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Cellular Minigene Models for the Study of Allelic Expression of the Tau Gene and Its Role in Progressive Supranuclear Palsy

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Cellular Minigene Models for the Study of Allelic Expression of the Tau Gene and Its Role in Progressive Supranuclear Palsy CELLULAR MINIGENE MODELS FOR THE STUDY OF ALLELIC EXPRESSION OF THE TAU GENE AND ITS ROLE IN PROGRESSIVE SUPRANUCLEAR PALSY Victoria Anne Kay Thesis submitted in the fulfilment of the degree of Doctor of Philosophy Reta Lila Weston Institute of Neurological Studies Institute of Neurology University College London March 2013 Declaration I, Victoria Kay, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Progressive supranuclear palsy (PSP) belongs to a group of neurodegenerative disorders that are characterised by hallmark pathology consisting of intra-neuronal aggregates of the microtubule-associated protein, tau. In PSP, these aggregates are almost exclusively composed of one of the two major tau protein isoform groups normally expressed at similar levels in the healthy brain, indicating a role for altered isoform regulation in PSP aetiology. Although no causal mutations have been identified, common variation within the gene encoding tau, MAPT, has been highly associated with PSP risk. The A-allele of the rs242557 single nucleotide polymorphism has been repeatedly shown to significantly increase the risk of developing PSP. Its location within a distal region of the MAPT promoter region is significant, with independent studies – including this one – demonstrating that the rs242557-A allele alters the function of a transcription regulatory domain. As transcription and alternative splicing processes have been shown to be co-regulated in some genes, it was hypothesised that the rs242557-A allele could directly affect MAPT alternative splicing through its differential effect on transcription. This project describes an investigation into the molecular mechanism linking the MAPT association with the tau isoform dysregulation characteristic of PSP. The design, construction and in vitro investigation of minigenes representing common MAPT variants will be presented in detail and will demonstrate that promoter identity plays an important role in regulating the alternative splicing of MAPT transcripts. The specific role of the rs242557 polymorphism in MAPT transcription and splicing are investigated and the two alleles of the polymorphism are shown to differentially influence these two molecular processes, providing a plausible mechanism linking the two phenomena known to be associated with PSP – a common genetic variant within the MAPT promoter region and detrimental changes to tau isoform production. 3 Acknowledgements There are many people who – knowingly or otherwise – made an important contribution to the completion of this thesis. I’d like to thank everyone at the Reta Lila Weston Institute and Queen’s Square Brain Bank for their support, advice and company over the past three and a half years. In particular, I am hugely indebted to Rohan de Silva and Roberto Simone, who taught me everything I know and were a valuable source of knowledge, experience and encouragement. Special mentions should also be given to Geshanti Hondhamuni, Adam Mamais, Rina Bandopadhyay and Badma Segarane, for making my PhD experience a very enjoyable one. I’d like to thank my wonderful family for their constant love and support, especially my two nephews, William and Jacob, for always making me smile. I would also like to thank Tessa Radwan, Lucy Dalton-Griffin and Sharon McLean, for their unflinching support and encouragement. The writing of this thesis was fuelled by endless cups of tea, often made by my friend and flatmate, Jenny Berlin. 4 Table of Contents Table of Figures ................................................................................................... 12 List of Tables ....................................................................................................... 14 1 Introduction ..................................................................................................... 15 1.1 Overview ................................................................................................ 15 1.2 The regulation of mammalian gene expression ...................................... 16 1.2.1 Genetic elements in the regulation of expression ............................. 16 1.2.2 The machineries involved in gene expression .................................. 17 1.2.2.1 Overview .................................................................................... 17 1.2.2.2 Transcription .............................................................................. 18 1.2.2.3 Splicing ...................................................................................... 21 1.2.2.4 Alternative splicing .................................................................... 23 1.2.3 Co-regulation of transcription and alternative splicing .................... 23 1.2.3.1 Overview .................................................................................... 23 1.2.3.2 Physical coupling ....................................................................... 24 1.2.3.3 Kinetic coupling ......................................................................... 25 1.2.3.4 Local regulation of co-transcriptional splicing by chromatin .... 27 1.2.3.5 The role of the promoter in alternative splicing regulation ....... 30 1.3 The tauopathies ....................................................................................... 32 1.3.1 Overview .......................................................................................... 32 1.3.2 Progressive supranuclear palsy (PSP) .............................................. 33 1.4 Microtubule-associated protein, tau ....................................................... 35 1.4.1 Function ............................................................................................ 35 1.4.2 Tau isoform expression .................................................................... 35 1.4.3 Tau phosphorylation ......................................................................... 38 1.4.4 Tau aggregation ................................................................................ 39 1.5 The MAPT gene ...................................................................................... 39 1.5.1 Structure ........................................................................................... 39 1.5.2 Exons 2 and 3 ................................................................................... 40 1.5.3 Exon 10 ............................................................................................ 41 1.5.4 Tau pathology ................................................................................... 42 1.6 The genetics of MAPT ............................................................................ 44 1.6.1 Genomic architecture ....................................................................... 44 1.6.2 Common MAPT variation and PSP .................................................. 46 1.6.2.1 Early association studies ............................................................ 46 1.6.2.2 The H1C haplotype and rs242557 ............................................. 46 1.6.2.3 The PSP genome-wide association study .................................. 47 1.6.2.4 MAPT haplotypes in other neurodegenerative disorders ........... 48 1.6.2.5 The effect of rs242557 on CSF tau levels ................................. 49 1.7 MAPT gene expression ........................................................................... 51 1.7.1 In vivo allele-specific expression studies ......................................... 51 1.7.2 In vitro luciferase reporter gene studies ........................................... 52 5 1.7.3 N-terminal exons 2 and 3 ................................................................. 53 1.8 Minigene studies of MAPT alternative splicing ..................................... 54 1.9 Project aims ............................................................................................ 57 2 Methods and Materials ................................................................................... 59 2.1 Methods .................................................................................................. 59 2.1.1 DNA/RNA sample extraction from tissue ....................................... 59 2.1.2 DNA/RNA quantification ................................................................ 59 2.1.3 Polymerase chain reaction ................................................................ 59 2.1.3.1 Standard PCR............................................................................. 59 2.1.3.2 Reverse transcription PCR ......................................................... 61 2.1.3.3 Agarose gel electrophoresis ....................................................... 62 2.1.3.4 Polyacrylamide gel electrophoresis ........................................... 63 2.1.4 Molecular biology: cloning .............................................................. 64 2.1.4.1 Purification of PCR products for use in cloning ........................ 64 2.1.4.2 Purification of DNA products by agarose gel electrophoresis .. 64 2.1.4.3 DNA ligation into plasmid vectors: pGEM-T Easy vector ....... 64 2.1.4.4 DNA ligation into plasmid vectors: Expression vectors ............ 65 2.1.4.5 Propagation of plasmid constructs in E.coli .............................. 65 2.1.4.6 Blue-white screening of pGEM-T Easy clones ......................... 66 2.1.4.7 Digestion by restriction enzyme ................................................ 67 2.1.4.8 DNA
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