University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 Unraveling The Beta Cell Translatome: Elucidation Of An Erk/hnrnpk/jund Axis Austin Lewis Good University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Good, Austin Lewis, "Unraveling The Beta Cell Translatome: Elucidation Of An Erk/hnrnpk/jund Axis" (2019). Publicly Accessible Penn Dissertations. 3327. https://repository.upenn.edu/edissertations/3327 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3327 For more information, please contact [email protected]. Unraveling The Beta Cell Translatome: Elucidation Of An Erk/hnrnpk/ jund Axis Abstract In type 2 diabetes, oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation, however, the mechanisms underlying this process in β cells are not fully understood. Here we use TRAP-seq to examine changes in the ribosome occupancy of mRNAs during conditions associated with β cell dysfunction, leading us to identify a cohort of translationally regulated genes with 3’UTR enrichment of a cytosine-rich motif. Of particular interest was the gene encoding JUND, a transcription factor with anti-oxidant functions in other cell types but whose role in cells is unknown. Interestingly, JUND is translationally upregulated in islets exposed to high glucose and free fatty acid levels, and depletion of JUND in β cells reduces oxidative stress and apoptosis caused by these conditions. Transcriptome assessment demonstrates that JUND regulates a cohort of genes that are commonly dysregulated during β cell dysfunction, including pro-oxidant and pro- inflammatory genes, consistent with this factor enhancing, rather than reducing, oxidative stress levels in cells. Further, hnRNPK, an RNA binding protein with specificity for cytosine-rich stretches, binds to the mRNA encoding JUND and is required for its post-transcriptional upregulation during metabolic stress. Although the absolute levels of hnRNPK do not change, there is a significant increase in hnRNPK phosphorylation during glucolipotoxicity. Importantly, this hnRNPK/JUND axis is activated in islets from diabetic db/db mice and in human islets exposed to metabolic stress. Finally, a series of mechanistic studies indicate that hnRNPK post- transcriptionally regulates JUND in a MEK/ERK- and DDX3X-dependent manner. Thus, a translation- centric approach uncovered hnRNPK and JUND as stress-responsive factors in β cells that contribute to redox imbalance and apoptosis during pathophysiologically relevant stress. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Doris A. Stoffers Keywords Beta cells, Diabetes, Oxidative stress, RNA binding proteins, Transcriptional regulation, Translation Subject Categories Cell Biology | Genetics | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3327 UNRAVELING THE β CELL TRANSLATOME: ELUCIDATION OF AN ERK/HNRNPK/JUND AXIS Austin L. Good 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 2019 Supervisor of Dissertation _________________ Doris A. Stoffers, M.D., Ph.D. Sylvan H. Eisman Professor of Medicine Graduate Group Chairperson _________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Klaus H. Kaestner, Ph.D. Thomas and Evelyn Suor Butterworth Professor in Genetics Patrick Seale, Ph.D. Associate Professor of Cell and Developmental Biology Stephen A. Liebhaber, M.D. Professor of Genetics Zissimos Mourelatos, M.D. Professor of Pathology and Laboratory Medicine ACKNOWLEDGMENTS I would like to thank the many people that have contributed to my scientific development and research endeavors during my graduate work. I am very grateful to my thesis advisor, Dr. Doris Stoffers, for her mentorship and support throughout this process. Her curiosity and excitement to more deeply understand scientific questions provide a wonderful model for her trainees. Throughout my training, Doris encouraged me to develop my own ideas and hypotheses while also serving as the guard rails to keep me on track when necessary. Her mentorship allowed me to grow as an investigator and gave me confidence to tackle complex problems. I am also very grateful for the opportunity to work alongside and interact with many bright and enthusiastic members of the Stoffers lab during my graduate work who have all contributed to my training. I especially want to thank Dr. Corey Cannon who served as a wonderful mentor for me when I joined the lab and who laid the foundation for all of the work presented here. I also want to thank Dave Groff and Dr. Andrea Rozo for putting up with all of my urgent order requests over the years. I also thank Drs. Christine Juliana, Diana Stanescu, Andrea Rozo, Jeff Raum, Ben Ediger, and Juxiang Yang for their many suggestions and advice on my project. I want to thank Matt Haemmerle and Alexis Oguh for their grad student camaraderie, and I wish them the best of luck in their thesis work and future endeavors. I would also like to thank Dr. Skip Brass for sharing his wisdom throughout the many stages of the MD-PhD program, and Maggie Krall for being a listening ear and giving perceptive advice. I am very lucky to have a family that provides me with unwavering support and encouragement. Amanda, Matt, Julian, and Camille, spending time together always provides a necessary escape from the world of science and gives me perspective to keep moving forward with a clear head. Mom and Dad, I can’t thank you enough for the love and support you have always given me. I can’t imagine better role models. And finally, I thank my wife Charly Ann for always making me smile at the end of a long day. You are my rock and I can’t wait for the many adventures ahead. ii ABSTRACT UNRAVELING THE β CELL TRANSLATOME: ELUCIDATION OF AN ERK/HNRNPK/JUND AXIS AUSTIN L. GOOD DORIS A. STOFFERS, M.D., Ph.D. In type 2 diabetes, oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation, however, the mechanisms underlying this process in β cells are not fully understood. Here we use TRAP-seq to examine changes in the ribosome occupancy of mRNAs during conditions associated with β cell dysfunction, leading us to identify a cohort of translationally regulated genes with 3’UTR enrichment of a cytosine-rich motif. Of particular interest was the gene encoding JUND, a transcription factor with anti-oxidant functions in other cell types but whose role in β cells is unknown. Interestingly, JUND is translationally upregulated in islets exposed to high glucose and free fatty acid levels, and depletion of JUND in β cells reduces oxidative stress and apoptosis caused by these conditions. Transcriptome assessment demonstrates that JUND regulates a cohort of genes that are commonly dysregulated during β cell dysfunction, including pro-oxidant and pro-inflammatory genes, consistent with this factor enhancing, rather than reducing, oxidative stress levels in β cells. Further, hnRNPK, an RNA binding protein with specificity for cytosine-rich stretches, binds to the mRNA encoding JUND and is required for its post-transcriptional upregulation during metabolic stress. Although the absolute levels of hnRNPK do not change, there is a significant increase in hnRNPK phosphorylation during glucolipotoxicity. Importantly, this hnRNPK/JUND axis is activated in islets from diabetic db/db mice and in human islets exposed to metabolic stress. Finally, a series of mechanistic studies indicate that hnRNPK post-transcriptionally regulates JUND in a MEK/ERK- and DDX3X-dependent manner. Thus, a translation-centric approach uncovered hnRNPK and JUND as stress-responsive factors in β cells that contribute to redox imbalance and apoptosis during pathophysiologically relevant stress. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ......................................................................................................... II ABSTRACT .............................................................................................................................. III LIST OF TABLES .................................................................................................................. VII LIST OF ILLUSTRATIONS .............................................................................................. VIII CHAPTER 1: INTRODUCTION ........................................................................................... 1 1.1 Paradigms of β cell demise in type 2 diabetes ....................................................................... 1 1.1.1 Impaired insulin secretion ........................................................................................................... 2 1.1.2 β cell apoptosis ............................................................................................................................. 4 1.1.3 Loss of β cell identify ..................................................................................................................