Publications by authors named "Neal A Englert"

10 Publications

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Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

Environ Health Perspect 2018 04 27;126(4):047015. Epub 2018 Apr 27.

Environmental Epigenomics and Disease Group, Immunity, Inflammation, and Disease Laboratory, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Research Triangle Park, North Carolina, USA.

Background: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure.

Objective: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes.

Method: We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [=38 from Clinical Research Unit (CRU) and =55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells (=19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA.

Results: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene () and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near , , and were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the SM-DMR appeared to up-regulate mRNA through activating the enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers.

Conclusions: Our findings suggest that SM-DMR up-regulates mRNA in a monocyte-specific manner by activating the enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.
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http://dx.doi.org/10.1289/EHP2395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071796PMC
April 2018

Pregnane X receptor (PXR) deficiency improves high fat diet-induced obesity via induction of fibroblast growth factor 15 (FGF15) expression.

Biochem Pharmacol 2017 10 27;142:194-203. Epub 2017 Jul 27.

Department of Preventive Medicine, School of Public Health and Management, Wenzhou Medical University, Wenzhou, Zhejiang 325035, PR China. Electronic address:

Obesity has become a significant global health problem, and is a high risk factor for a variety of metabolic diseases. Fibroblast growth factor (FGF) 15 plays an important role in the regulation of metabolism. Xenobiotic-sensing nuclear receptors pregnane X receptor (PXR/NR1I2) and constitutive androstane receptor (CAR/NR1I3) play important roles in xenobiotic detoxification and metabolism, and also are involved in the regulation of energy metabolism. However, the effects that PXR and CAR have on the regulation of FGF15 are unknown. Here, we found that body weight, hepatic triglyceride levels, liver steatosis, and hepatic mRNA expression levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1), the key enzymes in the bile acid classical synthesis pathway, were significantly decreased in high fat diet (HFD)-fed PXR knockout (KO) mice compared to HFD-fed wild-type mice. Interestingly, intestinal FGF15 expression levels were significantly elevated in HFD-fed PXR KO mice compared with HFD-fed wild-type mice. Additionally, serum total bile acid levels were significantly decreased in PXR KO mice than those in wild-type mice when fed a control diet or HFD. Total lipids in feces were significantly increased in HFD-fed PXR KO mice compared to HFD-fed wild-type mice. However, these alterations were not found in HFD-fed CAR KO mice. These results indicate that PXR deficiency improves HFD-induced obesity via induction of FGF15 expression, resulting in suppression of bile acid synthesis and reduction of lipid absorption, hepatic lipid accumulation and liver triglyceride levels. Our findings suggest that PXR may negatively regulate FGF15 expression and represent a potential therapeutic target for the treatment for metabolic disorders such as obesity.
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http://dx.doi.org/10.1016/j.bcp.2017.07.019DOI Listing
October 2017

Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes.

PLoS One 2016 9;11(12):e0166486. Epub 2016 Dec 9.

Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.

Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166486PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147832PMC
July 2017

Upregulation of human CYP2C9 expression by Bisphenol A via estrogen receptor alpha (ERα) and Med25.

Environ Toxicol 2017 Mar 8;32(3):970-978. Epub 2016 Jun 8.

Department of Preventive Medicine, School of Environmental Science and Public Health, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, PR China.

Bisphenol A (BPA) is an important industrial chemical, mainly used in the manufacture of polycarbonate plastic and epoxy resins. Due to its widespread use, humans have a high risk of exposure to BPA. BPA has been found to have adverse health effects such as interfering with hormone-related pathways and is well-known to act as an endocrine disruptor. The present study is the first to show the induction effect of BPA on gene expression and enzyme activity of CYP2C9, an important hepatic drug metabolizing enzyme in human. We further identify the mechanism of BPA upregulation of CYP2C9 expression. We show that BPA is able to transcriptionally activate CYP2C9 promoter through ERα and ERE site within the CYP2C9 promoter region in HepG2 cells, and can induce CYP2C9 gene expression and enzyme activity in human primary hepatocytes. Moreover, we demonstrate that Med25, a variable member of the Mediator complex, is a coactivator of ligand-activated ERα that interacts with ERα through its C-terminal LXXLL motif after BPA exposure, and is functionally involved in BPA-induced transcriptional regulation of CYP2C9 expression and enzyme activity. Our findings suggest that BPA exposure has a potential risk for adverse health effects in human liver metabolism by upregulation of CYP2C9 expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 970-978, 2017.
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http://dx.doi.org/10.1002/tox.22297DOI Listing
March 2017

Pyrene is a Novel Constitutive Androstane Receptor (CAR) Activator and Causes Hepatotoxicity by CAR.

Toxicol Sci 2015 Oct 8;147(2):436-45. Epub 2015 Jul 8.

*Department of Preventive Medicine, School of Environmental Science and Public Health, Wenzhou Medical University, Wenzhou, Zhejiang 325035, People's Republic of China and

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous persistent environmental pollutants which are primarily formed from the incomplete combustion of organic materials. Many potential sources of human exposure to PAHs exist, including daily exposures from the ambient environment or occupational settings. PAHs have been found to cause harmful effects on human health. Here, we evaluated the adverse effects of pyrene, a common PAH, on the liver. The present study demonstrates that pyrene is able to activate mouse constitutive androstane receptor (CAR). CAR protein, as measured by Western blot analysis, was observed to translocate into the nucleus from the cytoplasm in mouse liver after exposure to pyrene. Utilizing CAR null mice, we identified that CAR mediates pyrene-induced hepatotoxicity. Increased relative liver weight, hepatocellular hypertrophy, and elevated serum alanine aminotransferase levels were found in wild-type but not CAR null mice after orally administered pyrene. We further show that pyrene induced the expression of mouse liver metabolism enzymes including CYP2B10, CYP3A11, GSTm1, GSTm3, and SULT1A1, and caused hepatic glutathione depletion in wild-type but not CAR null mice. Moreover, by luciferase reporter assay and quantitative real-time PCR analysis, pyrene was found to be a potential inducer of CYP2B6 expression via activation of human CAR in HepG2 cells and human primary hepatocytes. Our observations suggest that pyrene is a novel CAR activator and that CAR is essential for mediating pyrene-induced liver injury.
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http://dx.doi.org/10.1093/toxsci/kfv142DOI Listing
October 2015

Epigenetic modification of histone 3 lysine 27: mediator subunit MED25 is required for the dissociation of polycomb repressive complex 2 from the promoter of cytochrome P450 2C9.

J Biol Chem 2015 Jan 12;290(4):2264-78. Epub 2014 Nov 12.

From the Laboratory of Toxicology and Pharmacology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.

The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator binds to nuclear receptors at target response elements and recruits chromatin-modifying enzymes and RNA polymerase II. Here, we examine the involvement of Mediator subunit MED25 in the epigenetic regulation of human cytochrome P450 2C9 (CYP2C9). MED25 is recruited to the CYP2C9 promoter through association with liver-enriched HNF4α, and we show that MED25 influences the H3K27 status of the HNF4α binding region. This region was enriched for the activating marker H3K27ac and histone acetyltransferase CREBBP after MED25 overexpression but was trimethylated when MED25 expression was silenced. The epigenetic regulator Polycomb repressive complex (PRC2), which represses expression by methylating H3K27, plays an important role in target gene regulation. Silencing MED25 correlated with increased association of PRC2 not only with the promoter region chromatin but with HNF4α itself. We confirmed the involvement of MED25 for fully functional preinitiation complex recruitment and transcriptional output in vitro. Formaldehyde-assisted isolation of regulatory elements (FAIRE) revealed chromatin conformation changes that were reliant on MED25, indicating that MED25 induced a permissive chromatin state that reflected increases in CYP2C9 mRNA. For the first time, we showed evidence that a functionally relevant human gene is transcriptionally regulated by HNF4α via MED25 and PRC2. CYP2C9 is important for the metabolism of many exogenous chemicals including pharmaceutical drugs as well as endogenous substrates. Thus, MED25 is important for regulating the epigenetic landscape resulting in transcriptional activation of a highly inducible gene, CYP2C9.
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http://dx.doi.org/10.1074/jbc.M114.579474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303677PMC
January 2015

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

Biochem Pharmacol 2012 Sep 21;84(5):722-35. Epub 2012 Jun 21.

Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.
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http://dx.doi.org/10.1016/j.bcp.2012.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965201PMC
September 2012

Persistent and non-persistent changes in gene expression result from long-term estrogen exposure of MCF-7 breast cancer cells.

J Steroid Biochem Mol Biol 2011 Feb 23;123(3-5):140-50. Epub 2010 Dec 23.

Laboratory of Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

Life-long estrogen exposure is recognized as a major risk factor for the development of breast cancer. While the initial events in the regulation of gene expression by estrogen have been described in detail, far less is known of the role of estrogen in the long-term regulation of gene expression. In this study, we investigated the effects of long-term exposure of MCF-7 breast cancer cells to 1nM 17β-estradiol on gene expression with the goal of distinguishing between gene expression that is continually reliant on estrogen receptor (ER) function as opposed to secondary and persistent effects that are downstream of ER. To assess the direct involvement of ER in the differential gene expression of long-term estrogen exposed (LTEE) cells in comparison with that of control cells, we exposed cultures to the selective estrogen receptor modulator raloxifene (RAL). cDNA microarray analysis showed that exposure to RAL inhibited expression of numerous characterized estrogen-regulated genes, including PGR, GREB1, and PDZK1. Genes that were increased in expression in LTEE cells yet were unaffected by RAL exposure included the aryl hydrocarbon receptor (AHR) and numerous other genes that were not previously reported to be regulated by estrogen. Epigenetic regulation was evident for the AHR gene; AhR transcript levels remained elevated for several cell passages after the removal of estrogen. Signal transducer and activator of transcription 1 (STAT1); STAT1-regulated genes including ISG15, IFI27, and IFIT1; and MHC class I genes were also up-regulated in LTEE cells and were unaffected by RAL exposure. STAT1 is commonly overexpressed in breast and other cancers, and is associated with increased resistance to radiation and chemotherapy. This is the first study to relate estrogen exposure to increased STAT1 expression in breast cancer cells, an effect that may represent an additional role of estrogen in the pathogenesis of breast cancer.
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http://dx.doi.org/10.1016/j.jsbmb.2010.12.010DOI Listing
February 2011

Long-term estrogen exposure promotes carcinogen bioactivation, induces persistent changes in gene expression, and enhances the tumorigenicity of MCF-7 human breast cancer cells.

Toxicol Appl Pharmacol 2009 Nov 18;240(3):355-66. Epub 2009 Jul 18.

Laboratory of Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor alpha (ERalpha) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERalpha and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERalpha- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17beta-estradiol (E(2)). With these LTEE cells and with parallel control cells cultured without E(2) supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E(2)-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E(2).
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http://dx.doi.org/10.1016/j.taap.2009.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180932PMC
November 2009