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The Role of BRF1 and BRF2 in the Immune Response

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The Role of BRF1 and BRF2 in the Immune Response The Role of BRF1 and BRF2 in the Immune Response Rebecca Crawford A thesis submitted for the degree of Doctor of Philosophy to the Imperial College Faculty of Medicine Imperial College London September 2008 The Kennedy Institute for Rheumatology Division Faculty of Medicine Imperial College London Chapter 1 Introduction Declaration This thesis is the result of my own work. Some figures were contributed with the help of other members of the laboratory. These figures are labeled as such. The work was carried out at the Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, London. 2 Chapter 1 Introduction Acknowledgements I would like to thank a number of people for their help, advice and patience over the past four years. First, my supervisor Dr. Andy Clark, who has provided continual support and advice particularly whilst writing this thesis. Secondly I would like to acknowledge the members of the lab who have provided me with ideas and technical assistance throughout my PhD. Finally, I would like to thank Alison, Tom and Liaquat without whose support and encouragement I would have struggled to reach this stage. 3 Chapter 1 Introduction Abstract Tristetraprolin (TTP), butyrate response factor 1 (BRF1) and butyrate response factor 2 (BRF2) are members of a family of zinc finger containing ARE binding proteins known as the Zfp36 family. They all possess a conserved tandem zinc finger domain, which facilitates their binding to mRNAs that contain adenosine/uridine rich elements (ARE) in their 3’ untranslated region (3’UTR). Binding to the target mRNA results in its destabilisation. Several mRNAs containing an ARE in their 3’UTR are stabilised by the mitogen activated protein kinase (MAPK) p38 pathway. Both the expression and the mRNA destabilising function of TTP are controlled by the p38 MAPK pathway. Much less is known about BRF1 and BRF2 functions, and it is not clear whether their expression or function is regulated by the p38 MAPK pathway. So far no difference has been seen in the binding specificities of the three proteins in vitro, however the phenotypes of knockout mice suggest that they have distinct functions, and may have different mRNA targets in vivo. Western blotting and quantitative PCR (qPCR) have been used to investigate the expression of members of the Zfp36 family. RNA interference was used to knock down the expression of BRF1 and BRF2 in HeLa cells, and the effects on p38-regulated inflammatory mediator expression were examined. BRF1-/- mouse embryonic fibroblast (MEF) cell lines were used to investigate the function of this family member. No evidence that BRF1 or BRF2 contributes to the post-transcriptional regulation of inflammatory mediators by the p38 MAPK pathway in HeLa cells or MEFs was found. BRF1 null MEFs over-expressed IL-6, protein, IL-6 mRNA and Cox-2 mRNA but did not over-express KC protein. The hypothesis that BRF1 is regulating IL-6 and Cox-2 by controlling mRNA stability was disproved. As a result investigation of the transcriptional regulation of these genes was researched. Primary transcript qPCR showed that both IL-6 and Cox-2 under-go more rapid transcription in BRF1-/- MEFs. This suggests that IL-6 and Cox-2 are indirect targets of BRF1 and that their regulation is through a transcription factor which is itself a target of BRF1. 4 Chapter 1 Introduction Contents Title page………………………………………………………………………………….....1 Declaration…………………………………………………………………………………..2 Acknowledgements……………………………………………………………………….....3 Abstract…………………………………………………………………………………...…4 Table of contents…………………………………………………………………………….5 List of figures and tables…………………………………………………………………….9 1) INTRODUCTION .....................................................................22 1.1) The immune system.................................................................................................. 22 1.1.1) Introduction........................................................................................................ 22 1.1.2) Innate Immunity and the inflammatory response- the initial response to infection ....................................................................................................................... 22 1.1.3) Leukocytes......................................................................................................... 23 1.1.4) Fibroblasts.......................................................................................................... 24 1.1.5) Cytokines ........................................................................................................... 25 1.1.5.1) Interleukin-1 (IL-1)..................................................................................... 26 1.1.5.2) Interleukin -6 (IL-6) and its receptor .......................................................... 27 1.1.5.3) Cyclooxygenase-1/2 (Cox-1/2)....................................................................... 29 1.1.5.4) Chemokine (CXC motif) family: Interleukin-8 (IL-8), growth-regulated oncogene (GRO)α and keratinocyte chemokine (KC)................................................. 31 1.2) Inflammatory signalling pathways ........................................................................... 33 1.2.1) Introduction........................................................................................................ 33 1.2.2) Interleukin-1 receptor (IL-1R) and the toll-like receptor (TLR) superfamily ... 33 1.2.3) IL-1 Signalling................................................................................................... 34 1.2.4) TLR Signalling .................................................................................................. 34 1.2.5) The mitogen activated protein kinase (MAPK) pathway .................................. 40 1.2.5.1) Extra cellular regulated kinase (ERK) pathway ......................................... 42 1.2.5.2) MAPKp38 Pathway .................................................................................... 43 1.2.5.3) The c- jun amino terminal kinase (JNK) pathway...................................... 45 1.2.6) The Phosphoinositide 3-kinase (PI3K) Pathway ............................................... 46 1.3) Gene Expression ....................................................................................................... 47 1.3.1) Introduction........................................................................................................ 47 1.3.2) Transcription...................................................................................................... 48 1.3.2.1) Chromatin structure .................................................................................... 48 1.3.2.2) Chromatin modifications ............................................................................ 48 1.3.2.3) Transcription activation and repression...................................................... 49 1.3.2.4) Initiation of transcription ............................................................................ 50 1.3.2.5) Elongation................................................................................................... 51 1.3.3) mRNA processing and translation..................................................................... 53 1.3.3.1) Introduction................................................................................................. 53 5 Chapter 1 Introduction 1.3.3.2) mRNA regulation- an important mechanism in the control of gene expression................................................................................................................. 55 1.3.3.3) mRNA decay .............................................................................................. 56 1.3.3.4) Processing bodies (PBs) and stress granules (SGs).................................... 58 1.3.3.5) mRNA surveillance: Nonsensense-mediated decay (NMD) ...................... 60 1.3.3.6) RNA interference (RNAi) pathway ............................................................ 61 1.3.4) Adenosine/ uridine rich element (ARE) mediated decay (AMD) ..................... 62 1.3.4.1) Introduction................................................................................................. 62 1.3.4.2) Classification of AREs ............................................................................... 63 1.3.5) Signalling pathways and mRNA decay ............................................................. 64 1.3.5.1) The p38 pathway and mRNA stabilisation................................................. 65 1.3.5.2) Mechanism of p38 mRNA mediated stabilisation...................................... 66 1.4) Adenosine/uridine rich element binding proteins (AUBPs)..................................... 68 1.4.1) Introduction........................................................................................................ 68 1.4.1.1) Human antigen R (HuR)............................................................................. 68 1.4.1.2) Heterogeneous ribonucleoprotein (hnRNP)................................................ 71 1.4.1.3) T cell –restricted antigen (TIA)-1 and TIA related protein (TIAR) ........... 74 1.4.1.4) The far upstream sequence element binding protein (FBP) family............ 76 1.4.1.5) The zinc finger 36 (Zfp36) family .............................................................. 79 1.4.1.5.1) Introduction.......................................................................................... 79 1.4.1.5.2) The tandem zinc finger (TZF) domain ...............................................
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