part of a larger clinical study. Interestingly according to microarray analysis, patients with the lowest levels of frataxin expression experienced down-regulation of protein synthesis and oxidative phosphorylation pathways which may play a role in disease severity.[unreadable] Previously we had found with a yeast system that reduced levels of frataxin led to a) iron accumulation within mitochondria; b) mitochondrial DNA damage/loss and protein damage; and c) nuclear damage. To assess the biological consequences of reduced frataxin levels in human cells, we developed two methods involving RNA interference. The first is a transient transfection of siRNAs that lowers frataxin levels 70 to 90%, similar to levels in FRDA. While effective, the knockdown is short lived, so in order to address longer-term issues we are developing stable cell lines that contain a tetracycline-responsive frataxin miRNA system. Preliminary results suggest comparable knockdown to the transient siRNA system. We are utilizing these RNA interference systems as well as many cell lines from FRDA patients and relatives in order to establish the relationship between level of frataxin and responsiveness to environmental toxicants.