Identification of the lipid droplet-associated protein adipophilin as a novel liver X receptor target gene Roger Mathisen Cand. Scient. thesis Department of Nutrition Faculty of Medicine, University of Oslo, 2004 2 3 Contents CONTENTS ..........................................................................................................................................3 ACKNOWLEDGMENTS....................................................................................................................6 ABBREVIATIONS...............................................................................................................................7 SUMMARY.........................................................................................................................................10 1. INTRODUCTION....................................................................................................................11 1.1 NUTRITION AND GENE REGULATION.......................................................................................11 1.2 THE BASAL TRANSCRIPTIONAL MACHINERY ...........................................................................13 1.3 NUCLEAR RECEPTORS ............................................................................................................15 1.4 PPARS AND LXRS.................................................................................................................18 1.5 LXRS IN CHOLESTEROL AND LIPID HOMEOSTASIS ..................................................................20 1.6 LIPID DROPLET-ASSOCIATED PROTEINS ..................................................................................25 2. OBJECTIVES...........................................................................................................................30 3. MATERIALS............................................................................................................................31 3.1 BACTERIA ..............................................................................................................................31 3.2 CELL LINES ............................................................................................................................31 3.3 CELL MEDIUM ........................................................................................................................31 3.4 CHEMICALS............................................................................................................................31 3.5 ENZYMES AND BUFFERS .........................................................................................................33 3.6 EQUIPMENT............................................................................................................................33 3.7 GROWTH MEDIA FOR BACTERIA .............................................................................................33 3.8 INSTRUMENTS ........................................................................................................................33 3.9 KITS .......................................................................................................................................34 4 3.10 PLASMIDS AND OLIGONUCLEOTIDES................................................................................. 34 3.11 SOFTWARE AND INTERNET RESOURCES ............................................................................ 35 4. METHODS............................................................................................................................... 36 4.1 ESSENTIAL DNA TECHNIQUES .............................................................................................. 36 4.1.1 Spectrophotometric quantitation of nucleic acid ....................................................... 36 4.1.2 Separation of DNA by agarose gel electrophoresis ................................................... 36 4.1.3 Preparation of cloning vectors................................................................................... 38 4.1.4 Agarose gel extraction ............................................................................................... 40 4.1.5 Ligation of DNA ......................................................................................................... 40 4.2 DNA CLONING IN PLASMID VECTORS .................................................................................... 42 4.2.1 Transformation of E. coli ........................................................................................... 42 4.2.2 Mini preparation of plasmid DNA ............................................................................. 44 4.2.3 Maxi preparation of plasmid DNA............................................................................. 45 4.3 GENERAL TECHNIQUES FOR CULTURING CELL LINES ............................................................. 48 4.3.1 Thawing of frozen cells .............................................................................................. 48 4.3.2 Cell counting .............................................................................................................. 48 4.3.3 Cell Cultures .............................................................................................................. 48 4.3.4 Differentiation of SGBS cells ..................................................................................... 50 4.4 PROMOTER ANALYSIS............................................................................................................ 51 4.4.1 Computer analysis...................................................................................................... 51 4.4.2 Cloning and mutagenesis of the adipophilin promoter.............................................. 52 4.4.3 Transient transfection assay ...................................................................................... 53 4.4.4 β-Galactosidase assay................................................................................................ 57 4.4.5 Green fluorescent protein transfection marker.......................................................... 58 5 4.5 DNA BINDING BY REGULATORY TRANSCRIPTION FACTORS....................................................59 4.5.1 Electrophoretic mobility shift assay (EMSA)..............................................................59 4.6 RNA ANALYSIS......................................................................................................................62 4.6.1 Isolation of total RNA from cell cultures with TRIZOL ..............................................62 4.6.2 Northern analyses .......................................................................................................63 4.7 STATISTICAL ANALYSIS..........................................................................................................67 5. RESULTS..................................................................................................................................68 5.1 CHARACTERIZATION OF THE HUMAN ADIPOPHILIN PROMOTER...............................................68 5.1.1 Identification of a putative LXRE................................................................................68 5.2 ELECTROPHORETIC MOBILITY SHIFT ASSAY ...........................................................................71 5.2.1 LXRα binds the human LXRE in the adipophilin promoter ........................................71 5.3 REGULATION OF THE ADIPOPHILIN PROMOTER IN COS-1 CELLS.............................................73 5.3.1 The adipophilin promoter is responsive to LXR activation ........................................73 5.3.2 Deletion construct shows elevated reporter activity...................................................75 5.3.3 Mutation of LXRE abolish induction by LXRα agonist...............................................77 5.4 REGULATION OF THE ADIPOPHILIN PROMOTER IN HEPG2 CELLS ............................................81 5.4.1 The adipophilin promoter is a target for regulation by LXR......................................81 6. DISCUSSION ...........................................................................................................................84 6.1 METHODOLOGY .....................................................................................................................84 6.2 GENERAL DISCUSSION............................................................................................................89 6.3 CONCLUSION..........................................................................................................................94 7. REFERENCES .........................................................................................................................95 APPENDIX........................................................................................................................................111 6 Acknowledgments This work was conducted at Department of Nutrition, University of Oslo from August 2002 to May 2004, in the laboratory of Associate Professor Hilde Irene Nebb. I would like to express my gratitude to supervisor Hilde Irene Nebb for including me in her group, for introducing me to the field of nutrigenomics, and for enthusiasm and support. I am also grateful to my co-supervisor Knut Tomas Dalen. Your detailed knowledge about molecular nutrition and enthusiastic interest in science has been a source for inspiration. I gladly express my gratitude for support, educational discussions and the challenges you have provided