UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Insights into Cellular Stress Revealed by SIRT7 Permalink https://escholarship.org/uc/item/415843w9 Author Liu, Yufei Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Insights into Cellular Stress Revealed by SIRT7 By Yufei Liu A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Danica Chen, Co-Chair Professor Ellen Robey, Co-Chair Professor George Martin Professor Astar Winoto Professor Amy Herr Spring 2014 Insights into Cellular Stress Revealed by SIRT7 ©2014 by Yufei Liu 1 Abstract Insights into Cellular Stress Revealed by SIRT7 by Yufei Liu Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Danica Chen, Co-Chair Professor Ellen Robey, Co-Chair Aging was once thought to be an inexorable process, driven by wear and tear that steadily accumulates in cells. However, we have now come to the realization that aging is subject to modulation by genetic pathways, including the insulin/IGF-1 pathway, the mTOR pathway, and sirtuins. A commonality among these pathways is that they exert their pro-longevity and pro-health effects through responding to stressors, such as diet, oxidative stress, or protein-folding stress, and initiating a self-protective program in the cell. Sirtuins have been shown to play a major role in the cellular response to calorie restriction and oxidative stress. However, their role in protein homeostasis and in particular, the response to stress from unfolded proteins has not been well- characterized. We explored this topic by studying the role of SIRT7, a nuclear member of the mammalian sirtuin family, in responding to protein-folding stress in the endoplasmic reticulum (ER) and in mitochondria. We found that SIRT7 plays a pivotal role in the response to ER stress and mitochondrial stress by inhibiting the transcription of ribosomal subunits and mitochondrial ribosomal subunits, which results in a reduction in translation, allowing more time for proper protein folding. We found that SIRT7 mediates its effects by binding to the transcription factors Myc and NRF-1, which are master regulators of ribosomal subunits and mitochondrial biogenesis respectively. Our study suggests that the stress response mediated by SIRT7 at the cellular level may have important implications for obesity-associated diseases and hematopoietic stem cell function. i Dedicated to... Mom and Dad, for your inexhaustible love and support… Yutong, may you never age… and Sandy, for filling my life with fun and warmth ii Table of Contents Chapter 1 An Introduction to Aging, Stress, and Sirtuins ........................................ 1 The Genetic Regulators of Aging ................................................................................. 1 Insulin/IGF-1 Signaling ............................................................................................. 1 mTOR ....................................................................................................................... 2 The Role of Sirtuins in Lifespan Extension and Stress Resistance .............................. 3 Oxidative Stress ........................................................................................................ 5 High Fat Diet ............................................................................................................. 6 The Relation Between Stress and Aging ..................................................................... 7 Oxidative Stress and Aging ...................................................................................... 7 Proteostasis and Aging ............................................................................................. 8 Concluding Themes ..................................................................................................... 9 Chapter 2 SIRT7 and the Cellular Response to Endoplasmic Reticulum Stress .. 11 Introduction ................................................................................................................ 12 Results ....................................................................................................................... 13 Deficiency of SIRT7 Results in Constitutive ER Stress ........................................... 14 SIRT7 Protects Against ER Stress ......................................................................... 15 SIRT7 Suppresses ER Stress Through a Myc-Dependent Mechanism .................. 15 Discussion .................................................................................................................. 16 Implications for Fatty Liver Disease ........................................................................ 17 Chapter 3 SIRT7 and the Mitochondrial Unfolded Protein Response .................... 29 Introduction ................................................................................................................ 31 Results ....................................................................................................................... 32 SIRT7 is Directed by NRF-1 to the Promoters of Mitochondrial Translation Genes and Mitochondrial Ribosomal Proteins ................................................................... 32 SIRT7 KD Cells have NRF-1-dependent Elevation of Mitochondrial Translation Genes ..................................................................................................................... 32 SIRT7 is Upregulated by mtUPR via ATF5 ............................................................. 33 SIRT7-deficient Cells have More Severe Mitochondrial Stress .............................. 33 NRF-1 KD Rescues Increased Mitochondrial Stress in SIRT7 KD Cells ................ 34 SIRT7 KD Cells are More Prone to Nutrient Starvation-Induced Cell Death ........... 34 SIRT7 Improves Hematopoietic Stem Cell Function by Combating Mitochondrial Stress ..................................................................................................................... 34 SIRT7 Combats Mitochondrial Stress in Aged HSCs and Improves Function ........ 35 Discussion .................................................................................................................. 35 A New Component of the mtUPR ........................................................................... 35 Implications for HSC Function and Aging ............................................................... 36 Chapter 4 Future Directions and Closing Remarks ................................................. 55 Specificity of the SIRT7 Response to Stress.............................................................. 55 iii SIRT7 and Longevity ................................................................................................. 56 The Connection Between SIRT7 and Cancer ............................................................ 57 Closing Remarks ........................................................................................................ 58 References ................................................................................................................... 63 Appendix: Materials and Methods ............................................................................. 75 Cell Culture ............................................................................................................... 75 RNAi .......................................................................................................................... 75 Co-immunoprecipitations and Immunoblots .............................................................. 75 Polysome Profiling ..................................................................................................... 76 ChIP and mRNA analysis .......................................................................................... 76 Flow Cytometry and Cell Sorting ............................................................................... 76 In-vitro Stimulation of HSCs ...................................................................................... 77 Lentiviral Transduction of HSCs ................................................................................ 77 Statistical Analysis ..................................................................................................... 77 iv List of Figures Fig. 1.1: Pathways regulating longevity ........................................................................ 11 Fig. 2.1: SIRT7 is induced by treatment with ER stress inducers. ................................ 18 Fig. 2.2: SIRT7 induction upon ER Stress is dependent on XBP1 ................................ 19 Fig. 2.3: Knockdown of SIRT7 leads to increased expression of ER Stress markers ... 20 Fig. 2.4: SIRT7 KO MEF cells have elevated ER stress ............................................... 21 Fig. 2.5: SIRT7 protects against ER stress induction ................................................... 22 Fig. 2.6: SIRT7 protects against ER stress-induced cell death ..................................... 23 Fig. 2.7: SIRT7 Binds to Myc ........................................................................................ 24 Fig. 2.8: Inhibition of Myc rescues the increased expression of SIRT7 ribosomal targets, but not other SIRT7 targets.