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Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2009 Hypoxia-inducible regulatory networks and their roles in aging in C. elegans Yi Zhang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Cell and Developmental Biology Commons, and the Genetics and Genomics Commons Recommended Citation Zhang, Yi, "Hypoxia-inducible regulatory networks and their roles in aging in C. elegans" (2009). Graduate Theses and Dissertations. 10914. https://lib.dr.iastate.edu/etd/10914 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Hypoxia-inducible factor regulatory networks and their roles in aging in C. elegans by Yi Zhang A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Molecular, Cellular and Development Biology Program of Study Committee: Jo Anne Powell-Coffman, Major Professor Kristen Johansen David Oliver Guru Rao Chris Tuggle Iowa State University Ames, Iowa 2009 ii TABLE OF CONTENTS CHAPTER 1. GENERAL INTRODUCTION.................................................................................. 1 LITERATURE REVIEW .............................................................................................................................2 AGING: LESSONS FROM STUDIES OF C. elegans ........................................................................................2 HYPOXIA RESPONSE AND HYPOXIA‐INDUCIBLE FACTOR................................................................11 THESIS ORGANIZATION....................................................................................................................... 17 REFERENCE.............................................................................................................................................. 18 FIGURE LEGEND ..................................................................................................................................... 32 CHAPTER 2. The HIF­1 hypoxia­inducible factor modulates lifespan in C. elegans ...........................................................................................................................................................34 ABSTRACT................................................................................................................................................ 34 INTRUDUCTION...................................................................................................................................... 36 RESULTS ................................................................................................................................................... 38 HIF‐1 over‐expression causes dose‐dependent extension of adult lifespan..............................38 HIF‐1 over‐expression acts in parallel to DAF‐16 and SKN‐1 to extend lifespan.....................39 Effects of hif­1 loss‐of‐function mutations on longevity......................................................................40 Resistance to t‐butyl‐peroxide and heat stress.......................................................................................42 DISCUSSION ............................................................................................................................................. 42 Effects of HIF‐1 over‐expression ...................................................................................................................43 Effects of hif­1 loss‐of‐function.......................................................................................................................44 HIF‐1 and hormesis .............................................................................................................................................45 Selective pressures to control HIF‐1 signaling........................................................................................46 MATERIALS AND METHODS............................................................................................................... 47 C. elegans strains...................................................................................................................................................47 hif‐1 minigenes and generation of transgenic animals........................................................................47 Lifespan and stress analyses ...........................................................................................................................48 Protein blots and Quantitative RT‐PCR ......................................................................................................49 ACKNOWLEDGEMENTS........................................................................................................................ 49 REFERENCES............................................................................................................................................ 50 FIGURE LEGENDS................................................................................................................................... 55 SUPPLEMENTAL INFORMATION....................................................................................................... 63 Table S1: Data from individual lifespan experiments ..........................................................................63 Table S2: Effects of hif­1 RNAi on the longevity of wildtype worms..............................................66 Table S3. Lifespan assays at 25 ºC on UV‐irradiated bacterial food (OP50)...........................68 CHAPTER3. HIF ­1­INDEPENDENT HYPOXIA RESPONSE SIGNALING MEDIATES AGING IN C.ELEGANS....................................................................................................................69 Abstract .................................................................................................................................................... 69 INTRODUCTION...................................................................................................................................... 70 iii RERULTS................................................................................................................................................... 71 HIF‐1 is not necessary for hypoxia‐mediated lifespan extension. ..................................................71 Hypoxia modulates longevity by a mechanism distinct from insulin‐like signaling...............73 IRE‐1 mediated unfolded protein response is required for hypoxia‐ mediated lifespan extension..................................................................................................................................................................74 DISCUSSION ............................................................................................................................................. 75 MATERIALS AND METHODS............................................................................................................... 77 C. elegans strains...................................................................................................................................................78 Life span assays.....................................................................................................................................................78 Hypoxia treatment...............................................................................................................................................78 Microscopy ..............................................................................................................................................................79 REFERENCES............................................................................................................................................ 79 FIGURE LEGENDS................................................................................................................................... 83 CHAPTER 4. SWAN­1 interacts with EGL­9 to regulate HIF­1 activity in C. elegans ...........................................................................................................................................................91 Abstract .................................................................................................................................................... 91 Introduction............................................................................................................................................ 92 Materials and Methods........................................................................................................................ 94 Results....................................................................................................................................................... 99 ia50 allele activates HIF‐1 activity in vhl­1 mutants .............................................................................99 ia50 is a loss‐of‐function mutation in swan­1(F53C11.8)................................................................. 101 SWAN‐1 suppresses HIF‐1 activity via a VHL‐1‐independent pathway.................................... 102 SWAN‐1 interacts with EGL‐9 to

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