ALCOHOL INDUCED HISTONE ACETYLATION MEDIATED BY HISTONE ACETYL TRANSFERASE GCN5 IN LIVER A Dissertation presented to the Faculty of the Graduate School University of Missouri-Columbia In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy by MAHUA CHOUDHURY Dr. Shivendra D. Shukla, Dissertation Supervisor AUGUST 2008 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled ALCOHOL INDUCED HISTONE ACETYLATION MEDIATED BY HISTONE ACETYL TRANSFERASE GCN5 IN LIVER presented by Mahua Choudhury, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Dr. Shivendra D. Shukla Dr. Ronald Korthuis Dr. Robert Lim Dr. Jamal Ibdah Dr. John F. Cannon ACKNOWLEDGEMENTS This work would not have been possible without the guidance of Dr. Shivendra D. Shukla, my dissertation supervisor. Dr. Shukla has been an exceptional advisor, not only as a professor but as a guardian in daily life. He was always inspiring, helpful and enriching. He had faith in me and allowed me to design my own experiment and also taught me to think rationally in every step. He took time to realize my limitations and guide me to go beyond that border and succeed in this implausible ride. I considered myself fortunate to have an advisor who paid attention to every professional and personal challenge and showed me the road to surpass them very easily. I am also very grateful to my doctoral committee members- Dr. Ronald Korthuis, Dr. Robert Lim, Dr. Jamal Ibdah, Dr. John F. Cannon and of course again Dr. Shivendra D. Shukla for their intellectual perspective, valuable time and encouragement. Dr. Lim significantly helped me over the processes of my experimental design and manuscript revision. Dr. Korthuis not only helped me in the dissertation process, he provided a real effort to find my future research career. I am also grateful to have a friendly and helpful Department of Medical Pharmacology and Physiology. In particular, I will thank Dr. Greg Sowa for helpful suggestions in research. I would also like to thank my fellow lab-mates and friends Taryn Typhaine James, Lana Yvonne Bruny, Dr. Pil Hoon Park, Dr.Youn ju Lee and Dr. Annayya Aroor and Dr. Phullara Shelat for their utmost support. I would also like to specially thank Daniel Jackson for all the technical assistance and his friendly support. ii I will never find adequate words of appreciation for my parents Kalpana and Madhu Sudan Choudhury. They supported my every decision and sheltered from every adversity. It would not have been possible without their continuous encouragement throughout the study in this foreign country. Finally, most significantly, I would like to thank my husband, Koushik. Without his unconditional love, support and encouragement, I would not be able to achieve my goal. He helped me in every adverse situation and is always there for me. I owe my every achievement to him. This research work was supported by RO1-AA 11962, RO1-AA 16347, and R21- AA 14852 from NIH. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................. ii LIST OF FIGURES ........................................................................................................ vii LIST OF TABLES ........................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................... x ABSTRACT ................................................................................................................... xiv Chapter I Introduction……………………………………………………………………1 I.1 Epigenetics.................................................................................................................. 1 I.1.1 Chromatin, Nucleosome and Histone ............................................................................ 2 I.2 Histone Modifications ................................................................................................ 5 I.2.1 Histone Modifying Enzymes ....................................................................................... 10 I.3 Aberrant Histone Modifications and Human Disease .......................................... …20 I.4 Alcohol (Ethanol) and Surrogate Alcohols ............................................................... 22 I.4.1 Alcohol Metabolism………………………………………………………...................26 I.4.2 Alcoholic Liver disease………………………..……………………………………….27 I.5 Alcohol, Oxidative Stress and Liver Injury………………………………………... 29 I.6 Primary Rat Hepatocytes and HepG2 Cell line (VA-13)…………………………...33 I.7 Hypothesis, Specific Aims and the Significance of the Dissertation Project...……..35 Chapter II Surrogate alcohols and their metabolites modify histone H3 acetylation: Involvement of histone acetyltransferase and histone deacetylase……...38 II.1 Abstract…………. ................................................................................................... 39 iv II.2 Introduction .............................................................................................................. 41 II.3 Materials and methods ............................................................................................. 43 II.4 Results ...................................................................................................................... 49 II.5 Discussion ................................................................................................................ 56 References ....................................................................................................................... 62 Chapter III Oxidative stress mediates ethanol induced histone H3 acetylation in rat hepatocytes……………………………………………………………..97 III.1 Abstract………… ................................................................................................... 98 III.2 Introduction ............................................................................................................. 99 III.3 Materials and Methods .......................................................................................... 102 III.4 Results ................................................................................................................... 106 III.5 Discussion ............................................................................................................. 110 References ..................................................................................................................... 115 Chapter IV Ethanol induced histone H3 acetylation is mediated by GCN5 in hepatoma cell………………………………………………………….133 IV.1 Abstract…………………………………………………………………………..134 IV.2 Inntroduction……………………………………………………………………..135 IV.3 Experimental Procedures……………………………………………………...…137 IV.4 Results……………………………………………………………………………139 IV.5 Discussion……………………………..…………………………………………141 References...……………………………………………………………………………143 v Chapter V Conclusions and Future Direction…………………………………………..152 References………………………………………………………………………………159 VITA……………………………………………………………………………………181 vi LIST OF FIGURES Figure Page I-1: Schmatic diagram of chromatin formation ................................................................ 4 I-2: Schematic Diagram of Histone Acetylation…………………………………………8 I-3 Schematic diagram of HATs and HDACs on Histone acetylation………...……….17 II-1: Effect of different carbon chain alcohols on acetylation of histone H3 at lys9…...67 II-2: Effect of different carbon chain alcohols on acetylation of histone H3 at lys 14, lys 18, lys 23, lys27............................................................................................ 69 II-3: Lactate dehydrogenase (LDH) release from primary rat hepatocytes treated with different alcohols. ...................................................................................... 72 II-4: Effect of different carbon chain alcohols on hepatocyte mitochondrial activity .... 74 II-5: Effect of different alcohols on histone acetyltransferase (HAT) activity ............... 76 II-6: Effect of different alcohols on histone deacetylase (HDAC) activity kinases…. 78 II-7: Effect of higher concentration of ethanol alone and in combination with surrogate alcohols on acetylation of histone H3 at lys9 .................................................... 80 II-8a: Effect of 4-mp on ethanol or 1-propanol-induced histone H3 acetylation at lys9………………………………………………………………………….....83 II-8b: Effect of cyanamide on ethanol or 1-propanol induced histone H3 acetylation at Lys9. ............................................................................................................. 85 II-8c: Effect of acetate and propionate on acetylation of histone H3 at lys9 .................. 87 II-9a: Effect of different alcohol metabolites on histone acetyltransferase (HAT) activity………………………………………………………………………....89 II-9b: Effect of different alcohols and metabolites added directly in HAT assay .......... 91 II-9c: Effect of different alcohols and metabolites added directly in HDAC assay ....... 93 II-9d: Effect of different alcohols and alcohol metabolites on HDAC activity in HeLa cell extract ............................................................................................... 95 vii III-1: Ethanol induced ROS generation in hepatocytes ................................................ 120 III-2: Effect of glutathione modulators on ethanol induced histone H3K9 acetylation in hepatocytes. .............................................................................