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This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Analysis of partner proteins of MeCP2 and their relevance to Rett syndrome Robert Ekiert Thesis presented for the degree of Doctor of Philosophy The University of Edinburgh 2012 Declaration I declare that this thesis was composed by myself and the research presented is my own unless otherwise stated. This work has not been submitted for any other degree or personal qualification. Robert Ekiert October 2012 2 Table of contents Declaration........................................................................................................... 2 Table of contents.................................................................................................. 3 List of Figures.........................................................................................................7 List of Tables ..........................................................................................................9 Acknowledgements .............................................................................................10 Abstract...............................................................................................................12 Abbreviations ......................................................................................................13 Chapter 1: Introduction .......................................................................................15 1.1 Modifications of DNA.....................................................................................15 1.1.1 DNA methylation and hydroxymethylation in eukaryotes ........................15 1.1.2 Processing of DNA modifications .............................................................18 1.1.3 The roles of DNA methylation ..................................................................19 1.2 Methyl-CpG binding proteins .........................................................................21 1.2.1 MBD proteins ..........................................................................................22 1.2.2 Kaiso and other proteins binding to methylated DNA ..............................23 1.2.3 Mouse models depleted of methyl-CpG binding proteins .........................25 1.3 MeCP2 ...........................................................................................................26 1.3.1 Gene and protein.....................................................................................26 1.3.2 Post-translational modifications of MeCP2 ..............................................29 1.3.3 MeCP2 binding to methylated DNA .........................................................30 1.3.4 MeCP2 binding to unmethylated DNA......................................................32 1.3.5 MeCP2 as a transcriptional repressor ......................................................34 1.3.6 Genes regulated by MeCP2......................................................................36 1.3.7 MeCP2 as a transcriptional activator.......................................................38 1.3.8 Other functions of MeCP2........................................................................39 1.4 Rett syndrome ...............................................................................................42 1.4.1 Rett syndrome in humans ........................................................................42 1.4.2 Mouse models of Rett syndrome..............................................................44 3 Aims of the thesis ................................................................................................47 Chapter 2: Materials and methods.......................................................................48 2.1 Materials........................................................................................................48 2.1.1 Standard buffers......................................................................................48 2.1.2 Antibodies ...............................................................................................49 2.1.3 Mice ........................................................................................................50 2.1.4 Cell lines ..................................................................................................51 2.1.5 PCR primers.............................................................................................51 2.2 Methods.........................................................................................................51 2.2.1 Isolation of nuclei from mouse brains ......................................................51 2.2.2 Nuclear protein isolation .........................................................................52 2.2.3 Protein electrophoresis............................................................................52 2.2.4 Western blotting......................................................................................52 2.2.5 Silver staining ..........................................................................................54 2.2.6 GFP immunoprecipitation ........................................................................54 2.2.7 Mass spectrometry ..................................................................................54 2.2.8 Mammalian cell culture...........................................................................55 2.2.9 Production of MeCP2 truncation mutants................................................56 2.2.10 DNA ligation and plasmid amplification.................................................56 2.2.11 DNA sequencing ....................................................................................57 2.2.12 HeLa cell transfections for FLAG immunoprecipitation...........................57 2.2.13 FLAG immunoprecipitation ....................................................................57 2.2.14 Cloning of CamKIIα gene........................................................................58 2.2.15 Live cell imaging ....................................................................................58 2.2.16 Immunocytochemistry of MeCP2 expressed in NIH-3T3 cells..................59 2.2.17 Immunohistochemistry in mouse brain ..................................................59 2.2.18 Picture analysis......................................................................................60 2.2.19 Biochemical fractionation of cytoplasmic and nuclear proteins..............60 2.2.20 Reverse-transcription quantitative PCR..................................................60 2.2.21 GAL4 luciferase reporter gene assays.....................................................61 4 2.2.22 ES cell culture ........................................................................................62 2.2.23 Neuronal differentiation system ............................................................62 2.2.24 Transfection of ES cell-derived neurons..................................................62 2.2.25 Methylation of DNA...............................................................................63 2.2.26 Methylated reporter assay in neurons ...................................................63 Chapter 3: Identification of proteins binding to MeCP2 ........................................65 3.1 Introduction ...................................................................................................65 3.2 Identification of MeCP2-interacting proteins by mass spectrometry ..............68 3.3 Verification of mass spectrometry data by Western blotting..........................72 3.3.1 Validation of interactions between MeCP2 and importins........................72 3.3.2 Validation of interactions between MeCP2 and kinases ...........................74 3.3.3 Validation of other interactions of MeCP2 ...............................................75 3.4 Mapping the confirmed interactions on the MeCP2 sequence .......................75 3.4.1 Generation of MeCP2 truncation mutants ...............................................75 3.4.2 Mapping the interactions of MeCP2 with importins α and subunits of NCoR/SMRT co-repressor complex ...................................................................77 3.4.3 Mapping the interaction of MeCP2 with casein kinase 2α........................77 3.4.4 Mapping the interaction of MeCP2 with CaMKIIα....................................78 3.5 Discussion ......................................................................................................80 Chapter 4: Analysis of MeCP2-importin interactions.............................................87 4.1 Introduction ...................................................................................................87 4.2 Mapping the MeCP2-importins interactions...................................................88
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