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) 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 have used ChIP-seq with p53, H3K4me3 and other chromatin factors as well as DNaseI seq in experiments to further examine the variables that impact the dynamics of p53 binding in cells. This work has revealed roles for chromatin context, p53 response element sequence content and evolution in stress-induced p53 genomic binding and transactivation. We have identified a polymorphic p53 response element in the KIT Ligand Gene that influences cancer risk and has undergone natural selection. We have identified that CSF1 is a novel p53 target gene affect by a polymorphism in p53 and whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest. We show that the CSF1 gene contains a conserved binding site for p53 and interestingly, that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). Conclusions/Significance. These studies reveal a functional link between variation in pp53RE 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 in press, Azzam et al in press). 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-responnsive 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 (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. This project is ongoing. 2) Characterize the role of new NRF2 target genes Background: Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like;NFE2L2) binding to DNA-regulatory sequences near stress responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2 in many biological pathways. 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. Activated NRF2 bound the NRF2 response element (TGActcaGC) in promoters of several known and novel target genes involved in iron hemostasis and heme metabolism, including known targets FTL and FTH1, and novel binding in the globin locus control region. Five hematopoietic genes that are candidates as novel NRF2 target genes were chosen for follow-up: AMBP, ABCB6, FECH, HRG-1 (SLC48A1), and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of KEAP1, negative regulator of NRF2 in K562 cells resulted in increased NQO1, AMBP and TBXAS1 expression. NF-E2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis. Future Plan: This project has been led by Michelle Campbell, Biologist. She will further verify a transcriptional role for NRF2 in erythroid cells, carrying out NRF2 ChIP-seq in erythroid cells treated with NRF2 activators. This is a collaboration with Michael Sporn, Dartmouth and with Drs. Ashley Chi and Marilyn Telen, Duke University, who are experts in hematology and sickle cell anemia. Importantly, this project has led to a funded clinical trial at Duke testing the use of NRF2 activators as therapeutic agents in sickle cell anemia. Conclusions: 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 and retinoid signaling. NRF2-directed manipulation of heme and RXR-mediated pathways could represent new therapeutic opportunities
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