The Role of Trim58 in Erythropoiesis
Total Page：16
File Type：pdf, Size：1020Kb
University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 The Role of Trim58 in Erythropoiesis Christopher Stephen Thom University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, Cell Biology Commons, and the Developmental Biology Commons Recommended Citation Thom, Christopher Stephen, "The Role of Trim58 in Erythropoiesis" (2014). Publicly Accessible Penn Dissertations. 1469. https://repository.upenn.edu/edissertations/1469 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1469 For more information, please contact [email protected]. The Role of Trim58 in Erythropoiesis Abstract Red blood cells (erythrocytes) deliver oxygen to all tissues of the body. Defects in red blood cell production (erythropoiesis) can cause disease. Mammalian erythropoiesis culminates in enucleation, an incompletely understood process that entails the physical separation of the nucleus and cytoplasm. The work in this thesis investigated the role of a previously uncharacterized protein named Trim58 in erythropoiesis. Human genetic studies suggested that TRIM58 played an important role in erythroid development. In humans and mice, Trim58 expression was found to be restricted to red blood cell precursors during late stage maturation. In fact, murine Trim58 was upregulated just prior to enucleation. Using short hairpin RNAs, Trim58 expression was inhibited in cultured murine erythroblasts. Through a variety of analyses, it was demonstrated that Trim58 is dispensable for early erythroid maturation. However, Trim58 knockdown impaired movement of the nucleus, thereby inhibiting enucleation. Trim58 is a member of the tripartite motif-containing family of proteins, many of which function as E3 ubiquitin ligases that can facilitate protein degradation. Protein interaction studies demonstrated that Trim58 bound directly to the molecular motor protein complex dynein. Consistent with its putative role as an E3 ubiquitin ligase, ectopic Trim58 expression in HeLa cells caused dynein degradation in a proteasome-dependent fashion. Furthermore, dynein loss and efficient enucleation were coincident and dependent upon Trim58 induction during erythroid culture maturation. Dynein mediates unidirectional nuclear movement toward the microtubule organizing center. Erythroid enucleation requires nuclear movement in the opposite direction. Hence, Trim58-mediated dynein degradation may be responsible for nuclear movement during enucleation. Our findings identify rim58T as the first erythroid-specific protein that regulates this process. More broadly, regulated proteolysis represents a previously unappreciated mode of regulation for dynein, which is critical for many cellular processes. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Mitchell J. Weiss Keywords Dynein, Enucleation, Erythropoiesis, Protein Quality Control, Tripartite Motif Protein, Ubiquitin Proteasome System Subject Categories Biochemistry | Cell Biology | Developmental Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1469 THE ROLE OF TRIM58 IN ERYTHROPOIESIS Christopher S Thom A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2014 Supervisor of Dissertation __________________________ Mitchell J Weiss, MD, PhD, Professor of Pediatrics Graduate Group Chairperson __________________________ Daniel S Kessler, PhD, Associate Professor of Cell and Developmental Biology Dissertation Committee Roger A Greenberg, MD, PhD, Associate Professor of Cancer Biology (Chair) Mark L Kahn, MD, Professor of Medicine Stephen A Liebhaber, MD, Professor of Genetics Serge Y Fuchs, MD, PhD, Professor of Cell Biology Michael Lampson, PhD, Associate Professor of Biology Erika L Holzbaur, PhD, Professor of Physiology THE ROLE OF TRIM58 IN ERYTHROPOIESIS COPYRIGHT 2014 Christopher Stephen Thom This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ iii DEDICATION I dedicate this thesis to my daughter Madeline and wife Colleen; my father Stephen, mother Linda, brother Matthew, and sister Megan; my scientific mentors Charles Lovett, Charles Roberts, and Mitchell Weiss. iv ACKNOWLEDGMENTS I am extremely grateful for the guidance and mentorship that I have received from Mitch Weiss over the course of my thesis work. He has been a role model as a physician-scientist in conducting interesting and important work, as well as everything else that comes with being an Investigator. He has supported me through the ups and downs of this project, through several “life events” during the course of my graduate studies, and has helped me to grow as a scientist. I am fortunate to have collaborated closely with unbelievably talented MD/PhD students during my time in the Weiss Lab. Eugene Khandros literally showed me where everything was in the lab and how to technically accomplish a variety of experiments. He is directly responsible for much of the success I have had during my graduate studies. On my first day in the lab, we pored over a list of proteins that Eugene identified in a screen. The number one hit was Trim58, which we thought might be interesting to study. For this and many other reasons, I am lucky to have followed in his footsteps. Elizabeth Traxler, another MD/PhD student, will continue work on Trim58 after my graduation. She has already made valuable contributions to our understanding of Trim58 biology, and I am sure she will do great things in the future. I appreciate all that she has taught me and I wish her all the success in the world. Yu Yao and Jenna Nickas provided technical assistance throughout the course of my thesis work. Their work was instrumental to the success of my projects, and it was a pleasure to work with both of them. v Without my family and friends, I would not be in this position today. My father Stephen is physician-scientist who has provided fatherly advice, scientific mentorship, and even specific suggestions related to my thesis projects. I am incredibly lucky to have him in my life as a devoted father and scientific role model. I am also grateful for support from my mother Linda, brother Matt, and sister Megan throughout all these years of higher education. I also thank my family members-in-law Angela, James Sr., Kathleen, James Jr., Mariela, and Isabella, and too many friends to specifically name here, for putting up with me and supporting me through good times and bad. Finally, and most importantly, to my daughter Madeline and wife Colleen: You make me the happiest person in the world. Your strength and determination inspire me to be a better person. I am also grateful to the following people for scientific support and helpful discussions: Weiss Lab Vikram Paralkar Orna Steinberg-Shemer Olivia Y Zhou Dolly Prabhu Duonan Yu Guowei Zhao Jiyoon Noh Maxim Pimkin Janine D’Souza Blobel Lab Gerd Blobel Stephan Kadauke Amy Campbell Rena Zheng vi Holzbaur Lab Jacob E Lazarus Meredith H Wilson Swathi Ayloo Mariko Tokito Kahn Lab David Rawnsley David Enis Patricia Mericko-Ishizuka Thesis Committee Roger A Greenberg Mark L Kahn Stephen A Liebhaber Serge Y Fuchs Erika L Holzbaur Michael A Lampson Collaborators Ana PG Silva and Joel P Mackay, University of Sydney Kazuhiko Adachi, Children’s Hospital of Philadelphia Medical Scientist Training Program Administration Lawrence F Brass Maggie Krall Maureen Kirsch Other individuals who supported the work presented in Chapter 3 Drs. Katherine S Ullman (University of Utah) and Laura Gutiérrez (Erasmus MC, Netherlands) provided helpful conversations, protocols and reagents. Dr. Min Min Lu and the University of Pennsylvania Molecular Cardiology Research Center Histology and Gene Expression Core assisted with radioactive in situ hybridization experiments. Dr. Andrea Stout and the University of Pennsylvania Cell and Developmental Biology Microscopy Core assisted with imaging. vii ABSTRACT THE ROLE OF TRIM58 IN ERYTHROPOIESIS Christopher S Thom Mitchell J Weiss, MD, PhD Red blood cells (erythrocytes) deliver oxygen to all tissues of the body. Defects in red blood cell production (erythropoiesis) can cause disease. Mammalian erythropoiesis culminates in enucleation, an incompletely understood process that entails the physical separation of the nucleus and cytoplasm. The work in this thesis investigated the role of a previously uncharacterized protein named Trim58 in erythropoiesis. Human genetic studies suggested that TRIM58 played an important role in erythroid development. In humans and mice, Trim58 expression was found to be restricted to red blood cell precursors during late stage maturation. In fact, murine Trim58 was upregulated just prior to enucleation. Using short hairpin RNAs, Trim58 expression was inhibited in cultured murine erythroblasts. Through a variety of analyses, it was demonstrated that Trim58 is dispensable for early erythroid maturation. However, Trim58 knockdown impaired movement of the nucleus, thereby inhibiting enucleation. Trim58 is a member of the tripartite motif-containing family of proteins, many of which function as E3 ubiquitin ligases that can facilitate protein degradation. Protein interaction