Thesis A kinome-wide RNAi screen to identify genes controlling membrane lipid homeostasis in human cells GEHIN, Charlotte Abstract The control of lipid homeostasis is a fundamental process that allows cells to maintain the unique lipid composition of their membrane compartments and to deal with the energetic fluxes from metabolism. If most of enzymes involved in lipid metabolism are characterized, the question of the genetic control of lipid homeostasis is still outstanding. In order to find genes that control the homeostasis of membrane lipids, I combined a large-scale RNAi screen targeting the human knome with the techniques of targeted lipidomic analysis by mass spectrometry to monitor lipid changes in HeLa cells. Data analysis of the screen allowed the characterization of candidate genes involved in the control of membrane lipid homeostasis. In parallel, in the context of the NCCR Chemical Biology, I developed a robotically-assisted siRNA transfection assay and screened a library of chemicals potentially able to transfect siRNA in Human cells at least as efficiently than commercially available compounds. Reference GEHIN, Charlotte. A kinome-wide RNAi screen to identify genes controlling membrane lipid homeostasis in human cells. Thèse de doctorat : Univ. Genève, 2014, no. Sc. 4670 URN : urn:nbn:ch:unige-380353 DOI : 10.13097/archive-ouverte/unige:38035 Available at: http://archive-ouverte.unige.ch/unige:38035 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de chimie et biochimie Département de biochimie Professeur H. Riezman A Kinome-Wide RNAi Screen to Identify Genes Controlling Membrane Lipid Homeostasis in Human cells THÈSE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie par Charlotte GEHIN de Hennebont (France) Thèse No4670 GENÈVE Atelier Repromail 2014 Table of contents TABLE OF CONTENTS SUMMARIES .................................................................................................................................. 2 1. English summary of the thesis ................................................................................................. 2 2. Résumé de la thèse en français ................................................................................................ 4 INTRODUCTION ............................................................................................................................ 6 1. Biology of membrane lipids .................................................................................................... 6 1.1. Lipids are essential components of life .................................................................................. 6 1.2. Membrane lipid composition in mammalian cells ................................................................. 8 1.2.1. Glycerophospholipids (GPLs) ...................................................................................... 11 1.2.2. Sphingolipids (SLs) ...................................................................................................... 17 1.2.3. Sterols ........................................................................................................................... 20 1.3. Insights into the canvas of membrane lipid metabolism ...................................................... 22 2. Control of membrane lipid homeostasis ................................................................................ 26 2.1. Importance of membrane lipid homeostasis ........................................................................ 26 2.2. Overview of mechanisms that control membrane lipid homeostasis .................................. 28 2.2.1 Energy sensors and regulatory mechanisms ................................................................. 28 2.2.2 Control of lipid trafficking by the lipid-transfer proteins (LTPs) ................................ 33 2.2.3 Control of sphingolipid homeostasis: review .............................................................. 37 3. Aim of the studies .................................................................................................................. 38 MATERIAL AND METHODS ..................................................................................................... 39 1. Experimental procedures ....................................................................................................... 39 2. Statistical analysis of the Kinome-wide RNAi screen .......................................................... 42 RESULTS ....................................................................................................................................... 44 1. Investigating the genetic control of membrane lipid homeostasis with RNA interference ... 44 1.1. Choice of siRNA as a tool for genetic perturbation screen in human cells. ........................ 44 1.2. Mass spectrometry targeted-lipidomics to quantify membrane lipid changes. ................... 45 i Table of contents 1.3. A pilot siRNA screen on lipid-related proteins identifies gene-specific lipid changes ....... 46 1.3.1. Choice of target genes for the pilot screen ................................................................... 47 1.3.2. Efficacy of siRNA-induced gene knockdown .............................................................. 47 1.3.3. siRNA gene knockdown is sufficient to detect significant lipid changes .................... 50 1.3.1. Cell-population context and gene knockdown effect: example of CERT .................... 56 1.4. Lessonss from the pilot screen experiment. ......................................................................... 59 2. Kinome-wide RNAi based screen in HeLa MZ cells ............................................................ 62 2.1. Experimental conditions ...................................................................................................... 62 2.2. Membrane lipid composition of HeLa MZ .......................................................................... 62 2.3. Data processing .................................................................................................................... 72 2.4. Quality controls .................................................................................................................... 77 2.1.1 Effects of transfection reagent and non-targeting siRNA controls .............................. 79 2.1.2 PLK1 siRNA induces an apoptotic lipid profile .......................................................... 79 2.1.1 CERT siRNA induces SL, PI and sterol changes ......................................................... 80 2.5. Kinome-wide RNAi screen results ...................................................................................... 86 2.2.4 Observation of lipid behavior. ...................................................................................... 86 2.2.5 The puzzle of hit identification .................................................................................... 87 a) Hit identification using threshold determination .......................................................... 88 b) Principal Component analysis (PCA)........................................................................... 91 c) Genes with patterns of lipid changes: correlation- vs HIS-based selection ................. 97 2.2.6 Validation of candidate genes based on literature ...................................................... 109 2.2.7 Hypothesis-driven analysis of lipid profiles ............................................................... 112 2.2.8 Ongoing research ........................................................................................................ 115 3. Screening of Dynamic Amphiphiles as siRNA Delivery System in Mammalian Cells ..... 116 3.1. Summary of the research ................................................................................................... 116 3.2. Articles ............................................................................................................................... 117 DISCUSSION .............................................................................................................................. 118 1. Analysis of candidate genes from the siRNA kinome-wide screen .................................... 118 2. How to interpret observed modifications of lipid profiles? ................................................ 122 3. Lessons from the screening “experience” and perspectives ................................................ 126 ii Table of contents APPENDIX .................................................................................................................................. 127 1. siRNAs ................................................................................................................................ 127 2. Oligonucleotide sequences of primers ................................................................................ 127 3. Discarded conditions ........................................................................................................... 127 4. Multiple reaction monitoring assay ..................................................................................... 127 5. Data ..................................................................................................................................... 127 ABBREVIATIONS .....................................................................................................................