STRUCTURAL AND BIOCHEMICAL INSIGHTS INTO METHYLATION SITE AND STATE SPECIFICITY OF JMJD2 LYSINE DEMETHYLASES by Swathi Krishnan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) in the University of Michigan 2012 Doctoral Committee: Associate Professor Raymond C. Trievel, Chair Professor Carol A. Fierke Assistant Professor Georgios Skiniotis Professor John J. G. Tesmer Associate Professor Xiaochun Yu © Swathi Krishnan 2012 To my family ii ACKNOWLEDGEMENTS It has been a valuable learning experience for me at the University of Michigan, Ann Arbor. Firstly, I thank Dr. Ray Trievel for giving me an opportunity to work in his lab. Ray has been helpful in many ways and been very generous with many expensive reagents and peptides that have been instrumental in my thesis research. I also thank him for encouraging me to apply for various grants and awards, writing review articles and reviewing manuscripts and helping me become a well-rounded researcher. I thank my thesis committee members for providing valuable suggestions and discussions and for making it all the way to the MSRB buildings for my 9am meetings. I thank Paul, for helping me with almost everything in the lab and for all the great conversations and Stacie, who taught me crystallography when I knew nothing about it, and helped me get my driver’s license! Thanks to Kim for being a vibrant presence in the lab and always encouraging me to smile through tough times. I also thank the Biochem students and staff, particularly, Beth for all the help and support. I would not be the person I am today without my mom and dad and I am immensely grateful to them for being my source of strength even when they were 9000 miles away. Thanks Preethi for being my moral support, friend and confidante for the past 25 years! You are the best sister anyone can ask for. Lastly, but by no means the least, I thank Rozario who, made me fall in love with New York City and him, showed me how baseball can be fun and changed my life in the most wonderful ways possible. As long as I am with you, I will consider myself the luckiest person in the world. iii TABLE OF CONTENTS DEDICATION........................................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................................... iii LIST OF FIGURES ................................................................................................................ vii LIST OF TABLES .................................................................................................................... x LIST OF ABBREVIATIONS ................................................................................................. xi ABSTRACT ............................................................................................................................ xiii CHAPTER 1: HISTONE LYSINE DEMETHYLASES ......................................................... 1 Histone Modifications and Regulation of Gene Expression ....................................... 1 Biological Functions of Histone Lysine Methylation ................................................. 3 Mechanisms of Histone Lysine Demethylation .......................................................... 4 JMJD2 KDMs in Biology and Disease ....................................................................... 9 JMJD2 KDMs as Drug Targets................................................................................. 11 Methylation Site and State Specificities in the JMJD2 KDMs ................................. 13 Objectives of This work ............................................................................................ 14 REFERENCES .............................................................................................................. 15 CHAPTER 2: OPTIMIZATION OF THE FORMALDEHYDE DEHYDROGENASE- COUPLED LYSINE DEMETHYLASE ASSAY FOR JUMONJI ENZYMES .................21 The Formaldehyde Dehydrogenase (FDH) Coupled Demethylase Assay ...............21 Inhibition of JmjC KDMs by Transition State Metals ..............................................23 MATERIALS AND METHODS .................................................................................24 Expression and Purification of Strep(II)-Tagged JMJD2A and JMJD2D ................24 Expression and Purification of Recombinant Formaldehyde Dehydrogenase .........25 Substrate Histone Peptides ........................................................................................28 Determination of Metal Content of JMJD2A and JMJD2D .....................................28 Reagents for the FDH- Coupled Demethylase Assay ...............................................29 Setting up the FDH- Coupled Demethylase Assay ...................................................29 NADH Calibration Assay .........................................................................................34 Determination of Kinetic Parameters ........................................................................36 RESULTS ......................................................................................................................39 Purity of Recombinant Formaldehyde Dehydrogenase ............................................39 Purity of Strep(II)-Tagged JMJD2A and JMJD2D ...................................................40 Metal Content Analysis of Strep(II)-tagged JMJD2A and JMJD2D ........................40 Kinetic Analysis ........................................................................................................42 Use of Methyllysine Analog Bearing Substrates ......................................................44 DISCUSSION ................................................................................................................50 iv ACKNOWLEDGEMENTS .....................................................................................................51 REFERENCES ..........................................................................................................................51 CHAPTER 3: METHYLATION SITE SPECIFICITY STUDIES-CRYSTAL STRUCTURE OF HUMAN JMJD2D ....................................................................................55 MATERIALS AND METHODS .................................................................................57 Cloning, Expression and Purification of His-JMJD2D.............................................57 Crystallization of JMJD2D•2-OG•H3K9me3 Ternary Complex .............................59 Optimization of Crystals by Surface Entropy Reduction .........................................62 X-Ray Diffraction Data Collection, Processing and Structure Determination .........62 RESULTS ......................................................................................................................64 Structure of JMJD2D Apoenzyme ............................................................................64 Structure of the JMJD2D•2-OG•H3K9me3 Complex ..............................................67 Interactions of JMJD2D with Substrate H3K9me3 ..................................................75 DISCUSSION ................................................................................................................77 ACKNOWLEDGEMENTS .........................................................................................78 REFERENCES ..............................................................................................................78 CHAPTER 4: METHYLATION SITE SPECIFICITY STUDIES-DIFFERENTIAL SPECIFICITY OF JMJD2D AND JMJD2A .........................................................................83 MATERIALS AND METHODS .................................................................................84 Protein Expression and Purification..........................................................................84 Histone Substrate Peptides ........................................................................................84 FDH-Coupled Demethylase Assay ...........................................................................85 Molecular Docking of JMJD2D and H3K36me3 .....................................................85 RESULTS ......................................................................................................................87 Comparison of H3K9me3 Recognition by JMJD2D and JMJD2A ..........................87 Role of H3T11ph in the Recognition of H3K9me3 by JMJD2 KDMs ....................93 Mode of H3K36me3 Occlusion by JMJD2D............................................................95 Activity of JMJD2D and JMJD2A toward hybrid peptides....................................108 DISCUSSION ..............................................................................................................115 Mode of H3K9me3 recognition and H3K36me3 occlusion by JMJD2 KDMs ......115 Comparing the JMJD2 and UTX Enzyme Families ...............................................116 ACKNOWLEDGEMENTS .......................................................................................119 REFERENCES ............................................................................................................121 CHAPTER 5: SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS................123 Methods for Purifying and Assaying Fe(II)-dependent Dioxygenases...................124 Structural Basis for Methylation Site Specificity in JMJD2 KDMs .......................125