Transcriptional Regulation of the Human Microsomal
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The Pennsylvania State University The Graduate School The Huck Institutes of the Life Sciences TRANSCRIPTIONAL REGULATION OF THE HUMAN MICROSOMAL EPOXIDE HYDROLASE GENE (EPHX1) DRIVEN BY A FAR UPSTREAM ALTERNATIVE PROMOTER A Dissertation in Molecular Toxicology by Shengzhong Su 2013 Shengzhong Su Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2013 ii The dissertation of Shengzhong Su was reviewed and approved* by the following: Curtis J. Omiecinski Professor of Veterinary and Biomedical Sciences and Hallowell Chair Dissertation Advisor Chair of Committee Chair of Intercollege Graduate Degree Program in Molecular Toxicology Adam B. Glick Associate Professor of Veterinary and Biomedical Sciences K. Sandeep Prabhu Associate Professor of Immunology and Molecular Toxicology John Vanden Heuvel Professor of Molecular Toxicology Joshua D. Lambert Associate Professor of Food Science *Signatures are on file in the Graduate School iii ABSTRACT Microsomal epoxide hydrolase (mEH) is an important metabolizing enzyme that plays roles in both detoxification and bioactivation of xenobiotics. Human mEH gene expression is subjected to the regulation of alternative promoter usage generating multiple transcripts, including the most prevalent, termed E1b and E1. These transcripts possess distinct untranslated first exons but encode identical mEH protein given that the second exon contains the translation initiation site. E1b is ubiquitously expressed at high levels in all tissues while E1 is selectively expressed in the liver. Although several liver-specific transcription factors were characterized previously as involved in the regulation of E1 transcription, little is known regarding the molecular mechanism regulating E1b expression. Study of those underlying processes is the principle focus of these investigations. Initially in these studies we sought to identify the key transcription factors responsible for controlling the constitutive expression of the E1b transcript. Sequence analysis of E1b proximal promoter revealed several potential Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs established their roles in regulating E1b promoter activities. Chromatin immunoprecipitation (ChIP) analyses demonstrated that both Sp1 and Sp3 are bound to the proximal promoter region of E1b. Silencing, or knockdown of Sp1 expression using siRNA had no detectable effect on the endogenous E1b transcriptional level. However, knockdown of Sp3 greatly diminished E1b expression in several different human cell lines. These results demonstrated that Sp3 in particular was involved in regulating the basal expression patterns of the mEH E1b variant transcript. Secondly, following analysis of DNase I hypersensitivity data available in the ENCODE project, we identified and characterized two intronic DNA elements in the mEH genomic region. This led to the discovery that the master oxidative stress regulator, Nuclear factor erythroid-derived 2 (NF-E2)-related factor 2 (Nrf2) functioned as a mediator of E1b upregulation in lung cancer-derived cells. Results obtained from both luciferase gene reporter and ChIP assays indicated that Nrf2 interacts with the 2nd intronic DNA element following its activation with the antioxidants, sulforaphane or tBHQ. DNA sequence analysis of the enhancer region together with electrophoretic mobility shift assays (EMSA) enabled the identification of a conserved antioxidant-response element within the enhancer that appeared to mediate these transcriptional responses. Finally in these studies, we sought to characterize differences in the transcriptional responses of the E1b and E1 promoters in hepatoma cell lines and human normal hepatocytes to chemical mediators. Nrf2 siRNA knockdown studies in hepatoma C3A cells were performed to identify the Nrf2 signaling pathway as functional in mediating the activation effects contributed by the monofunctional inducers, sulforaphane and tBHQ, to both of E1b and E1 promoters. Luciferase reporter assays demonstrated that these effects were localized to the 2nd intronic enhancer element. However, bifunctional inducers exhibited considerable differences in their regulation of E1b and E1 transcript expression. Treatment with 3-MC, an aryl hydrocarbon receptor (AhR) agonist, induced E1b expression but inhibited E1. Another bifunctional inducer, β-naphthoflavone (β-NF) upregulated both E1b and E1 whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment resulted in decreased E1 expression iv (similar to 3-MC), yet had no discernible effect on expression of E1b. Further studies with 6,2',4'- trimethoxyflavone (TMF), an AhR antagonist, together with Nrf2 siRNA, suggested that both AhR and Nrf2 signaling pathways contribute to the regulation of the E1b and E1 promoters mediated by bifunctional AhR agonists. As well, these effects were comparatively evaluated in human hepatoma HepG2 and Huh7 cells, in human primary hepatocytes and in human lung BEAS-2B cells. Overall, this thesis research successfully identified and defined multiple levels of molecular interaction that contribute to the regulation of the dual gene promoter usage characterizing human mEH gene expression. The results contributed through these investigations provide important advances in our core understanding of the complex genetic regulatory schemes controlling the dual promoter usage and tissue-selective expression character underlying the functional biology and toxicological roles of human mEH. v TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................................................. vii LIST OF TABLES ................................................................................................................................................. viii ABBREVIATIONS .................................................................................................................................................. ix ACKNOWLEDGEMENTS ..................................................................................................................................... xi DEDICATION ........................................................................................................................................................ xii Chapter 1 Introduction ................................................................................................................................................1 GENERAL INTRODUCTION TO XENOBIOTIC METABOLISM ...............................................................1 EPOXIDE HYDROLASE ..................................................................................................................................2 The physiological functions of mEH ..........................................................................................................4 Gene organization of human mEH gene (EPHX1) .....................................................................................4 Polymorphisms and their clinical implications ...........................................................................................5 Transcriptional regulation of Human mEH gene ........................................................................................7 Tissue-specific regulation of human EPHX1 gene by the selective use of alternative promoters ..............7 TRANSCRIPTIONAL REGULATION OF GENE EXPRESSION ..................................................................9 The core promoter .......................................................................................................................................9 Focused versus dispersed core promoters .................................................................................................10 CpG islands (CGI) ....................................................................................................................................11 Proximal Promoter Elements ....................................................................................................................12 Transcription factor Sp1 ...........................................................................................................................13 Distal regulatory elements ........................................................................................................................13 Identification of distal enhancer elements ................................................................................................15 TRANSCRIPTION FACTORS REGULATING DRUG-METABOLIZING ENZYMES..............................16 OXIDATIVE STRESS AND NRF2-ARE SIGNALING PATHWAY ............................................................17 The structures of Nrf2 and Keap1 .............................................................................................................18 Keap1 and Nrf2 mutation in cancers ........................................................................................................20 The crosstalk between Nrf2 and AhR signaling pathways .......................................................................20 HYPOTHESES AND AIMS ............................................................................................................................21 Chapter 2 Transcription factors Sp1 and Sp3 contribute to the basal expression of human microsomal epoxide hydrolase alternative E1b mRNA variant by interacting with the proximal E1b promoter .....................................31