Circadian Clock Regulation of the Glycogen Metabolism In
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Circadian clock regulation of the glycogen metabolism in Neurospora crassa A Dissertation Submitted to the Faculty of The Graduate School of the University of Cincinnati In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Lily (Mokryun) Baek Committee Chair: Christian I. Hong, Ph.D. Committee Members: Nelson Horseman, Ph.D. Sookkyung Lim, Ph.D. Sean Moore, M.S., M.D. Yana Zavros, Ph.D. i ABSTRACT Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Industrialized modern society forces our work and activity to be outside of the conventional day, leading to adverse health effects, such as obesity and metabolic disorders. Therefore, identifying molecular pathways responsible for the development of diseases in the circadian-disturbed conditions is critical to find ways for alleviating the negative consequence of night shift work with keeping its societal demands. Glucose homeostasis depends on extracellular signaling and allosteric control; however, the molecular mechanisms linking the circadian clock to glucose homeostasis remain largely unknown. Here we investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in a filamentous fungal model Neurospora crassa. We found that glycogen synthase (gsn) mRNA, glycogen phosphorylase (gpn) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Furthermore, we identified that the core clock component, WCC directly binds to the gsn promoter, regulating periodic changes in GSN protein, phosphorylation and glycogen products. In addition, the WCC-controlled TFs, CSP-1 and VOS-1 cooperatively modulate the phase and amplitude of rhythmic expression of glycogen metabolic genes, and glycogen accumulation. Finally, the night preferred growth was disrupted in ∆gsn, ∆gpn, and clock mutant strains, demonstrating a potential physiological role for the clock in glycogen metabolism. ii iii TABLE OF CONTENTS PAGE ABSTRACT ...........................................................................................................ii TABLE OF CONTENTS………………………………………………………...iv ACKNOWLEDGMENTS.....................................................................................vii LIST OF FIGURES..............................................................................................viii LIST OF TABLES ..................................................................................................x CHAPTER I BACKGROUND AND LITERATURE REVIEW………………1 1.1 Circadian rhythms..……………………………………………….…........1 1.1.1 The circadian clock in mammals ..…………………………………..5 1.1.1.1 Hierarchical entrainment of the circadian rhythm in mammals…5 1.1.1.2 Molecular structure of circadian clock in mammal………………7 1.2 Neurospora crassa and its circadian clock……………………………….9 1.2.1 Neurospora crassa and its life cycle…………………………………9 1.2.2 Neurospora crassa as a model organism for circadian studies…..…11 1.2.2.1 The molecular mechanisms of circadian rhythms in Neurospora crassa………………………………………………………..…13 1.2.2.2 Entrainment of the Neurospora crassa circadian clock………….14 1.3 Circadian rhythms and metabolism: How the clock regulates metabolism and vice versa………………………………..……………15 1.3.1. Metabolic dysfunctions in circadian disturbances………………..16 iv 1.3.1.1 Shift work (non-genetic clock disturbance)...………………….16 1.3.1.2 Metabolic phenotypes in clock mutants…..…………………….17 1.3.2 Clock-controlled metabolism………………………...……….……19 1.3.2.1 Rhythms in genes encoding metabolic enzymes…………….…20 1.3.2.2 Non-transcriptional regulations of the clock in metabolism……22 1.3.2.3 Circadian rhythm in metabolic hormones………………………23 1.3.3 Metabolic entrainment of the circadian clock…………….….……..24 1.3.3.1 Feeding and food-entrainable oscillators……………………….24 1.3.3.2 Circadian clocks responding to metabolism……………… …...25 1.4 The circadian clock and glucose homeostasis ……………………………27 1.4.1 A brief review of glucose metabolism in mammals…………………27 1.4.2 The clock-controlled glucose metabolism in mammals…………….28 1.4.3 Glycogen metabolism……………………………………………….30 1.4.3.1 Glycogen synthesis…………………………………………….31 1.4.3.2 Glycogen degradation………………………………………….33 1.4.3.3 Glycogen metabolism in Neurospora crassa…………………..35 1.4.3.4 Clock-controlled glycogen metabolism………………………..38 PROBLEM STATEMENT……………………………………………………....40 CHAPTER II CIRCADIAN RHYTHMS IN GLYCOGEN METABOLISM IN NEUROSPORA CRASSA……………….……………………...41 OVERVIEW…………………………………………………………………..41 2.1 Introduction………………………………………………………………42 2.2 Materials and methods…………………………………………………...44 2.3 Results……………………………………………………………………49 v 2.4 Discussion……..…………………………………………………………51 Acknowledgement…………………………………………………………...63 CHAPTER III IDENTIFICATION OF TRANCRIPTION FACTORS CONNECTING THE CLOCK AND GLYCOGENMETABOLISM...64 OVERVIEW…………………………………………………………………...64 3.1 Introduction………………………………………………………………65 3.2 Materials and methods………...…………………………………………68 3.3 Results……………………………………………………………………73 3.4 Discussion………………………………………………………………..76 Acknowledgement…………………………………………………………...78 CHAPTER IV GLYCOGEN METABOLISM AND ITS CONSEQUENCE ON GROWTH AT NIGHT…………………..92 OVERVIEW………………………………………………………………..…92 4.1 Introduction………………………………………………………………93 4.2 Materials and methods…………...………………………………………95 4.3 Results……………………………………………………………………96 4.4 Discussion………………………………………………………………..98 CHAPTER V CONCLUSIONS/FUTURE DIRECTIONS…………………….103 Summary…………………………………….……………………………...103 Conclusions/Future directions…………….………………………………..104 Final thoughts………………………………………………………………113 Supplementary information………………………………………………...117 REFERENCES…………………………………………………………………122 vi ACKNOWLEDGMENTS I would like to thank my PhD advisor, Dr. Christian Hong, for his continued support, encouragement and guidance. I would also like to thank my committee members, Dr. Nelson Horseman, Dr. Sookkyung Lim, Dr. Sean Moore, and Dr. Yana Zavros for their constructive suggestions and support. I am thankful to lab members, former and present, and special thanks to Dr. Toru Matsu-ura and Kaoru Matsu-ura for their support, kindness, and friendship through my long years in HongLab. I would also like to thank all the collaborators and the personnel involved in my research project-Drs. Deborah Bell-Pedersen, Teresa Lamb (Texas A&M, USA), Maria Celia Bertolini, Stela Virgilio (UNESP, Brazil). Thanks also go to my colleagues and the department faculty and staff for making my time at UC a good experience. Lastly, I dedicated this work to my family, without whom I would never be the person I am today, and thank for their unconditional love and endless support. vii LIST OF FIGURES .....................................................................................PAGE Figure 1.1 Circadian oscillators are governed by a common mechanism ……….4 Figure 1.2 Race tube assay to detect circadian rhythm in Neurospora crassa ….12 Figure 1.3 Schematic representation of glycogen synthesis and degradation in Neurospora crassa ………………………………………………......37 Figure 2.1 Clock control of gsn and gpn mRNA levels, and rhythmic glycogen accumulation………………………………………………………....54 Figure 2.2 Glycogen accumulation and core clock gene expression in knockout strains lacking either gsn or gpn …………………………………….56 Figure 2.3 Circadian rhythm in GSN protein levels, and its phosphorylation…..58 Figure S2.1 Raw data of bioluminescence from gsn::luciferase and gpn::luciferase………………………………...................................61 Figure S2.2 Raw data of bioluminescence from frq::luciferase…………………62 Figure 3.1 CSP-1 regulates the rhythmic expression of gpn.................................79 Figure 3.2 VOS-1 binds rhythmically to the gsn promoter.……………………..81 Figure 3.3 VOS-1 influences rhythmic gsn, gpn, and glycogen levels………….83 Figure 3.4 Δcsp-1;Δvos-1 alters the phase and amplitude of rhythm in glycogen accumulation, not rhythmicity………………..…………...84 Figure 3.5 WCC directly binds to gsn promoter………………...………….……85 Figure 3.6 WCC controls gsn expression, promoting rhythmic glycogen Accumulation…………………………………………………………87 Figure S3.1 Raw data of bioluminescence from gsn::luciferase and gpn::luciferase……………...............................................................89 Figure 4.1 Schematic diagram of glycogen exhausted cultures for growth rate measurements……………………………………………………….100 Figure 4.2 Glycogen metabolism impacts rhythmic Neurospora crassa growth rates………………………………………………………...……….101 viii Figure 5.1 Maintenance of phase is important for the rhythm of glycogen accumulation……………………………………………………….. 114 Figure 5.2 Schematic diagram of clock-controlled glycogen metabolism and its physiological consequence in Neurospora crassa………………116 ix LIST OF TABLES .......................................................................................PAGE TABLE 2.1 Strains used in this study…………………………………................59 TABLE 2.2 Primers used in this study...…………………………………...........60 TABLE 3.1 Strains used in this study…………………………………................90 TABLE 3.2 Primers used in this study...…………………………………...........91 TABLE 4.1 Strains used in this study…………………………………..............102 TABLE S5.1 Strains used in this study…………………………………...........117 TABLE S5.2 Primers used in this study...………………………………….......117 x CHAPTER I. BACKGROUNDS AND LITERATURE REVIEW 1.1 Circadian rhythms Circadian rhythm (Latin circa diem, meaning ‘about a day’) is an anticipatory internal time-keeping mechanism conserved in a wide range of living organisms from bacteria to human.