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The Role of the MLL-HOXA9 Axis in Normal and Malignant

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The Role of the MLL-HOXA9 Axis in Normal and Malignant The Role of the MLL-HOXA9 Axis in Normal and Malignant Hematopoiesis by Jingya Wang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Cellular Pathology) in the University of Michigan 2013 Doctoral Committee: Professor Jay L. Hess, Chair Assistant Professor Tomasz Cierpicki Professor Eric R. Fearon Assistant Professor Maria E. Figueroa Professor Tom K. Kerppola Associate Professor Xiaochun Yu © Jingya Wang 2013 To My parents and My husband ii Acknowledgement This dissertation would not have been possible without the tremendous support and guidance from my advisor, Dr. Jay L. Hess. Jay’s enthusiasm and dedication towards scientific research have always been a great source of inspiration. His scientific vision and openness to novel ideas greatly influenced my scientific career. I would also like to thank Jay for encouraging me to pursue my interest in statistics and the opportunity to apply my knowledge to biomedical research. I deeply appreciate Jay’s invaluable advice and generous support for the next stage of my career. My sincere appreciations also go to Drs. Andrew Muntean and Maria (“Ken”) Figueroa. Throughout my graduate study, Andy was actively involved in many aspects of my projects and my scientific career, including providing technical assistance and scientific thinking, and most importantly, sharing his insights of science and being a better scientist. I could not have been where I am without his generous help. Ken led me into the field of high throughput sequencing data analysis, and patiently discussed experimental design to algorithm in details. Her presence also contributes greatly to the completion of this thesis. I would like to thank my thesis committee members, Drs. Eric R. Fearon, Tom K. Kerppola, Xiaochun Yu, Maria (“Ken”) E. Figueroa, and Tomasz Cierpicki for iii generously offering their time and invaluable advice for my research, especially Dr. Xiaochun Yu for his help with my ubiquitination assays. I also thank all of the investigators of the hematology group, including Drs. ali Dou, Jolanta rembecka, Jean-Francois ual, ifan Liu, and Zaneta Nikolovska-Coleska who helped me in every aspect with my projects. Especially, I thank Dr. Yali Dou and Dr. Jolanta Grembecka for letting me involved in their research, which are great learning opportunities. Finally, I thank Dr. Richard McEachin and Kevin Cauchy at The U-M Center for Computational Medicine and Bioinformatics (CCMB) for their help with my data analysis. I would like to express my appreciation to all the current and past members of the Hess lab, including Joel Bronstein, Sara Monroe, Jim Connelly, Yongsheng Huang, Stephanie Jo, Surya Nagaraja, Daniel Sanders, Jane Tan, Cailin Collins, Yuqing Sun, Peilin Ma, Humaira Nawer and Hongzhi Miao, as well as members of the Dou lab, especially Fang Cao and Shirley Lee, who made my stay in the Hess lab a fantastic and enjoyable experience. A special thank you goes to Laura Wu, the summer intern student who assisted me with the biochemical assays of E3 ligase activity of MLL PHD2 (Chapter 3). In addition, I would like to thank Cailin Collins and Joel Bronstein for their continuous effort on the Hoxa9 project, which is a great source of inspiration (Chapter 4). I would like to thank Ms. Lynn McCain for her administrative assistance in and out of the lab. I am also grateful to the Molecular and Cellular Pathology iv program director Dr. Nicholas Lukacs and administrative specialists Laura Hessler and Laura Labut, and the Statistics dual master degree program advisor Dr. Ji Zhu and administrative specialist Judy McDonald for their strong support the entire time. I would like to express my deepest gratitude to my parents, Mr. Quanxin Wang and Mrs. Fengling Huang, and my beloved husband Geng Yu, for their unconditional love and support through the last four years. I could have never accomplished this work without them and I hope I can continue to be their pride. v Preface This dissertation comprises the research I conducted in Dr. Jay Hess’ lab from 2009 to 2013. The objective was to better understand the role of the MLL-HOXA9 axis in normal and malignant hematopoiesis. Specifically, I wanted to explore: How is the activity of MLL regulated during hematopoiesis? What are the mechanisms of MLL fusion mediated transformation? And how does HOXA9 regulate gene expression and mediate leukemogenesis? The first two questions were tackled by studying the function of the PHD/Bromo region of MLL, which is invariably disrupted or deleted in oncogenic MLL fusion proteins and is incompatible to MLL fusion-mediated transformation. My studies revealed that this region facilitates MLL ubiquitination in multiple ways. Chapter 2 describes the role of MLL bromodomain in recruitment of the ECSASB2 E3 ligase complex. This work was published in Blood, 2012 (Wang, J., Muntean, A.G., and Hess, J.L. ECSASB2 mediates MLL degradation during hematopoietic differentiation). Chapter 3 describes the intrinsic E3 ligase activity of the second PHD finger. This work was published in The Journal of Biological Chemistry, 2012 (Wang, J., Muntean, A.G., Wu, L., and Hess, J.L. A subset of mixed lineage leukemia proteins has PHD domain-mediated E3 ligase activity). vi The third question was tackled in Chapter 4 by studying the cooperativity between Hoxa9 and its potential cofactors C/ebpα and Pu.1. This on-going project is highly collaborative: Joel Bronstein focused on protein biochemistry assays; Cailin Collins performed all the molecular and cellular assays and prepared libraries for ChIP-sequencing and RNA-sequencing; and I performed analysis of the high throughput sequencing data, which is presented in this dissertation. Chapter 4 also serves as the writing component to partially fulfill the requirements for a dual Master’s degree in Statistics. vii Table of Contents Dedication……………………………………………………………………………..ii Acknowledgement ........................................................................................................ iii Preface………………………………………………………………………………...vi List of Figures ............................................................................................................. xiv List of Tables .............................................................................................................. xix List of Appendices ....................................................................................................... xx Chapter1 Introduction ............................................................................................... 1 1.1 MLL in normal and malignant hematopoiesis ............................................... 1 1.1.1 Protein structure and molecular function of wild-type MLL ............. 1 1.1.2 MLL in normal hematopoiesis ........................................................... 3 1.1.3 MLL fusion mediated leukemia ......................................................... 4 1.1.4 PHD finger ....................................................................................... 12 1.1.5 Bromodomain .................................................................................. 23 viii 1.2 HOXA9 is a key target gene of MLL .......................................................... 27 1.2.1 HOXA9 in MLL mediated leukemia ............................................... 27 1.2.2 Molecular function of HOXA9 ........................................................ 28 1.3 Function of MLL family proteins and their role in human diseases ............ 30 1.4 ECSASB (Elongin B/C-Cullin–SOCS box protein) E3 ligase complex ........ 32 Chapter2 ECSASB2 Mediates MLL Degradation during Hematopoietic Differentiation .............................................................................................................. 35 2.1 Abstract ........................................................................................................ 35 2.2 Introduction .................................................................................................. 36 2.3 Experimental Procedures ............................................................................. 38 2.3.1 Cell culture ....................................................................................... 39 2.3.2 Vector construction .......................................................................... 39 2.3.3 Immunoprecipitation and Western blotting ..................................... 40 2.3.4 Protein identification by LC-Tandem Mass Spectroscopy .............. 40 2.3.5 Dual luciferase assay........................................................................ 41 2.3.6 Chromatin immunoprecipitation (ChIP) .......................................... 41 ix 2.3.7 Real-time quantitative reverse transcription PCR (RT-qPCR) ........ 43 2.3.8 shRNA knockdown and Characterization of cell differentiation ..... 43 2.3.9 Protein purification .......................................................................... 44 2.3.10 In vitro binding assay ....................................................................... 45 2.3.11 In vitro ubiqutination assay .............................................................. 46 2.4 Results .......................................................................................................... 46 2.4.1 ASB2 interacts with the PHD/Bromo region of MLL ..................... 46 2.4.2 ASB2 degrades MLL and reduces MLL transactivation ability ...... 50 2.4.3 The bromodomain and 4th PHD finger of MLL mediate interaction with
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