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 the 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. 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 2011). We have used ChIP-seq experiments to further validate differential binding in cells and are carrying out additional ChIP-seq experiments to examine how chromatin structure is affected by activation of p53. 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, Millau et al 2011, Bandele et al, 2011). 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 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 2011). 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. Results: We found 244 high-confidence, NRF2-bound genomic regions and 99% of these regions contained NRF2-regulatory sequences. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated about two thirds of the candidates are likely to be NRF2-dependent including ferrochelatase (FECH) and retinoid X receptor alpha (RXRA). FECH was induced by the NRF2 inducer SFN in several cell lines and in human blood. NRF2- mediated regulation of FECH, the gene defective in human erythropoetic protoporphyria, suggests the possibility of NRF2-targeted therapy for this disease. NRF2 regulation of RXRA has implications for numerous cellular responses including response to retinoids and adipogenesis. SFN treatment affects Rxra expression early in 3T3-L1 adipogenesis and knockdown of Nrf2 delays Rxra expression, both leading to impaired adipogenesis. 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 retinoid X receptor-mediated pathways could represent a new therapeutic opportunity.
Showing the most recent 10 out of 18 publications
