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)(Levings et al) NRF2 (encoded by the gene NFE2L2) is a redox-responsive transcription factor that regulates expression of cytoprotective genes via its interaction with regulatory DNA sequences known as antioxidant response elements (AREs). NRF2 activity is induced by oxidative stress, but oxidative stress is not the only context in which NRF2 can be activated. Mutations that disrupt the interaction between NRF2 and KEAP1, an important inhibitor of NRF2, lead to NRF2 hyperactivation and promote oncogenesis. The exact mechanisms underlying NRF2s oncogenic properties remain unclear, but likely involve aberrant expression of select NRF2 target genes. We tested this possibility using an integrative genomics approach to get a precise view of the direct NRF2 target genes dysregulated in tumors with NRF2 hyperactivating mutations. This approach revealed a core set of 32 direct NRF2 targets that are consistently upregulated in NRF2 hyperactivated tumors. This set of NRF2 cancer target genes includes canonical redox-related NRF2 targets (NQO1, TRX, GCLC, etc.), as well as several target genes that have not been previously linked to NRF2 activation. Importantly, NRF2-driven upregulation of this gene set is largely independent of the organ system where the tumor developed. One of the key distinguishing features of these NRF2 cancer target genes is that they are regulated by high affinity AREs that fall within genomic regions possessing a ubiquitously permissive chromatin signature. This finding implies that the NRF2 cancer target genes are highly responsive to oncogenic NRF2 in most tissues because they lack the regulatory constraints that restrict expression of most other NRF2 target genes. Notably, this NRF2 cancer target gene set also serves as a reliable proxy for NRF2 activity. High NRF2 activity is associated with significant decreases in survival in multiple cancer types. Overall, the pervasive upregulation of these NRF2 cancer targets across multiple cancers, and their association with negative prognostic outcomes, suggests that these will be central to dissecting the functional implications of NRF2 hyperactivation in several cancer contexts. 2.(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). 3. 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. (Bergens et al)Nucleated red blood cells (nRBCs/CD235a+ cells) are developmentally immature RBCs that display genome-wide hypomethylation and are observed at increased frequency in the cord blood of smoking mothers. We tested if AHRR methylation levels in CD235a+ nRBCs or nRBC counts influenced AHRR methylation in whole cord blood. Prenatal smoke exposure was highly significantly associated with AHRR methylation in cord blood, CD14+ monocytes, and CD235a+ nRBCs. AHRR methylation levels in nRBCs and nRBC counts had minimal effect on cord blood measurements. However, regression models using estimated nRBCs or actual nRBC counts outperformed those lacking these covariates.

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Liu, Yongmei; Reynolds, Lindsay M; Ding, Jingzhong et al. (2017) Blood monocyte transcriptome and epigenome analyses reveal loci associated with human atherosclerosis. Nat Commun 8:393
High, Monica; Cho, Hye-Youn; Marzec, Jacqui et al. (2016) Determinants of host susceptibility to murine respiratory syncytial virus (RSV) disease identify a role for the innate immunity scavenger receptor MARCO gene in human infants. EBioMedicine 11:73-84
Stracquadanio, Giovanni; Wang, Xuting; Wallace, Marsha D et al. (2016) The importance of p53 pathway genetics in inherited and somatic cancer genomes. Nat Rev Cancer 16:251-65
Wang, Xuting; Campbell, Michelle R; Lacher, Sarah E et al. (2016) A Polymorphic Antioxidant Response Element Links NRF2/sMAF Binding to Enhanced MAPT Expression and Reduced Risk of Parkinsonian Disorders. Cell Rep 15:830-842
Lacher, Sarah E; Lee, Joslynn S; Wang, Xuting et al. (2015) Beyond antioxidant genes in the ancient Nrf2 regulatory network. Free Radic Biol Med 88:452-465
Su, Dan; Wang, Xuting; Campbell, Michelle R et al. (2015) Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation. PLoS Genet 11:e1004885
Ciencewicki, Jonathan M; Wang, Xuting; Marzec, Jacqui et al. (2014) A genetic model of differential susceptibility to human respiratory syncytial virus (RSV) infection. FASEB J 28:1947-56
Azzam, Gregory; Wang, Xuting; Bell, Douglas et al. (2013) CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest. PLoS One 8:e74297
Zeron-Medina, Jorge; Wang, Xuting; Repapi, Emmanouela et al. (2013) A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection. Cell 155:410-22
Campbell, Michelle R; Karaca, Mehmet; Adamski, Kelly N et al. (2013) Novel hematopoietic target genes in the NRF2-mediated transcriptional pathway. Oxid Med Cell Longev 2013:120305

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