P53 project. 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 identify functional SNPs in p53 transcription factor binding sites ? 2) functionally assess p53 response element and candidate SNPs in molecular and cellular assays under p53-control in yeast and mammalian cells.? ? Accomplisments: ? ? 1) Background. The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation, p53 binding and transactivation functions of p53, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. ? Methodology/Principal Findings. Using MAPD we measured sequence specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for 8 well-established human REs. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. We also adapted MAPD to measure interactions between estrogen receptor alpha and its cognate REs. ? Conclusions/Significance. This microsphere assay system utilizes biologically meaningful cell extracts, is a quantitative alternative to qualitative, laborious mobility shift assays and provides a powerful experimental tool for elucidating the functional impact of sequence and protein variation in transcriptional networks (manuscript submitted). ? ? NRF2 Oxidative Stress Project. 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 SNPs 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:? ? 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.? In addition we have developed methodologies for functionally assessing allelic imbalance and these are reviewed in Chorley et al. (2008).
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