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: A) Develop bioinformatic tools that identify and predict p53 transcription factor binding sites and identify SNPs in these sites. B) functionally assess p53 response elements and candidate SNPs in molecular and cellular assays, as well as in vivo human tissues. Accomplishments: 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 have evaluated an additional 64 SNPs in known or putative p53 response elements and validated differences using expression in lymphocyte cell lines (Bandele et al 2010). We have intitiated ChIP-seq experiments to further validate differential binding in cells. Conclusions/Significance. This microsphere assay system utilizes biologically meaningful cell extracts and is a quantitative alternative to qualitative, laborious mobility shift assays. It provides a powerful experimental tool for elucidating the functional impact of sequence and protein variation in transcriptional networks (Noureddine et al 2009, Bandele et al, 2010). NRF2 Oxidative Stress Project. Computational discovery and functional validation of polymorphisms in the ARE/NRF2 response pathway. 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 NRF2 binding sites and SNPs that modulate expression of ARE/NRF2-responnsive genes in human tissues (i.e. one allele weakens or abolishes the ARE/NRF2-dependent response of the adjacent gene).
Aims : A) Computationally evaluate 13 million human single nucleotide polymorphisms (SNPs) to identify SNPs in ARE/NRF2 responsive genes; B) Screen and prioritize the top candidates after analyzing available functional data, validation of genotype frequency, and evaluating expression in relevant human tissues; C) Characterize functional differences (i.e. luciferase, chromatin immunoprecipitation) between polymorphic alleles in NRF2-responsive genes identified in Aims above. 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: 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. Our integrated computational system for discovering functional SNPs and predicting their impact on the expression of target genes is accomplished by: (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. Use NRF2 ChIP-seq or ChIP on ChIP to demonstrate bone fide binding sites. We have identified functional polymorphisms in the antioxidant response element (ARE), found in the promoter region of NRF2 regulated genes including detoxification enzymes/proteins. We have identified a set of polymorphic AREs with functional evidence, and are carrying out experimental validation of these SNPs using ChIP, gene expression assays and ChIP-seq and ChIP on ChIP. In collaboration with Dr. Avrum Spira, Boston University Medical Center, we have identified SNPs that impact the expression of NRF2 regulated genes in bronchial epithelial cells (Wang et al 2010). We are examining oxidant-induced responses in umbilical cord blood of premature and full term neonates.
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