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.
Aim : 1) Develop bioinformatic tools that identify and predict p53 transcription factor binding sites and identify SNPs in these sites. Accomplishment: We have used an integrated approach to combine genome-wide maps of p53 occupancy with disease risk signals previously identified in genome-wide association studies to find potential functional p53RE SNPs that are associated with disease. We further integrate these data with diverse publicly available transcriptional functional datasets to provide evidence that several p53RE SNPs affect the ability of p53 to regulate transcription, and we propose plausible mechanisms for their influence on cancer (Stracquantio et al).
Aim 2) Assess functional variation in p53 response including binding, epigenetics, response elements and candidate SNPs in molecular and cellular assays, as well as in vivo human tissues. Accomplishments: Background. 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 and use ChIP-sequencing to determine binding strength. Methodology/Principal Findings. We analyzed stress-induced changes of p53 binding, chromatin state, and gene expression after treating human lymphoblastoid cells with the DNA-damaging agent doxorubicin and then mapping p53 binding and the chromatin activation mark, H3K4me3, by ChIP-seq. We discovered that p53-responsive genes showing the largest changes in expression had low levels of H3K4me3 and were repressed at baseline. Binding sites with greater similarity to p53RE consensus sequence correlated with increased p53 occupancy, however, chromatin landscape strongly influenced the relationship between occupancy and gene induction. Surprisingly, p53 strongly bound to thousands of DNA elements located in repressed chromatin that have recently evolved from human retroviral transposons. Characterizing the chromatin-mediated p53 stress response and the deregulation of transposons may prove to be clinically relevant for understanding outcomes in cytotoxic therapy for cancer (Su et al PLoS Gen). In collaboration with Maureen Murphy, Wistar Institute, we have previously identified CSF1 as a novel p53 target gene affected by a polymorphism in p53 that affects apoptosis and p53-mediated growth arrest (Azzam et al, 2013). Our current work has identified an African-specific polymorphic variant in the TP53 gene that impairs tumor suppressor function. A nonsynonymous single nucleotide polymorphism at codon 47 in the p53 tumor suppressor gene (P47S; rs1800371) exists in African populations. Here we report that the S47 variant has impaired tumor suppressor function and may affect cancer risk. We generated a mouse model for the P47S polymorphism and show that mice expressing the S47 form of p53 are susceptible to spontaneous cancers . We posit that the P47S polymorphism in p53 contributes to disparities in cancer risk and mortality in African-descent populations (Jennis et al, Genes and Dev). Conclusions/Significance. These studies reveal a functional link between variation in p53RE sequence and chromatin accessibility that seems to have been tuned via evolutionary selection pressure. (Millau et al 2011, Bandele et al, 2011, Zeron-Medina et al 2013, Azzam et al 2013, Jennis et al 2016, Stracquadanio et al 2016). 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. The overall objective of our proposal is to identify NRF2 binding sites and SNPs 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 : 1A) Computationally evaluate >20 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 (eQTLs) between alleles in NRF2-responsive genes identified in Aims above. Significance: The ARE/NRF2 response element SNPs identified here may be risk factors for oxidative stress related disease. Accomplishments: We have used an integrated approach to combine genome-wide maps of NRF2/MAF occupancy with eQTLs and disease risk signals previously identified in genome-wide association studies to find potential functional AREs SNPs that are associated with disease. The NRF2/sMAF protein complex regulates the oxidative stress response by occupying cis-acting enhancers containing an antioxidant response element (ARE). Integrating genome-wide maps of NRF2/sMAF occupancy with disease-susceptibility loci, we discovered 8 polymorphic AREs linked to 14 highly-ranked disease-risk SNPs in Caucasians. Among these SNPs was rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple experiments and its strong-binding allele associated with higher mRNA levels in cell lines and human brain tissue. Induction of MAPT transcription by NRF2 was confirmed using a human neuroblastoma cell line and a Nrf2-deficient mouse model. Most importantly, rs242561 displayed complete linkage disequilibrium with a highly protective allele identified in multiple GWASs of progressive supranuclear palsy, Parkinsons disease, and corticobasal degeneration. These observations suggest a potential role for NRF2/sMAF in tauopathies and a possible role for NRF2 pathway activators in disease prevention. 2) Characterize the role of new NRF2 target genes To identify human NRF2-regulated genes, we conducted ChIP-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates (Campbell et al, 2014) using gene expression to identify direct NRF2 target genes in Drosophila and humans. These data have allowed us to construct the deeply conserved ancient NRF2 regulatory network target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways, metabolism, and a number of less expected genes (Lacher et al). Genes regulated by NRF2 binding extend well beyond oxidative stress to those involved in heme metabolism, cell cycle, proteasome degradation, glucose metabolism, neurological function and retinoid signaling. NRF2-directed manipulation of these pathways could represent new therapeutic opportunities.
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