The p53 tumor suppressor protein is a master regulatory transcription factor that coordinates cellular responses to DNA damage and other sources of cellular stress. Besides mutations in p53, or in proteins involved in the p53 response pathway, genetic variation in promoter response elements (REs) of individual p53 target genes are expected to alter biological responses to stress. ? p53 project aims: ? 1) Develop bioinformatic tools that identifies functional SNPs in p53 transcription factor binding sites ? 2)functionally assess candidate SNPs in molecular and cellular assays under p53-control in yeast and mammalian cells.? ? Computational discovery and functional validation of polymorphisms in the ARE/NRF2 response pathway? Project Summary: The antioxidant response element (ARE) is a cis-acting enhancer sequence found in the promoter region of many genes encoding anti-oxidative and Phase II detoxification enzymes. In response to oxidative stress, the transcription factor NRF2 binds to AREs, mediating transcriptional activation of responsive genes and thereby modulating in vivo defense mechanisms against oxidative damage. Although studies identifying new genes in the ARE/NRF2 pathway have given insights into potential mechanisms of environmentally induced human disease, little is known about sequence variants that affect gene expression levels or that have functional phenotypic impact on exposure response. The overall objective of our proposal is to identify sets of single nucleotide polymorphism (SNP) allele pairs that modulate expression of ARE/NRF2-responsive genes in human tissues (i.e. one allele weakens or abolishes the ARE/NRF2-dependent response of the adjacent gene).
Aims :? 1) Computationally evaluate 10.5 million human single nucleotide polymorphisms (SNPs) to identify polymorphisms in ARE/NRF2 responsive genes; ? 2) Screen and prioritize the top candidates after analyzing available functional data, validation of genotype frequency, and evaluating expression in relevant tissues;? 3) Characterize functional differences (i.e. luciferase, chromatin immunoprecipitation) between polymorphic alleles in NRF2-responsive genes identified in Aims 1 and 2. ? ? Significance: The ARE/NRF2 response element SNPs identified here may be risk factors for developing oxidant-induced injury and may be predictive of clinical outcome following injury. This knowledge will be useful for identifying high-risk individuals and for developing novel prevention and treatment strategies.? ? Accomplishments:? ? 1. The p53 tumor suppressor is a sequence-specific pleiotropic transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell cycle arrest or triggering apoptosis. Although the human p53 binding site sequence (or response element, RE) is well-characterized, some genes have consensus-poor REs that are nevertheless both necessary and sufficient for transactivation by p53. Identification of new functional gene regulatory elements under these conditions is problematic and evolutionary conservation is often employed. We evaluated the comparative genomics approach for assessing evolutionary conservation of putative binding sites by examining conservation of 83 experimentally-validated human p53 REs against mouse, rat, rabbit and dog genomes, and detected pronounced conservation differences among p53 response elements and p53-regulated pathways. Bona fide NRF2 and NFB binding sites, which direct oxidative stress and innate immunity responses, were used as controls and both exhibited high interspecific conservation. Surprisingly, the average p53 RE was not significantly more conserved than background genomic sequence, and p53 REs in apoptosis genes as a group showed very little conservation. The common bioinformatics practice of filtering RE predictions by 80% rodent sequence identity would not only give a false positive rate of 19%, but miss up to 57% of true p53 REs. Examination of interspecific DNA base substitutions as a function of position in the p53 consensus sequence reveals an unexpected excess of diversity in apoptosis-regulating REs versus cell cycle controlling REs (rodent comparisons: p-values <1.0 E-12). While some p53 REs show relatively high levels of conservation, REs in many genes such as BAX, FAS, PCNA, CASP6, SIVA1, and P53AIP1 show little if any homology to rodent sequences. This difference suggests that among mammalian species, evolutionary conservation differs among p53 response elements, with some having ancient ancestry and others of more recent origin. Overall our results reveal divergent evolutionary pressure among the binding targets of p53 and emphasize that comparative genomics methods must be used judiciously and tailored to the evolutionary history of the targeted functional regulatory regions.? 2)Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes, and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system we: (1) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (2) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (3) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (4) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed; and (5) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.
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