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Regulation of Rat CYP2B1 Gene Expression: the Role of Nuclear UCL Regulation of rat CYP2B1 gene expression The role of nuclear receptors Roongsiri Muangmoonchai A dissertation submitted to the Faculty of Life Sciences in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the University of London Department of Biochemistry and Molecular Biology University College London June 2000 ProQuest Number: U642761 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642761 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Cytochrome P450 (GYP) plays a central role in the metabolism of endogenous and exogenous compounds. This thesis aims to investigate the molecular mechanism which underlies CYP2B1 induction in response to xenobiotics, particularly phénobarbital (PB), by characterisation of the phenobarbital-responsive element (PBRE) of the CYP2B1 gene and identification of nuclear regulatory proteins, which bind to the PBRE. In vivo transfection studies by biolistic particle delivery, showed that the 5’-upstream region between -2301 to -2142 (159 bp) conferred PB responsiveness on a reporter gene. Within this region, there are two nuclear receptor binding sites; NR1 and NR2, flanking a nuclear factor-1 binding site. Gel mobility shift assays showed the heterodimerisation of constitutive androstane receptor-p (CAR-p) and retinoid X receptor a (RXRa) onto the NR1 site, but not the NR2 site. Co­ transfection of the expression vector for CAR-p, with a CYP2B1 PBRE-Luc reporter construct confirmed both constitutive and xenobiotic-mediated transactivation of reporter gene expression in transfected liver and primary hepatocytes. However, HepG2, Mela, and CV-1 cell lines support only constitutive activation of gene expression by CAR-p. The co-activator SRC-1 enhances both constitutive and xenobiotic-mediated transactivation of a CYP2B1 PBRE-Luc reporter gene via CAR-p in primary hepatocytes. The SRC- 1 stimulation of CAR-p transactivation is not observed when a reporter construct containing only the NR1 site was used. The regulation of the CYP2B1 gene in response to xenobiotics is therefore mediated by a nuclear receptor mechanism and involves the interaction between several transcription factors and co-activators. PB and pregnenolone 16a-carbonitrile (PCN) increase the expression of both CYP2B1 and CYP3A1 genes. PCN-induction of CYP3A1 is mediated by a pregnane X receptor (PXR). Both CAR-|3 and PXR were shown to bind to and transactivate via the same DNA element, either the CYP2B1 NR1 or the CYP3A1 PXRE, in primary hepatocytes and in vivo. Thus, the versatility of these promiscuous nuclear receptors in regulating of more than one CYP may enable an organism to efficiently respond to xenobiotics. To my dear parents, Ruangsak & Sangwian Muangmoonchai Acknowledgements This thesis could not have been fulfilled without support from a number of people to whom I am deeply grateful. Throughout these years, it must have been a very hard time for my parents while I have pursued my degree in a foreign country. Although we were apart, they have never failed to give their continual love, support and encouragement, not only the past years, but for all my life. My grand parents, my brother and relatives have also shared the sacrifices made by my parents. In particular, to my husband, Wirote Chotpativetkul - I owe him a great appreciation for his patience and sacrifices. W e have shared all the good and hard times together. I could not have completed my degree without his love, care and understanding. My supervisor. Prof. Elizabeth Shephard, has given me the opportunity to work as a Ph.D. student under her care. She has taken full responsibility during my studies. I am very grateful for her help, guidance, and encouragement. I hope to do the same for my students. I also would like to thank Prof. Ian Phillips for his helpful discussions. Special thanks to Dr. Mina Edwards for her kind assistance in the liver perfusion and cell culture work. She always cheered me up and gave me lots of support. I also would like to extend my deep gratitude to my internal monitor. Prof. Jeremy Brockes, who encouraged and advised me throughout my research. The facility for using the gene gun equipment was provided by his laboratory. I am very grateful for the assistance of Cristiana Velloso and the people in his group. My colleagues have been very helpful and supportive. I owe a great deal to Dr. Slew Cheng Wong and Despina Smirlis for their help, friendship, and encouragement. I cherished all the times we spent together. I am also thankful to the help of Dr. Azara Janmohamed and Dr. Diana Hernandez. Many thanks to my friends and colleagues in Thailand and England, especially to Palap Lekmanee, Dr. Pornchan Saitongdee and Dr. Preprame Pattanamahakul, for all their friendship and support. There are many other people whose names have not been mentioned here, who had been very kind and offered me help throughout my research. This degree was financially supported by the Royal Thai government. Table of Contents A b s tra c t .............................................................................................................................. 2 Acknowledgements ......................................................................................................5 Table of contents .......................................................................................................... 7 List of figures ................................................................................................................. 13 List of tables ................................................................................................................... 16 Abbreviations ................................................................................................................. 17 INTRODUCTION .............................................................................................................. 20 1.1 O ver view of xenobiotic metabolism ..............................................................20 1.2 T h e c y t o c h r o m e P450 superfamily ..............................................................23 1.3 Evolution and nomenclature of cytochrome P 4 5 0 ................................25 1.4 R e g u l a t io n o f C Y P g e n e e x p r e s s io n ............................................................27 1.5 T ranscriptional activation of C Y P genes by different classes OF INDUCERS....................................................................................................................30 1.5.1 Aromatic hydrocarbon-inducible CYP1A family .................................. 30 1.5.2 The phenobarbital-inducible CYP2B family ......................................... 31 1.5.3 Steroid-inducible CYP3A family ............................................................... 35 1.5.4 Peroxisome proliferator-inducible CYP4A fam ily ................................36 1.6 Achievements in the study of the molecular mechanism UNDERLYING CYP2B GENE EXPRESSION BY PB ........................................................38 1.6.1 CYP induction in Bacillus megaterium as a model for mammalian CYP gene regulation ..................................................................... 39 1.6.2 Proposed phenobarbital-responsive elements within the proximal promoter....................................................................................................40 1.6.3 Distal phenobarbital-responsive enhancer elements ........................42 1.7 T he nuclear receptor superfamily ..............................................................46 1.7.1 Retinoid X receptor ...................................................................................... 51 1.7.2 Nuclear orphan receptors .......................................................................... 52 1.7.3 Regulation of CYP gene expression by nuclear receptors .............. 56 1.8 A im s a n d s c o p e o f t h is t h e s is .......................................................................... 58 MATERIALS AND METHODS......................................................................60 2.1 Chemicals AND REAGENTS....................................................................................60 2.2 DNA CLONES, EXPRESSION VECTORS AND OLIGONUCLEOTIDES.....................60 2.3 A nimals and cell l in e s ....................................................................................... 61 2.4 Cloning of DNA F ragm ents .........................................................................62 2.4.1 Linearisation of the plasmid vector ...................................................... 62 2.4.2 Dephosphorylation of linearised plasmid DNA ...................................62 2.4.3 Preparation of DNA fragments for subcloning ....................................63 2.4.4 Ligation reaction ...................................................................................... 64 2.4.5 Transformation of bacteria ....................................................................
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