Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 Genome-wide analyses of expression data and HIF-1 binding sites provide insights to the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans Dingxia Feng Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biology Commons Recommended Citation Feng, Dingxia, "Genome-wide analyses of expression data and HIF-1 binding sites provide insights to the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans" (2013). Graduate Theses and Dissertations. 13324. https://lib.dr.iastate.edu/etd/13324 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]. Genome-wide analyses of expression data and HIF-1 binding sites provide insights to the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans by Dingxia Feng A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Jo Anne Powell-Coffman, Major Professor Thomas Peterson Yanhai Yin Peng Liu Drena Dobbs Jeffrey Essner Iowa State University Ames, Iowa 2013 Copyright © Dingxia Feng, 2013. All rights reserved. ii TABLE OF CONTENTS CHAPTER 1. GENERAL INTRODUCTION ..................................................................... 1 Literature Review ............................................................................................................... 4 C. elegans HIF-1 and low oxygen adaptation ................................................................... 4 HIF-1 has other functions besides hypoxia response in C. elegans .................................. 5 Regulation of HIF-1 stability and activity in C. elegans .................................................. 8 Other key stress responsive pathways in C. elegans....................................................... 10 Thesis Organization .......................................................................................................... 11 References .......................................................................................................................... 13 Figure Legends .................................................................................................................. 22 CHAPTER 2. SKN-1 INHIBITS THE HIF-1 HYPOXIA-INDUCIBLE FACTOR ...... 23 Abstract .............................................................................................................................. 23 Introduction ....................................................................................................................... 24 Materials and Methods ..................................................................................................... 26 Strains ............................................................................................................................. 26 RNAi screen .................................................................................................................... 26 Hypoxia stress and oxidative stress assays ..................................................................... 27 Pegl-9:GFP expression constructs ................................................................................. 27 Protein Blots.................................................................................................................... 28 RNA extraction and real-time quantitative RT-PCR ...................................................... 28 Results ................................................................................................................................ 28 skn-1 attenuates HIF-1 protein levels and HIF-1 function .............................................. 30 Differential requirements for skn-1 and hif-1 ................................................................. 30 SKN-1/NRF promotes egl-9 expression ......................................................................... 31 Separate and overlapping functions for SKN-1 and HIF-1 ............................................ 33 Discussion........................................................................................................................... 34 SKN-1 promotes egl-9 expression, thereby inhibiting HIF-1 ......................................... 34 RNAi screen identifies genes that regulate nhr-57 expression ....................................... 36 References .......................................................................................................................... 37 Figure Legends .................................................................................................................. 41 CHAPTER 3. TRANSCRIPTOME ANALYSES OF HIF-1 OVER-ACTIVATION .... 48 Abstract .............................................................................................................................. 48 Introduction ....................................................................................................................... 48 Materials and Methods ..................................................................................................... 52 Strains ............................................................................................................................. 52 Gene expression microarray experiment and data analysis ............................................ 52 Heat map ......................................................................................................................... 53 Microarray datasets comparisons .................................................................................... 54 iii Hypoxia survival and development assay ....................................................................... 54 Results ................................................................................................................................ 54 Transcriptional phenotypes of vhl-1, rhy-1, egl-9, and swan-1; vhl-1 ............................ 54 Convergence of HIF pathway and pathogen immune response pathways ..................... 57 HIF-1 and DAF-16 function synergistically in hypoxia resistance ................................ 59 Discussion........................................................................................................................... 61 RHY-1, EGL-9 and SWAN-1 function in common pathway(s) to regulate HIF-1 ........ 62 The diverse cellular processes regulated by HIF-1 may account for its diversified functions .......................................................................................................................... 62 Insights to other SWAN-1 and EGL-9 functions ............................................................ 63 HIF-1 interacts with DAF-16 to regulate hypoxia survival ............................................ 63 References .......................................................................................................................... 64 Figure Legends .................................................................................................................. 69 CHAPTER 4. GENOME-WIDE PROFILING OF HYPOXIA-INDUCED GENES ..... 79 Abstract .............................................................................................................................. 79 Introduction ....................................................................................................................... 79 Materials and Methods ..................................................................................................... 81 Strains ............................................................................................................................. 81 Gene expression microarray experiment and data analysis ............................................ 81 Consensus motifs at the promoters of HIF-1 dependent genes under hypoxia............... 83 HIF-1 chromatin immunoprecipitation (ChIP) ............................................................... 83 ChIP-Seq library preparation and sequencing ................................................................ 86 ChIP-Seq data analysis ................................................................................................... 86 Heat map ......................................................................................................................... 87 Microarray datasets comparisons .................................................................................... 88 RNA interference ............................................................................................................ 88 Hypoxia survival and development assay ....................................................................... 88 Results ................................................................................................................................ 89 Identification of genes that respond to 2 hours hypoxia (0.5% oxygen) ........................ 89 HIF-1 regulates metabolism, stress response and other functions under hypoxia .......... 93 Effects of HIF-1-dependent hypoxia-responsive genes in hypoxia adaptation .............. 95 Genes regulated by HIF-1 overlap with genes regulated