University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Circadian Regulation Of Hepatic Metabolism By Nuclear Receptors Rev-Erb And Ror Yuxiang Zhang University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Genetics Commons, Pharmacology Commons, and the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Zhang, Yuxiang, "Circadian Regulation Of Hepatic Metabolism By Nuclear Receptors Rev-Erb And Ror" (2017). Publicly Accessible Penn Dissertations. 2665. https://repository.upenn.edu/edissertations/2665 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2665 For more information, please contact [email protected]. Circadian Regulation Of Hepatic Metabolism By Nuclear Receptors Rev-Erb And Ror Abstract Circadian and metabolic physiology are intricately intertwined. Although the liver clock is entrained by the central clock, it also directly controls metabolic gene expression. Hormone responsive nuclear factors are hypothesized to be the major clock components that regulate metabolism, which include Rev-erbs and RORs. In this study, we explored the mechanism of regulation of liver circadian gene expression by Rev- erbs and RORs. We found that Rev-erbalpha modulates the clock and metabolism by different genomic mechanisms. Clock control requires Rev-erbalpha to bind directly to the genome at its cognate sites, where it competes with activating ROR TFs. By contrast, Rev-erbalpha regulates metabolic genes primarily by recruiting the HDAC3 corepressor to sites to which it is tethered by cell type-specific transcription factors. Thus, direct competition between Rev-erbalpha and ROR TFs provides a universal mechanism for self-sustained control of molecular clock across all tissues, whereas Rev-erbalpha utilizes�lineage-determining factors to convey a tissue-specific epigenomic rhythm that egulatesr metabolism tailored to the specific need of that tissue. In addition, we also investigate the circadian regulation of hepatic metabolism by nuclear receptors RORalpha and RORgamma. We discovered that hepatic depletion of RORs increased expressions of genes involved in lipogenesis as well as the hepatic triglyceride levels, specifically at ZT22 (5 AM) and during feeding, but not at ZT10 (5 PM) nor during fasting. GRO-seq analysis suggested that regulation of lipid metabolism by RORs was mediated by SREBP1-c. Indeed, ROR depletion significantly up-regulated nuclear SREBP1-c protein level specifically at ZT22. Overall, our study reveals a time-specific egulationr of liver lipid metabolism by RORs, and suggests a potential role of chronotherapy targeting circadian factors in the treatment of metabolic disorders. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Mitchell A. Lazar Second Advisor Klaus H. Kaestner Keywords Circadian Clock, Epigenomics, Liver, Metabolism, Nuclear Receptor, Transcriptional Regulation Subject Categories Genetics | Pharmacology | Pharmacy and Pharmaceutical Sciences This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2665 CIRCADIAN REGULATION OF HEPATIC METABOLISM BY NUCLEAR RECEPTORS REV-ERB AND ROR Yuxiang Zhang A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation ______________ Mitchell A. Lazar, M.D., Ph.D. Willard and Rhoda Ware Professor in Diabetes and Metabolic Diseases Graduate Group Chairperson ______________ Julie A. Blendy, Ph.D. Professor of Pharmacology Dissertation Committee Klaus H. Kaestner, Ph.D., Thomas and Evelyn Suor Butterworth Professor in Genetics Garret A. FitzGerald, M.D., Robert L. McNeil, Jr., Professor in Translational Medicine and Therapeutics Amita Sehgal, Ph.D., John Herr Musser Professor of Neuroscience To my wife Sheng Zhang, my son Stephen J. Zhang and our parents. ii ACKNOWLEDGMENT I would like to thank my mentor Dr. Mitchell A. Lazar, first and foremost, for his guidance and support. His insightful advice always will leads to a deeper scientific thinking, and exciting topics to explore. He has also created a vibrant and collaborative lab environment, in which everyone is happy to share thoughts, reagents and protocol. I would like to thank him and every members of the Lazar lab for the mutual help and encouragement. I would also like to thank my committee members: Dr. Klaus H. Kaestner who serves as chair, Dr. Garret A. FitzGerald and Dr. Amita Seghal for their advice, constructive criticism and time to join my committee meeting. I would also like to Dr. Peter F. Davies for his guidance during my Master program study at UPenn. Davies Lab was the first lab I worked for in UPenn and he taught me and helped my family a lot when I first came to U.S. He also encouraged me to pursue a career in science. I would like to acknowledge the Pharmacology Graduate Group (PGG). I particularly want to thank Dr. Julie A. Blendy for their advice and support, Sarah Squire, PGG coordinator, for her coordination and organization of the graduate group activities, and support and help during my thesis studying process. iii I am also grateful to all the people who helped me with the work presented in this dissertation. I would like to thank Dan Feng, Zheng Sun for their helpful advice and experimental guidance when I rotated and started my project in Lazar lab. I would like to thank Bin Fang and Manashree for the bioinformatics analysis. I would like to thank David J. Steger, Matthew J. Emmett, Romeo Papazyan, Sean M. Armour, Jarrett R. Remsberg, Jennifer Jager, Dongyin Guan, Yong Hoon Kim, Raymond E. Soccio, Wenxiang Hu, Lindsey Peed, and Erika Briggs for their useful advice and technical assistance, and staffs in the Next-Generation Sequencing Core at University of Pennsylvania Perelman School of Medicine. I am indebted to my family for all the love and support while I worked on this dissertation and always. My wife Sheng Zhang has assisted me in every aspect of my life and taken good care of my son Stephen J. Zhang. This work could not be made possible without her efforts. I would also like to thank our parents who supported us and inspired us to pursue our dream. iv ABSTRACT CIRCADIAN REGULATION OF HEPATIC METABOLISM BY NUCLEAR RECEPTORS REV-ERB AND ROR Yuxiang Zhang Mitchell A. Lazar Circadian and metabolic physiology are intricately intertwined. Although the liver clock is entrained by the central clock, it also directly controls metabolic gene expression. Hormone responsive nuclear factors are hypothesized to be the major clock components that regulate metabolism, which include Rev-erbs and RORs. In this study, we explored the mechanism of regulation of liver circadian gene expression by Rev-erbs and RORs. We found that Rev-erbα modulates the clock and metabolism by different genomic mechanisms. Clock control requires Rev-erbα to bind directly to the genome at its cognate sites, where it competes with activating ROR TFs. By contrast, Rev-erbα regulates metabolic genes primarily by recruiting the HDAC3 corepressor to sites to which it is tethered by cell type-specific transcription factors. Thus, direct competition between Rev- erbα and ROR TFs provides a universal mechanism for self-sustained control of molecular clock across all tissues, whereas Rev-erbα utilizes lineage-determining factors to convey a tissue-specific epigenomic rhythm that regulates metabolism tailored to the specific need of that tissue. v In addition, we also investigate the circadian regulation of hepatic metabolism by nuclear receptors RORα and RORγ. We discovered that hepatic depletion of RORs increased expressions of genes involved in lipogenesis as well as the hepatic triglyceride levels, specifically at ZT22 (5 AM) and during feeding, but not at ZT10 (5 PM) nor during fasting. GRO-seq analysis suggested that regulation of lipid metabolism by RORs was mediated by SREBP1-c. Indeed, ROR depletion significantly up-regulated nuclear SREBP1-c protein level specifically at ZT22. Overall, our study reveals a time-specific regulation of liver lipid metabolism by RORs, and suggests a potential role of chronotherapy targeting circadian factors in the treatment of metabolic disorders. vi TABLE OF CONTENTS ACKNOWLEDGMENT ......................................................................................... III ABSTRACT .......................................................................................................... V TABLE OF CONTENTS ..................................................................................... VII LIST OF TABLES ................................................................................................ XI LIST OF ILLUSTRATIONS ................................................................................ XII CHAPTER 1 BACKGROUND AND INTRODUCTION .......................................... 1 1.1 Nuclear Receptors Rev-erbs and RORs Link Circadian to Metabolism ... 2 1.1a Nuclear Receptors in Core Molecular Clock System ................................. 2 1.1b Expression and Functions of Rev-erbs and RORs ..................................... 3 1.1c Post-Translational Modification of Rev-erbs and RORs ............................. 4 1.2 Rev-erbs and RORs as Regulators of Central Clock and Brain Function 5 1.3 Role of Rev-erbs and RORs in Peripheral Tissues ...................................