UNDERSTANDING CYP6D1 TRANSCRIPTION: IMPLICATIONS FOR XENOBIOTIC INDUCTION AND INSECTICIDE RESISTANCE by George Guan-Hua Lin This thesis/dissertation document has been electronically approved by the following individuals: Scott,Jeffrey Graham (Chairperson) Lis,John T (Minor Member) Lazzaro,Brian (Minor Member) UNDERSTANDING CYP6D1 TRANSCRIPTION: IMPLICATIONS FOR XENOBIOTIC INDUCTION AND INSECTCIDE RESISTANCE A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by George Guan-Hua Lin August 2010 © 2010 George Guan-Hua Lin UNDERSTANDING CYP6D1 TRANSCRIPTION: IMPLICATIONS FOR XENOBIOTIC INDUCTION AND INSECTICIDE RESISTANCE George Guan-Hua Lin, Ph. D. Cornell University 2010 House fly cytochrome P450 CYP6D1 carries out the metabolism of xenobiotics. Expression of house fly CYP6D1 is induced in response to the prototypical P450 inducer, phenobarbital (PB), in the insecticide susceptible strains, CS and aabys. In the permethrin resistant LPR strain, increased transcription of CYP6D1 confers the metabolism-mediated resistance. CYP6D1 is constitutively overexpressed without significant PB induction in LPR. A series of experiments were conducted to understand the transcriptional regulation of CYP6D1. The core promoter of CYP6D1 is a dispersed type, as two transcription start sites were identified. Assays of the CYP6D1v2 promoter from the CS strain in Drosophila S2 cells identified promoter regions critical for basal transcription and for PB induction. Using RNAi treatment of Drosophila S2 cells, HR96 (hormone receptor-like in 96) and BR-C (broad-complex) were identified to be transcription factors critical for PB induction of CYP6D1v2. HR96 and BR-C were an activator and repressor, respectively, of PB induction of CYP6D1v2. The same promoter region of CYP6D1v1 from LPR was examined and shown to mediate PB induction to similar levels as CYP6D1v2 from CS. This indicates variations in promoter sequences are not responsible for the lack of PB induction of CYP6D1v1. Therefore, constitutive overexpression without PB induction of CYP6D1 in LPR is due to an unidentified trans acting factor. HR96 was cloned and sequenced to examine if it is this trans acting factor. Multiple HR96 alleles were identified and alleles v8-v10 were found to encode E28V and G110D amino acid substitutions in LPR. Permethrin selection of LPR showed HR96 alleles v8-v10 were not associated with permethrin resistance. Quantitative real-time RT-PCR showed no difference of HR96 expression levels between LPR and CS. Thus HR96 is not the trans acting factor responsible for constitutive overexpression of CYP6D1 in LPR. The molecular basis of constitutive overexpression of CYP6D1 in LPR is attributed to a trans acting factor responsible for PB induction in susceptible strains, but this trans acting factor remains unidentified. BIOGRAPHICAL SKETCH George Guan-Hua Lin was born on March 15, 1976 in Taipei, Taiwan. He was the first child in a family with two children. He showed strong interests in music and in book reading in his early childhood. He completed elementary, middle, and high school education in Taipei, Taiwan. In high school, he was selected for an advanced program in life sciences at Academia Sinica and he started showing strong enthusiasm for science and biology. Afterward, he entered National Taiwan University with a major in Plant Pathology and Entomology. At college, he received the National Taiwan University Presidential Award for his outstanding academic performance. In his sophomore year, he joined Dr. How-Jing Lee’s lab and participated in a long-term ecological research project investigating the dynamic of aquatic insect populations in Hapen Nature Reserve in northern Taiwan for three years. He then joined Drs. How-Jing Lee and Ruey-Fen Liou to study for his Master’s degree. There, he studied circadian clock gene, period, in German cockroaches. In 2002, after two years mandatory military service, George continued his journey in scientific research and worked as a research assistant in the following three years in National Taiwan University and in Academia Sinica. He married Yu-Chin Yu on November 2002 and their first son, Dong-Ciao, was born on September 2004, in Taipei, Taiwan. In fall 2005, George came to Department of Entomology at Cornell University and joined Dr. Jeff Scott’s lab to study for his Ph.D. His studies focused on the molecular basis dictating the constitutive overexpression of cytochrome P450 CYP6D1 in the permethrin resistant LPR strain of house fly. He was a teaching assistant for a variety of courses and served as the Jugatae Seminar Coordinator. At the 2009 ESA meeting, he received the President’s Prize for his ten- minute paper entitled “HR96 and BR-C modulate phenobarbital induced transcription iii of cytochrome P450 CYP6D1 in Drosophila S2 cells. Besides progress in scientific research, his second son, Jasper Yu-Sheng, was born in Ithaca, NY, on May 2009. During these five years of Ph.D. study, George was a Ph.D. student in the lab and was a husband and a father at home. The development of multiple lines in George’s life and enormous support from his major advisor Dr. Jeff Scott and from Cornell provided George great and valuable lessons to build up his own management skill and philosophy. These five years of Ph.D. study and life in Ithaca have laid out the fundamental stepping stones which will lead George and his family to the land full of happiness in their following journey. iv To my wife and children who have always believed in and supported me. v ACKNOWLEDGMENTS I thank my major advisor, Dr. Jeff Scott, for his enormous support during these five years of Ph.D. study. Jeff is the key person who made these five years wonderful and inspiring. There are so many dimensions of Jeff which I have learned and which have given my Ph.D. training not only limited to scientific research but also expanded into other dimensions in my life. I appreciate the wonderfulness and the inspiration from him. I thank Drs. John Lis and Brian Lazzaro for serving in my Ph.D. committee and giving different and precious perspectives to my research work. I would never forget the words from John, “do the right thing, get it solid, and have an impact in your field of study.” I realized the long-term effect and power of these words when I saw the exit result for my Ph.D. study. Special thank goes to Brian for his in depth philosophical advice and comments on my manuscripts and dissertation. Many thanks go to members I have worked with in Scott lab; Cheryl Leichter, Frank Rinkevich, Daisuke Hanai, Drs. Ronda Hamm, Melissa Hardstone, Ian Scott, Toshinori Kozaki, Jian-Rong Gao, and Jianwei Gao. I always remember the help and the joy from any and all of you. Special thank goes to Dr. Ann Hajek for providing critical facilities for my research work and also many thanks go to members in her lab for their friendships. Thanks to Entomology Department and funding opportunities including Sarkaria Fellowship, Griswold Research Award, and Palmer Fellowship. Also thanks to all friends in Entomology Department or at Cornell who have brought so many joys to me and my family. Lastly, great thanks go to my wife, Yu-Chin, and my two boys, Dong-Ciao and Jasper Yu-Sheng. Thanks also go to my parents and young brother in Taiwan. Without out any of you, my life would never be joyful and meaningful. vi TABLE OF CONTENTS BIOGRAPHICAL SKETCH………………………………………………………. iii DEDICATION……………………………………………………………………... v ACKNOWLEDGEMENTS………………………………………………………... vi TABLE OF CONTENTS………………………………………………………….. vii LIST OF FIGURES………………………………………………………………... xiii LIST OF TABLES………………………………………………………………… xv LIST OF ABBREVIATIONS……………………………………………………… xvi CHAPTER 1: LITERATURE REVIEW………………………………………...… 1 1.1 Introduction…………………………………………………………….……. 1 1.2 Metabolism of xenobiotics…………………………………………………… 1 1.2.1 Phase I and phase II metabolism…………………………………… 1 1.2.2 Detoxification enzymes………………..………………………….. 3 1.2.2.1 Cytochrome P450s…………………..…………………………... 3 1.2.2.1.1 Biochemistry………………………………………………..…. 3 1.2.2.1.2 Structure……………...………………………………………… 4 1.2.2.1.3 Substrate selectivity…………..……………………………….. 5 1.2.2.1.4 Nomenclature and classification…………………..…………... 6 1.2.2.1.5 Biological functions…………………………………...……….. 7 1.2.2.1.6 Evolution of insect P450s………………...……………………. 8 1.2.2.2 Hydrolases………………………………………………………... 10 1.2.2.3 Glutathione S-transferases…………………………………...…... 10 1.3 Induction of P450s by phenobarbital……………………………...…………. 12 vii 1.3.1 PB induction of P450s in bacteria………………………………….. 12 1.3.2 PB induction of P450s in mammals………………………..……… 13 1.3.2.1 PB induction of CYP2B genes by PBREM and CAR…..……….. 13 1.2.2.2 PB induction of CYP3A genes by XREM and PXR……...……… 14 1.2.3 PB induction in insects……………………..……………...……... 16 1.4 Insecticide resistance………………………………………………………… 17 1.5 Major mechanisms of insecticide resistance………..……………………….. 18 1.5.1 Metabolism-mediated resistance…………………………………… 18 1.5.1.1 Upregulated transcription………………………………………… 19 1.5.1.1.1 D. melanogaster Cyp6a2…………………………….………… 19 1.5.1.1.2 D. melanogaster Cyp6a8…………….………………………… 19 1.5.1.1.3 D. melanogaster Cyp6g1……………………………………… 20 1.5.1.1.4 M. domestica CYP6A1………………………………………… 20 1.5.1.1.5 Other detoxification enzymes…………..……………………... 22 1.5.1.2 Gene duplication………………..……………………………….. 22 1.5.1.3 Changes in the catalytic activity of a detoxification enzyme……. 23 1.5.2 Target site insensitivity…………………….……………………... 24 1.5.3 Reduced penetration………………………………………………... 24 1.6 Increased transcription of CYP6D1 in the LPR strain of the house fly…..…. 25 1.6.1 The house fly……………………………………………………….. 25 1.6.2 The permethrin resistant LPR strain………..……………………... 25 1.6.3 Mechanisms of resistance and genetic linkage study………..……. 26 1.6.4 Overexpression of P450lpr in the LPR strain…………………….…. 26 1.6.5 Cloning CYP6D1………………….……………………………….. 27 1.6.6 Expression profile of CYP6D1……………………………………... 28 1.6.7 Molecular basis of increased transcription of CYP6D1 in the LPR viii strain................................................................................................... 28 1.6.8 Overexpression of other PB inducible P450s in LPR strain……....