This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Functional characterization of Rasd1 : a novel interaction between Rasd1 and Ear2 (NR2F6) is involved in the regulation of renin transcription Tan, Jen Jen 2012 Tan, J. J. (2012). Functional characterization of Rasd1 : a novel interaction between Rasd1 AND Ear2 (NR2F6) is involved in the regulation of renin transcription. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/48679 https://doi.org/10.32657/10356/48679 Downloaded on 05 Oct 2021 00:01:35 SGT FUNCTIONAL CHARACTERIZATION OF RASD1: A NOVEL INTERACTION BETWEEN RASD1 AND EAR2 (NR2F6) IS INVOLVED IN THE REGULATION OF RENIN TRANSCRIPTION TAN JEN JEN School of Biological Sciences, Nanyang Technological University A thesis submitted to the Nanyang Technological University in partial fulfilment of the requirement for the degree of Doctor of Philosophy 2012 ACKNOWLEDGEMENTS There are several important people I have to thank, without whom, the completion of my project and thesis would not have been possible. I express my heartfelt gratitude to my supervisor, Prof Chen Ken-Shiung. His advice, generosity, understanding, encouragement and support have been extremely important in keeping me focused and motivated. His expertise, patience and willingness to teach are very inspiring. I thank him for his invaluable insight and guidance, especially during the drafting of my manuscript and during thesis writing. Working in his lab has proved to be an immensely enjoyable and intellectually satisfying learning experience. To all professors who have provided me with constructive advice and assistance throughout - especially Dr Koh Cheng Gee, Dr Li Hoi Yeung, Dr Feng Zhiwei, A/P Liu Ding Xiang and Prof Peter Droge. Thank you. I thank my lab mates and colleagues, past and present- Angeline, Hai Loon, Wai Loon, Daren, Lixia, Aihua, Shiyun and Lifang for their assistance in every way, from sharing of buffers to the numerous discussions on troubleshooting of failed experiments. They are the people who made coming to work fun and who made my entire journey less bumpy. The Agency of Science and Technology and Nanyang Technological University, School of Biological Sciences, to whom I am indebted to for providing me with the opportunity and funds to pursue my research, and to attend several conferences all over the world. I am eternally grateful to my parents and brother for their unconditional love. They have been a constant source of strength. And to my fiancé, Kenneth. He has been extremely supportive and understanding, the best anyone can ever ask for. He is far more important to me than he ever realizes. These are the people who never let me forget what the most important things in life are. I dedicate this thesis to them. 1 | P a g e TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................... 1 TABLE OF CONTENTS ........................................................................... 2 LIST OF FIGURES AND TABLES ....................................................... 9 ABBREVIATIONS ..................................................................................... 11 SUMMARY ................................................................................................... 15 Introduction ........................................................................................................ 15 Aims ................................................................................................................... 15 Results ................................................................................................................ 16 Conclusions ........................................................................................................ 17 CHAPTER 1: INTRODUCTION ......................................................... 18 1.1. Ras superfamily of proteins ..................................................................... 18 1.1.1. Biochemical activities of Ras proteins ......................................... 18 1.1.2. Structural characteristics of Ras proteins ................................... 20 1.1.3. Biological roles of Ras proteins ................................................... 22 1.2. Rasd1- a member of the Ras superfamily of GTPases ............................ 22 1.2.1. Rasd1 and its involvement in signal transduction ....................... 23 1.2.2. Rasd1 and the circadian clock ..................................................... 25 1.3. Renin-angiotensin system ........................................................................ 26 1.3.1. Renin-angiotensin system: The main players ............................... 27 1.4. Local Renin-angiotensin systems ............................................................ 29 2 | P a g e 1.4.1. Functions of the local RASs ......................................................... 30 1.4.2. RAS in the central nervous system ............................................... 31 1.5. Renin ....................................................................................................... 32 1.6. Transcriptional regulation of renin .......................................................... 34 1.6.1. Renin promoter and its regulatory elements ................................ 34 1.6.2. Renin promoter ............................................................................ 34 1.6.3. Renin enhancer............................................................................. 35 1.6.4. Regulation of renin transcription by Ear2 ................................... 37 1.7. Orphan nuclear receptors ......................................................................... 38 1.7.1. What are orphan nuclear receptors? ........................................... 38 1.7.2. Biological functions of orphan nuclear receptors ....................... 39 1.7.3. COUP-TFs and Ear2 ................................................................... 40 1.8. Ear2 ......................................................................................................... 42 CHAPTER 2: AIMS AND SIGNIFICANCE .................................... 45 CHAPTER 3: MATERIALS AND METHODS .............................. 47 3.1. Yeast two-hybrid assay............................................................................ 47 3.1.1. Yeast strains and vectors used in yeast two-hybrid assay............ 47 3.1.2. Yeast two-hybrid screening .......................................................... 48 3.1.3. Confirming the specificity of interaction ..................................... 49 3.1.4. Yeast transformation using LiAc/PEG method ............................ 50 3.1.5. Plasmid isolation from yeast ........................................................ 51 3.1.6. Transforming E.coli with yeast plasmids ..................................... 51 3.2. Making of electrocompetent E.coli cells ................................................. 52 3 | P a g e 3.3. Plasmids construction .............................................................................. 53 3.3.1. Plasmids expressing Rasd1 and Rasd1 mutants .......................... 53 3.3.2. Plasmids expressing Ear2 and Ear2 truncated constructs .......... 54 3.3.3. Reporter plasmids used in luciferase assays ................................ 54 3.3.4. General cloning procedures ......................................................... 55 3.4. DNA extraction from bacterial artificial chromosome (BAC) clone ...... 56 3.5. Mammalian cell cultures ......................................................................... 56 3.6. Mammalian cell transfection ................................................................... 57 3.7. Protein extraction from mouse brain ....................................................... 58 3.8. Protein binding assays and Western blotting .......................................... 59 3.8.1. GST pull-down assay.................................................................... 59 3.8.2. Co-precipitation assay ................................................................. 60 3.8.3. Co-immunoprecipitation (coIP) ................................................... 60 3.8.4. Ni-NTA pull-down ........................................................................ 61 3.8.5. Western blotting ........................................................................... 61 3.9. Indirect immunofluorescence staining .................................................... 62 3.10. RNA interference experiments .............................................................. 63 3.10.1. Design of shRNA expressing plasmids ....................................... 63 3.10.2. shRNA Knockdown ..................................................................... 64 3.11. Luciferase reporter assay ....................................................................... 64 3.12. Real time RT-PCR ................................................................................. 65 3.13. Semi-quantitative RT-PCR .................................................................... 66 3.14. Chromatin immunoprecipitation (ChIP) ............................................... 66 3.15. Electromobility Shift Assay (EMSA) .................................................... 68 4 | P a g e CHAPTER 4: RESULTS ......................................................................... 70 4.1. Yeast two-hybrid ..................................................................................... 70 4.2.