Brian N. Chorley

Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha

Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. 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 that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.

Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies

The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease.

Nrf2-regulated PPARγ Expression Is Critical to Protection against Acute Lung Injury in Mice

RATIONALE The NF-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is essential for protection against oxidative injury and inflammation including hyperoxia-induced acute lung injury. Microarray expression profiling revealed that lung peroxisome proliferator activated receptor gamma (PPARgamma) induction is suppressed in hyperoxia-susceptible Nrf2-deficient (Nrf2(-/-)) mice compared with wild-type (Nrf2(+/+)) mice. PPARgamma has pleiotropic beneficial effects including antiinflammation in multiple tissues. OBJECTIVES We tested the hypothesis that PPARgamma is an important determinant of pulmonary responsivity to hyperoxia regulated by Nrf2. METHODS A computational bioinformatic method was applied to screen potential AREs in the Pparg promoter for Nrf2 binding. The functional role of a potential ARE was investigated by in vitro promoter analysis. A role for PPARgamma in hyperoxia-induced acute lung injury was determined by temporal silencing of PPARgamma via intranasal delivery of PPARgamma-specific interference RNA and by administration of a PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) in mice. MEASUREMENTS AND MAIN RESULTS Deletion or site-directed mutagenesis of a potential ARE spanning -784/-764 sequence significantly attenuated hyperoxia-increased Pparg promoter activity in airway epithelial cells overexpressing Nrf2, indicating that the -784/-764 ARE is critical for Nrf2-regulated PPARgamma expression. Mice with decreased lung PPARgamma by specific interference RNA treatment had significantly augmented hyperoxia-induced pulmonary inflammation and injury. 15 Deoxy-Delta(12,14)-prostaglandin J(2) administration significantly reduced hyperoxia-induced lung inflammation and edema in Nrf2(+/+), but not in Nrf2(-/-) mice. CONCLUSIONS Results indicate for the first time that Nrf2-driven PPARgamma induction has an essential protective role in pulmonary oxidant injury. Our observations provide new insights into the therapeutic potential of PPARgamma in airway oxidative inflammatory disorders.

Genetic Variation and Antioxidant Response Gene Expression in the Bronchial Airway Epithelium of Smokers at Risk for Lung Cancer

Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.

Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

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—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. 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 (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

Nuclear factor- (erythroid-derived 2) like 2 (NFE2L2, NRF2) is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN) to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq) identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE) in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: 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 NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 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.

A Polymorphic Antioxidant Response Element Links NRF2/sMAF Binding to Enhanced MAPT Expression and Reduced Risk of Parkinsonian Disorders

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 eight polymorphic AREs linked to 14 highly ranked disease-risk SNPs in individuals of European ancestry. 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.

The cellular and genomic response of rat dopaminergic neurons (N27) to coated nanosilver

This study examined if nanosilver (nanoAg) of different sizes and coatings were differentially toxic to oxidative stress-sensitive neurons. N27 rat dopaminergic neurons were exposed (0.5-5 ppm) to a set of nanoAg of different sizes (10nm, 75 nm) and coatings (PVP, citrate) and their physicochemical, cellular and genomic response measured. Both coatings retained their manufactured sizes in culture media, however, the zeta potentials of both sizes of PVP-coated nanoAg were significantly less electronegative than those of their citrate-coated counterparts. Markers of oxidative stress, measured at 0.5-5 ppm exposure concentrations, indicated that caspase 3/7 activity and glutathione levels were significantly increased by both sizes of PVP-coated nanoAg and by the 75 nm citrate-coated nanoAg. Both sizes of PVP-coated nanoAg also increased intra-neuronal nitrite levels and activated ARE/NRF2, a reporter gene for the oxidative stress-protection pathway. Global gene expression on N27 neurons, exposed to 0.5 ppm for 8h, indicated a dominant effect by PVP-coated nanoAg over citrate. The 75 nm PVP-coated material altered 196 genes that were loosely associated with mitochondrial dysfunction. In contrast, the 10nm PVP-coated nanoAg altered 82 genes that were strongly associated with NRF2 oxidative stress pathways. Less that 20% of the affected genes were shared by both sizes of PVP-coated nanoAg. These cellular and genomic findings suggest that PVP-coated nanoAg is more bioactive than citrate-coated nanoAg. Although both sizes of PVP-coated nanoAg altered the genomic expression of N27 neurons along oxidative stress pathways, exposure to the 75 nm nanoAg favored pathways associated with mitochondrial dysfunction, whereas the 10nm PVP-coated nanoAg affected NRF2 neuronal protective pathways.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Abstract B51: Discovery of novel genomic targets in the NRF2‐mediated antioxidant response pathway by ChIP‐on‐chip and ChIP‐seq

Sulforaphane is a dietary isothiocyanate that has been shown to have anticancer, antidiabetic and antimicrobial properties. Found in cruciferous vegetables, sulforaphane acts primarily as a phase II enzyme inducer through the pathway mediated by nuclear factor (erythroid‐derived 2)‐like 2 (NRF2). NRF2 is the key transcriptional activator in this pathway, binding as a heterodimer with small Maf proteins to cis ‐acting antioxidant response elements (AREs) found in the promoter regions of downstream oxidative response genes. In Nrf2 knockout mice, oxidative stress exposure causes increased damage to lung, liver and neurological tissue compared to wild type mice. Conversely, an aggressive cancer phenotype has been linked to permanently activated NRF2; indicating NRF2 may directly regulate other pathways unrelated to oxidant defense. In order to investigate NRF2 binding and gene regulation on a genome wide scale, we analyzed chromatin immunoprecipitated (ChIP) NRF2‐bound DNA using ChIP‐on‐chip and ChIP‐Seq technologies. Specifically, a human lymphoblastoid cell line (HapMap GM11994) was exposed in triplicate to 10uM sulforaphane, crosslinked, sonicated and immunoprecipitated with NRF2 antibody. NRF2‐bound DNA was amplified and hybridized to the Agilent Human Promoter chip, which provides −5.5 to +2.5 kilobase coverage around the majority of gene transcriptional start sites (ChIP‐on‐chip). Additionally, ChIP NRF2‐bound DNA was sequenced using the Solexa GAII second‐generation sequencing platform (ChIP‐seq). ChIP‐on‐chip intensity data was input to Agilent9s DNA analytics software and 1056 probes in promoter regions of 277 genes displayed sulforaphane ‐ induced NRF2 binding, 20 of which had been previously identified as NRF2‐regulated genes. Ingenuity pathway analysis found these 277 genes were involved in oxidative stress, cellular growth and proliferation, cellular development, small molecule biochemistry and other signaling pathways. We cross referenced the ChIP‐on‐chip data with results from gene expression (induction>1.2‐fold/suppression −6 ) in 60 sulforaphane‐exposed HapMap lymphoblastoid cells lines as determined by Illumina microarray. We identified 32 differentially expressed genes with NRF2 binding, 17 that had not previously been described as regulated by NRF2. Preliminary analysis of the ChIP‐seq data has mapped 2.1 million uniquely aligned sequencing reads resulting in 945 bound regions (high quality peaks). Of these, 5% are located near known NRF2‐binding regions, and approximately 20% of the regions overlap with the ChIP‐on‐chip platform. Using both the ChIP‐on‐chip and ChIP‐seq platform in this study has allowed us to investigate NRF2 on a genome‐wide scale and identify novel sulforaphane‐activated NRF2 gene targets. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B51.