Transcriptional Regulation of the Col1a2 Gene in Kidney

TRANSCRIPTIONAL REGULATION OF THE COL1A2 GENE IN KIDNEY FIBROBLASTS A thesis submitted to Imperial College London in candidature for the degree of Doctor of Philosophy by: MARIA FRAGIADAKI Imperial College London, Renal Section, Division of Medicine Commonwealth Building, Du Cane Road London, W12 0NN JULY 2009 1 ABSTRACT Renal tubulointerstitial fibrosis is a major predictor of progressive glomerular disease. It occurs as a result of persistent inflammation and is characterised by excessive deposition of extracellular matrix (ECM) proteins, including accumulation of type I collagen, the most abundant protein of the ECM. Type I collagen is encoded by two genes, COL1A1 and COL1A2, that are tightly regulated at a transcriptional level. A key aim of this study was to use the previously identified COL1A2 promoter and enhancer sequences in order to identify novel regulatory cis- acting elements and the relevant transcription factors that regulate collagen transcription in cells derived from healthy or diseased kidneys. Moreover, the effects of hypoxia and transforming growth factor beta (TGFβ), which are both pro- fibrotic stimuli, on collagen transcription were studied. TGFβ is known to activate CDP/cux transcription factors which can suppress gene activation; based on this finding the role of CDP/cux in COL1A2 transcriptional regulation was assessed. In conclusion, the work presented in this thesis provides an insight into the complex control mechanisms that regulate collagen transcription in both physiological and pathological conditions. 2 ACKNOWLEDGEMENTS I would like to thank my supervisor Dr George Bou-Gharios for his discussion, throughout my PhD. Moreover Professor Patrick Maxwell for the intellectual stimulation and guidance and Dr Paul C Evans for reviewing my thesis. Numerous members of the laboratory, past and present, that have become friends and provided help and assistance, especially Hiroyusi Nakamura, Tetsuro Ikeda and Jenny Smith. I would also like to thank Kidney Research UK for funding my study. Finally, I would like to dedicate this thesis to all my family and friends for their continued belief and unfailing confidence in my abilities. 3 CONTENTS ABSTRACT 2 ACKNOWLEDGEMENTS 3 CONTENTS 4 LIST OF FIGURES 8 ABBREVIATIONS 10 PUBLISHED AND PRESENTED WORK 14 CHAPTER 1- GENERAL INTRODUCTION 16 1.1 INTRODUCTION 16 1.2 TISSUE FIBROSIS: THE KIDNEY 20 The nephron 20 TGFβ and Epithelial- to mesenchymal transition (EMT) 26 Chronic hypoxia hypothesis 33 1.3 EXTRACELLULAR MATRIX AND THE COLLAGEN FAMILY OF PROTEINS 38 Collagen, ECM and fibrosis 38 Cells that synthesise collagen 44 The collagen family of proteins 52 Type I Collagen 55 Genetic disease and type I Collagen 57 1.3 EUKARYOTIC TRANSCRIPTIONAL CONTROL 59 The pre-initiation complex 61 Enhancers and co-ordinately expressed genes 64 The collagen genes 66 Transcriptional control of type I collagen 71 1.5 PROJECT AIMS 79 CHAPTER 2- METHODS AND MATERIALS 81 2.1 MAMMALIAN CELL CULTURE 83 Cell lines 83 Maintenance of cells 83 Cryopreservation 84 2.2 TRANSIENT DNA TRANSFECTIONS 84 Seeding of the cells 84 Transfection protocol 85 Reporter gene assay 86 4 2.3 PREPARATION OF NUCLEIC ACIDS 87 Small-scale DNA extraction 87 Large-scale DNA extraction 89 Total RNA extraction and quantification 90 Oligonucleotides 91 2.4 STANDARD MANIPULATIONS OF DNA 92 Agarose gel electrophoresis 92 DNA isolation from agarose gel 93 Restriction enzyme digestion of plasmid DNA 93 2.5 RECOMBINANT DNA PROCEDURES 94 Blunt-ended DNA ligation 94 Competent DH5α cell preparation 95 Transformation of DH5α 96 Screening for recombinant plasmid 96 2.6 POLYMERASE CHAIN REACTION 97 Standard PCR 97 Colony PCR 99 cDNA synthesis 100 Real-time PCR 100 2.7 CLONING VECTORS 101 pBluescript 101 pβgal-Basic 101 2.8 GENERATION OF TRANSGENIC ANIMALS 104 Mice and microinjection 104 Preparation of embryos and detection of the transgene 106 2.9 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) 107 Nuclear extract preparation 107 Quantification of nuclear protein 107 Radio-labelling of the DNA 108 Binding reaction and SDS-PAGE 108 Autoradiography 109 2.10 WESTERN BLOTTING 110 Preparation of lysates 110 Electrophoresis, transfer and developing 110 2.10 STATISTICAL ANALYSIS 112 CHAPTER 3- INVESTIGATION OF THE HUMAN COL1A2 FAR- UPSTREAM ENHANCER 113 3.1 INTRODUCTION 114 3.2 IDENTIFICATION OF THE MINIMAL DNA SEQUENCE REQUIRED TO DRIVE KIDNEY-SPECIFIC EXPRESSION OF COL1A2 IN VITRO 118 Aims & and experimental design 118 Results 119 3.3 THE ROLE OF AP1 AND TGFβ IN COL1A2 ENHANCER ACTIVATION 130 Aims & experimental design 130 Results 131 5 3.4 GENERATION OF MOUSE TRANSGENIC EMBRYOS USING THE COL1A2 ENHANCER REGION 136 Aims & experimental design 136 Results 137 3.5 DISCUSSION 140 CHAPTER 4- REGULATION OF COL1A2 BY CDP/CUX: A ROLE FOR TGFΒ 148 4.1 INTRODUCTION 149 4.2 SUPPRESSION OF COL1A2 mRNA BY CDP/CUX 150 Aim and Experimental design 150 Results 151 4.3 ANTAGONISTIC RELATIONSHIP BETWEEN CDP/CUX AND TGFβ ON COL1A2 mRNA PRODUCTION 159 Aim and Experimental design 159 Results 160 4.4 IN SILICO ANALYSIS OF COL1A2 PUTATIVE CDP/CUX CIS-ACTING ELEMENTS AND BINDING ASSAYS 165 Aim and Experimental design 165 Results 167 CHAPTER 5: HYPOXIC REGULATION OF COLLAGEN 182 5.1 INTRODUCTION 183 5.2 HYPOXIA INDUCES INCREASED mRNA EXPRESSION OF THE COL1A2 GENE 186 Aim and Experimental design 186 Results 187 5.3 BIOINFORMATICS ANALYSIS OF COL1A2 5’ UTR FOR PUTATIVE HYPOXIA RESPONSE ELEMENTS AND SP1 191 Aim and Experimental design 191 Results 192 5.4 MINIMAL DNA REGION REQUIRED FOR THE HYPOXIA-INDUCED COL1A2 INCREASED EXPRESSION 195 Aim and Experimental design 195 Results 195 5.5 DISCUSSION 199 CHAPTER 6- GENERAL DISCUSSION 207 6.1 DISCUSSION 207 6.2 FUTURE DIRECTIONS 221 6.3 LIMITATIONS OF THE STUDY 224 REFERENCE LIST 227 6 APPENDIX A- SUPPLIERS 261 APPENDIX B- BUFFERS AND SOLUTIONS 263 APPENDIX C- TF EXTENDED LIST 266 APPENDIX D- CONSERVATION OF THE HUMAN COL1A2 5’ UTR 289 7 LIST OF FIGURES Figure 1.1: A simplified schematic illustration of a kidney nephron. 22 Figure 1.2: Normal versus fibrotic kidney. 25 Figure 1.3: The canonical TGFβ signalling pathway. 29 Figure 1.4: Cellular modifications associated with EMT. 32 Figure 1.5: The chronic hypoxia hypothesis. 34 Figure 1.6: Representation of the constituents of ECM. 41 Figure 1.7: The roles of different components of the ECM. 43 Figure 1.8: Origins of collagen-producing cells. 50 Figure 1.9: Normal versus activated fibroblasts. 51 Figure 1.11: Cartoon emphasizing the importance of collagen. 53 Figure 1.12: The collagen triple helix. 53 Figure 1.13: The collagen structure. 56 Figure 1.14: Fundamental elements of eukaryotic transcriptional control. 63 Figure 1.15: Representation of the trans-acting factors and cis-acting regulatory elements of COL1A2. 79 Figure 2.1: Schematic representation of the cloning strategy. 103 Figure 2.2: Generation of transgenics. 105 Figure 3.1: Transgenic animal analysis of human COL1A2 upstream DNA sequence. 116 Figure 3.2: Part of the human COL1A2 enhancer can drive-kidney specific expression in vivo. 117 Figure 3.4: Testing COL1A2 promoter and promoter with full length activity in mammalian collagen-producing cells. 124 Figure 3.5: Further characterisation of COL1A2 enhancer activity by testing sequential deletion constructs. 127 Figure 3.6: Identification of minimal DNA sequence required for promoter/enhancer activity. 128 Figure 3.7: TGFβ stimulation enhanced basal COL1A2 promoter/enhancer activity. 133 Figure 3.8: Site-directed mutagenesis revealed a cis-acting element important in regulation of basal COL1A2 expression. 135 Figure 3.9: Generation of COL1A2-reporter transgenics. 139 Figure 4.1: Validation, by qPCR, of CDP/cux overexpression in fibroblasts. 153 Figure 4.2: Overexpression of CDP/cux suppressed COL1A2 mRNA expression in fibroblasts. 156 Figure 4.4: Overexpression of CDP/cux suppresses COL1A2 promoter activity in renal fibroblasts, though the proximal promoter. 158 8 Figure 4.5: Stimulation of fibroblasts with TGFβ induced an increased production of CDP/cux which correlated with reduced expression of COL1A2. 163 Figure 4.6 CDP/cux overexpression suppresses COL1A2 transcriptional activity through the promoter and reverses TGFβ-induced COL1A2 activation. 164 Figure 4.7: In silico analysis revealed 5 putative CDP/cux binding sites 170 Figure 4.8: CBF binds to the COL1A2 proximal promoter at position -80bp relative to the transcriptional start site. 172 Figure 4.9: CDP binds to the COL1A2 proximal promoter through a cis-acting element located -200bp relative to the transcriptional start site. 173 Figure 4.10: Overexpression of CDP/cux with binding to the -80bp site. 174 Figure 5.1: HIF response in the presence and absence of oxygen. 185 Figure 5.2: COL1A2 expression in kidney and lung cells. 189 Figure 5.3: VEGF expression and HIF activity in human kidney fibroblasts in response to hypoxia. 190 Figure 5.4: Putative hypoxia response elements and SP1 motifs on COL1A2 regulatory DNA sequences. 194 Figure 5.5: The COL1A2 promoter/enhancer is activated in response to hypoxia. 197 Figure 6.1: TGFβ mediates COL1A2 transcription in a dose-dependent manner via induction of CDP/cux. 219 9 ABBREVIATIONS α-SMA alpha-smooth muscle actin AMP ampicillin AP1 activator protein-1 APS ammonium persulphate BMP-7 bone morphogenic protein 7/ osteogenic protein-1 bp base pair BSA bovine serum albumin CAT chloroamphenicol acetyl-transferase CBF CCAAT binding factor CDP/cux CCAAT displacement protein CME collagen modulating element COL1A1 / 2 collagen 1 alpha 1 / 2 COL4A3/4/5 collagen

Load more