TRANSCRIPTIONAL REGULATION OF THE HUMAN ALCOHOL DEHYDROGENASES AND ALCOHOLISM Sirisha Pochareddy Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University September 2010 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Howard J. Edenberg, Ph.D., Chair Maureen A. Harrington, Ph.D. Doctoral Committee David G. Skalnik, Ph.D. Ann Roman, Ph.D. July 30, 2010 ii This work is dedicated to my parents and my brother for their unwavering support and unconditional love. iii ACKNOWLEDGEMENTS I would like to sincerely thank my mentor Dr. Howard Edenberg, for his guidance, support throughout the five years of my research in his lab. It has been an amazing learning experience working with him and I am confident this training will help all through my research career. I would like to thank members of my research committee, Dr. Maureen Harrington, Dr. David Skalnik and Dr. Ann Roman. I am grateful to them for their guidance, encouraging comments, time and effort. I greatly appreciate Dr. Harrington’s questions during the committee meeting that helped me think broadly about my area of research. I am very thankful to Dr. Skalnik for reading through my manuscript and giving his valuable comments. My special thanks to Dr. Ann Roman for staying on my committee even after her retirement. I am also thankful to Dr. Jeanette McClintick for her patience in answering my never ending list of questions about the microarray analysis. She also had been a great support during the tough times in the lab. I would like to thank her making an effort to remember birthdays of all lab members and baking her awesome brownies. I would like to thank other lab members, Ron Jerome, Jun Wang and Sowmya Jairam. It was a great pleasure to know Ron during the last year of my stay. He made the toughest years of Ph.D. less stressful and more fun. Jun was always helpful in the lab. I am also thankful to Sowmya for sharing her ideas with me and helping me think more about ADH transcriptional regulation. I would also like to thank Dr. Xiaoling Xuei and Dr. Yunlong Liu for all their help. iv I would like to thank my best friends, Dr. Sirisha Asuri and Dr. Raji Muthukrishnan for their beautiful, unconditional friendship. I am also thankful to my other friends Sulo, Aditi, Heather, and Chandra for all the fun. Finally, I would like to thank my family members. My mom Prabhavathy and my dad P.S. Reddy have been there for me always, supporting all my decisions. They have been with me through the highs and the lows and always made me believe that everything is going to be fine. My dream of doing research and getting a Ph.D. would not have been possible without their strong emotional support. Another pillar of support in my life is my brother Subhash. He is my guide, teacher, friend, brother and has been a great source of strength in the most difficult times. Anna, thank you so much for everything. I would also like to thank my sister-in-law, Jhansi for being a sister I never had and a great friend. Lastly, I would like to thank cute little ones - my nephew Arjun, my niece Megha, Nishant, Niha and Charan, for lifting my spirits with their innocent smiles. v ABSTRACT Sirisha Pochareddy TRANSCRIPTIONAL REGULATION OF THE HUMAN ALCOHOL DEHYDROGENASES AND ALCOHOLISM Alcohol dehydrogenase (ADH) genes encode proteins that metabolize ethanol to acetaldehyde. Humans have seven ADH genes in a cluster. The hypothesis of this study was that by controlling the levels of ADH enzymes, cis- regulatory regions could affect the risk for alcoholism. The goal was thus to identify distal regulatory regions of ADHs. To achieve this, sequence conservation across 220 kb of the ADH cluster was examined. An enhancer (4E) was identified upstream of ADH4. In HepG2 human hepatoma cells, 4E increased the activity of an ADH4 basal promoter by 50-fold. 4E was cell specific, as no enhancer activity was detected in a human lung cell line, H1299. The enhancer activity was located in a 565 bp region (4E3). Four FOXA and one HNF-1A protein binding sites were shown to be functional in the 4E3 region. To test if this region could affect the risk for alcoholism, the effect of variations in 4E3 on enhancer activity was tested. Two variations had a significant effect on enhancer activity, decreasing the activity to 0.6-fold. A third variation had a small but significant effect. The effect of variations in the ADH1B proximal promoter was also tested. At SNP rs1229982, the C allele had 30% lower activity than the A allele. vi In addition to studying the regulatory regions of ADH genes, the effects of alcohol on liver-derived cells (HepG2) were also explored. Liver is the primary site of alcohol metabolism, and is highly vulnerable to injuries due to chronic alcohol abuse. To identify the effects of long term ethanol exposure on global gene expression and alternative splicing, HepG2 cells were cultured in 75 mM ethanol for nine days. Global gene expression changes and alternative splicing were measured using Affymetrix GeneChip® Human Exon 1.0 ST Arrays. At the level of gene expression, genes involved in stress response pathways, metabolic pathways (including carbohydrate and lipid metabolism) and chromatin regulation were affected. Alcohol effects were also observed on alternative transcript isoforms of some genes. Howard J. Edenberg, Ph.D. Committee Chair. vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xii LIST OF FIGURES ............................................................................................. xiii ABBREVIATIONS ............................................................................................... xiv I. INTRODUCTION ............................................................................................... 1 1. Alcohol dehydrogenases .............................................................................. 1 2. Human ADH cluster ...................................................................................... 5 3. Additional pathways of alcohol metabolism .................................................. 6 4. Alcoholism .................................................................................................... 7 5. ADHs and alcoholism ................................................................................... 9 6. Transcriptional regulation of ADHs ............................................................. 11 7. Identification of cis-regulatory regions ........................................................ 17 8. Transcription factors ................................................................................... 18 8.a. FoxA family ......................................................................................... 19 8.b. HNF-1A ............................................................................................... 20 9. Alcohol and the liver ................................................................................... 21 10. Alternative transcript isoforms and diseases ............................................ 24 11. Global transcriptional profiling .................................................................. 27 12. Research objectives ................................................................................. 32 viii II. MATERIALS AND METHODS ........................................................................ 34 1. Identification of putative distal regulatory elements .................................... 34 2. Cloning of test fragments ............................................................................ 34 3. Transient transfections and reporter gene assays ...................................... 38 4. Electrophoretic mobility shift assays (EMSA) ............................................. 40 5. Site directed mutagenesis .......................................................................... 42 6. Generation of the 4E haplotypes ................................................................ 42 7. Long-term treatment of HepG2 cells with ethanol....................................... 44 8. RNA extraction, labeling and hybridization ................................................. 44 9. Exon array data analysis ............................................................................ 45 10. Validation of differential gene expression by qRT-PCR ............................ 51 11. Validation of alternative splicing by qRT-PCR .......................................... 52 III. RESULTS ....................................................................................................... 54 1. Identification of an enhancer in the ADH cluster ......................................... 54 2. Characterization of the enhancer element 4E ............................................ 58 2.a. Effect of 4E on heterologous promoters .............................................. 58 2.b. Function of 4E in non-hepatoma cells ................................................. 58 2.c. Localization of sequences required for 4E enhancer activity ............... 59 2.d. Identification of potential protein binding sites in 4E ........................... 61 2.e. Effect of mutations on enhancer activity.............................................. 66 ix 3. Effects of regulatory variations on gene expression