Discovery And Functional Genomics Of Environmental Response Genes

National Institute Of Environmental Health Sciences

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Abstract

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. 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. To examine the variables that impact the dynamics of p53 binding in cells 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, 2015). In a collaboration with Maureen Murphy, Wistar Institute, we have previously identified that CSF1 is a novel p53 target gene affected by a polymorphism in p53 and whose protein product functions in a feed-forward manner to suppress apoptosis and enhance 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 contributes to increased 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 . In breast cancer samples from pre-menopausal African American women, we show that the P47S variant is significantly over-represented in cancer cases versus controls. We posit that the P47S polymorphism in p53 is an emerging and important contributor to disparities in cancer risk and mortality in African-descent populations (Jennis et al Submitted 2015). 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. (Noureddine et al 2009, Millau et al 2011, Bandele et al, 2011, Zeron-Medina et al Cell 2013, Azzam et al 2013). 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 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 (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 disease risk signals previously identified in genome-wide association studies to find potential functional AREs SNPs that are associated with disease. We further integrate these data with diverse publicly available transcriptional functional datasets to provide evidence that several ARE SNPs affect the ability of NRF2 to regulate transcription, and we propose a plausible mechanism for their influence on diseases. This project is ongoing. 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. We have also used enhancer reporter assays and electrophoretic mobility shift assays to confirm NRF2-mediated regulation of ARE (antioxidant response element) activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that and NRF2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear NRF2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the NRF2-ARE pathway, and indicate that NRF2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis have been central to this pathway since its inception (Lacher et al, 2015). Genes regulated by NRF2 binding extend well beyond the oxidative stress and phase 2 metabolism genes to those involved in heme metabolism, cell cycle control proteasome degradation, glucose metabolism and retinoid signaling. NRF2-directed manipulation of these pathways could represent new therapeutic opportunities.