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. 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. 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). 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 Cell, 2013, Azzam et al 2013, Jennis et al 2016, Stracquadanio et al 2016). In FY18 we are not working on p53. 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 project 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). Accomplishments: 1)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, we discovered 8 polymorphic AREs linked to 14 highly-ranked disease-risk SNPs in Caucasians (Wang et al 2016). 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 (Lacher et al) 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. Late onset Alzheimer's disease (AD) is a multifactorial disorder, with AD risk influenced by both environmental and genetic factors. Recent genome-wide association studies (GWAS) have identified genetic loci associated with increased risk of developing AD. The MS4A (membrane-spanning 4-domains subfamily A) gene cluster is one of the most significant loci associated with AD risk, and MS4A6A expression is correlated with AD pathology. We identified a single nucleotide polymorphism, rs667897, at the MS4A locus that creates an antioxidant response element and links MS4A6A expression to the stress responsive Cap-n-Collar (CNC) transcription factors NRF1 (encoded by NFE2L1) and NRF2 (encoded by NFE2L2). The risk allele of rs667897 generates a strong CNC binding sequence that is activated by proteostatic stress in an NRF1-dependent manner, and is associated with increased expression of the gene MS4A6A. Together, these findings suggest that the cytoprotective CNC regulatory network aberrantly activates MS4A6A expression and increases AD risk in a subset of the population. (Lacher et al). 3) (Rooney et al) Computational approaches were developed to identify factors that modulate Nrf2 in a mouse liver gene expression compendium. Forty-eight Nrf2 biomarker genes were identified using profiles from the livers of mice in which Nrf2 was activated genetically in Keap1-null mice or chemically by a potent activator of Nrf2 signaling. The rank-based Running Fisher statistical test was used to determine the correlation between the Nrf2 biomarker genes and a test set of 81 profiles with known Nrf2 activation status demonstrating a balanced accuracy of 96%. For a large number of factors examined in the compendium, we found consistent relationships between activation of Nrf2 and feminization of the liver transcriptome through suppression of the male-specific growth hormone (GH)-regulated transcription factor STAT5b. The livers of female mice exhibited higher Nrf2 activation than male mice in untreated or chemical-treated conditions. In male mice, Nrf2 was activated by treatment with ethinyl estradiol, whereas in female mice, Nrf2 was suppressed by treatment with testosterone. Nrf2 was activated in 5 models of disrupted GH signaling containing mutations in Pit1, Prop1, Ghrh, Ghrhr, and Ghr. Out of 59 chemical treatments that activated Nrf2, 36 exhibited STAT5b suppression in the male liver. The Nrf2-STAT5b coupling was absent in in vitro comparisons of chemical treatments. The enhanced basal and inducible levels of Nrf2 activation in females relative to males provides a molecular explanation for the greater resistance often seen in females vs. males to age-dependent diseases and chemical-induced toxicity (Rooney et al 2018). 4. DNA methylation studies. (Wan et al 2018) Using an innovative high-resolution reduced representation bisulfite sequencing (RRBS) technique, NIEHS researchers found novel modifications in DNA methylation patterns present in circulating immune cells from smokers in two independent human studies from the NIEHS Clinical Research Unit and the Multi-Ethnic Study of Atherosclerosis (MESA). DNA methylation plays an essential role in gene regulation in response to environmental and developmental stress. RRBS revealed novel smoking-associated differentially methylated regions (SM-DMRs) and a poised enhancer region of the aryl-hydrocarbon receptor repressor (AHRR) gene in the strongest of the SM-DMRs seen in CD15+ granulocytes and CD14+ monocytes. Surprisingly these were also easily detected in DNA from saliva cells, which are composed mostly of leukocytes similar to blood composition. Methylation of the AHRR CpG site, cg05575921, has been associated with smoking and subclinical atherosclerosis. In smokers, the AHRR SM-DMR activates the AHRR enhancer region, which increases enhancer non-coding RNA in monocytes and upregulates AHRR messenger RNA. This novel finding suggests that AHRR activation may be a risk factor in atherosclerosis and saliva DNA may be useful for detecting alterations in AHRR methylation.