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Chapter 1: Introduction The role of lamin A and emerin in mediating genome organisation A thesis for the degree of Doctor of Philosophy by Lauren Sarah Godwin School of Health Sciences and Social Care Brunel University July 2010 Abstract The nuclear matrix (NM) is proposed to be a permanent network of core filaments underlying thicker fibres, present regardless of transcriptional activity. It is found to be both RNA and protein rich; indeed, numerous important nuclear proteins are components of the structure. In addition to mediating the organisation of entire chromosomes, the NM has also been demonstrated to tether telomeres via their TTAGGG repeats. In order to examine telomeric interactions with the NM, a technique known as the DNA halo preparation has been employed. Regions of DNA that are tightly attached to the structure are found within a so-called residual nucleus, while those sequences forming lesser associations produce a halo of DNA. Coupled with various FISH methodologies, this technique allowed the anchorage of genomic regions by the NM, to be analysed. In normal fibroblasts, the majority of chromosomes and telomeres were extensively anchored to the NM. Such interactions did not vary significantly in proliferating and senescent nuclei. However, a decrease in NM-associated telomeres was detected in quiescence. Since lamin A is an integral component of the NM, it seemed pertinent to examine chromosome and telomere NM-anchorage in Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts, which contain mutant forms of lamin A. Indeed, genome tethering by the NM was perturbed in HGPS. In immortalised HGPS fibroblasts, this disrupted anchorage appeared to be rescued; the implications of this finding will be discussed. This study also suggested that telomere-NM interactions are aberrant in X-linked Emery-Dreifuss Muscular Dystrophy (X-EDMD), which is caused by mutant forms of emerin, another NM-associated protein. The positioning of selected genes in control and X-EDMD cell lines was examined in un- extracted nuclei using 2D and 3D FISH. Subtle shifts in the organisation of these genes were detected in diseased cells; however, their expression levels remained unaltered. Furthermore, in order to examine the architectural integrity of the nuclear lamina in lamin A and emerin mutant cell lines, scanning electron microscopy (SEM) was i employed. This work revealed that such structures were indeed compromised in disease. The findings presented in this thesis highlight the importance of lamin A and emerin in mediating the organisation of the genome and taken together, promote the hypothesis that dysfunctional NM dynamics may well contribute to disease pathology. ii Declaration I declare that all the work presented within this thesis was produced by me except from where stated. Lauren Godwin iii Acknowledgements First and foremost, I am extremely grateful for the encouragement, direction and support of my PhD supervisor, Dr. Joanna Bridger. Throughout my studies, she has allowed me to fulfil my potential as a PhD scientist. I would also like to thank Dr. Ian Kill, my second supervisor, for his help, training and guidance. I am highly thankful to Dr. Joanna Bridger for arranging the financial support which funded my PhD research. In terms of funding, I would like acknowledge and thank the EU FP6 Programme (Eurolaminopathies), Brunel University and the Progeria Research Fund. I would like to thank Professor Christopher Hutchison and Dr. Martin Goldberg for welcoming me into their laboratories as a visiting scientist. Dr. Martin Goldberg’s electron microscopy training was invaluable; without which, I could not have produced the data presented in chapter 6 of this thesis. I am grateful to numerous scientists for providing a number of cell lines which I have used during my PhD: Professor CJ Hutchison (AP, KK, TR, 99P0598, 99P0599, Y259X), Professor Manfred Wehnert (G12660), Professor Glenn Morris (ED5364), Professor Richard Faragher (TAG06297) and Dr. Christopher Parris (UACC 903). I would like to acknowledge the laboratory support I received during my PhD studies. Firstly to Dr. Ishita Mehta, who assisted in the maintenance of various HGPS cultures. I would also like to thank Miss Elisa Garimberti for showing me how to generate single- gene probes. For general laboratory support during my studies, I am grateful to Dr. Helen Foster, Ms. Denise May and Ms. Helen Cox. I could not have survived my PhD without the support of my friends at Brunel, especially Dr. Helen Foster, Miss Elisa Garimberti and Mr Andrew McVean. Last, but by no means least, I would like to thank my family for providing infinite love and support throughout my PhD studies. In particular, I will always be endebted to my husband, David, for putting up with my erratic working hours and moods! Thank you. iv Abbreviations 2D = 2 dimensional 3D = 3 dimensional 4C = Chromosome conformation capture-on-chip 53BP1 = p53 binding protein 1 A = Adenine AD = Autosomal dominant APC = Adenomatous polyposis coli APD = Accumulated population doublings APL = Acquired Partial Lipodystrophy BAC = Bacterial Artificial Chromosome BAF = Barrier to autointegration BCIP = 5-bromo-4-chloro-3-indolyl-phosphate Biotin-16-dUTP = Biotin-16-2'-deoxy-uridine-5'-triphosphate bp = Base pair BrdU = Bromodeoxyuridine BSA = Bovine serum albumin Btf = Bcl-2-associated transcription factor C = Cytosine CCD = Charge-coupled device cDNA = Complementary DNA ChiP = Chromatin immunoprecipitation CMT = Charcot-Marie Tooth CT = Chromosome territory CTE = Chromosome territory edge Cy3 = Indocarbocyanine DAPI = 4', 6-diamidino-2-phenylindole DEPC = Diethyl pyrocarbonate DHB = DNA halo buffer DMEM = Dulbecco’s Modified Eagle Medium DNA = Deoxyribonucleic acid DNase = Deoxyribonuclease v dNTP = Deoxynucleoside-triphosphate DOP-PCR = Degenerate oligonucleotide primed polymerase chain reaction DSB = Double strand breaks E = Exon EDMD = Emery-Dreifuss Muscular Dystrophy EDTA = Ethylenediaminetetra-acetic acid EM = Electron microscopy ER = Endoplasmic reticulum FACE = Farnesylated protein-converting enzyme 2 FBS = Fetal bovine serum feSEM = Field emission scanning electron microscopy FISH = Fluorecence in situ hybridisation FITC = Fluorescein isothiocyanate FLPD = Dunnigan-Type Familial Partial Lipodystrophy FrdU = Fluorodeoxyuridine FT/FTase = Farnesyltransferase FTI = Farnesyltransferase inhibitor HGPS = Hutchinson-Gilford progeria syndrome g = g-force G = Guanine G0 = Quiescence GCL = Germ cell-less GFP = Green fluorescent protein GGT = Geranylgeranyltransferase GGTI = Geranylgeranyltransferase inhibitor HDF = Human dermal fibroblast HIV = Human immunodeficiency virus HP1 = Heterochromatin protein 1 HSC = Haematopoetic stem cell ICD = Interchromatin domain INM = Inner nuclear membrane kb = Kilobase LAD = Lamina-associated domain vi LAP = Lamina-associated polypeptide LBR = Lamin B Receptor LEM = LAP2-emerin-MAN1 LINC = linker of nucleoskeleton and cytoskeleton LIS = Lithium di-iodosalicylic acid MAD = Mandibuloacral Dysplasia MAPK = Mitogen-activated protein kinase MAR = Matrix attachment region MEF = Mouse embryonic fibroblast MMCT = Microcell-mediated chromosome transfer mRNA = Messenger ribonucleic acid MSC = Mesenchymal stem cells n = Number NBP = Aminobisphosphonate NCS = Newborn calf serum NCT = Nitroblue tetrazolium NE = Nuclear envelope/ edge NHEJ = Non-homologous end joining NL = Nuclear lamina nm = Nanometer NM = Nuclear matrix NMP = Nuclear matrix protein NPC = Nuclear pore complex NS = Nucleoskeleton OsO4 = Osmium tetroxide ONM = Outer nuclear membrane PBS = Phosphate buffered saline PCR = Polymerase chain reaction PFA = Paraformalydehyde PHA = Pelger-Huet Anomaly PNA = Peptide nucleic acid pRb = Retinoblastoma protein RD = Restrictive Dermopathy vii RIDGE = Regions of increased gene expression RNA = Ribonucleic acid RNase = Ribonuclease rRNA = Ribosomal RNA RT= Revese transcription SAF-A = Scaffold attachment factor-A SAF-B = Scaffold attachment factor-B SAR = Scaffold attachment region SDS = Sodium dodecyl sulphate SEM = Scanning electron microscopy/ Standard error mean snRNA = Small nuclear ribonucleic acid SREBP1 = Sterol response element binding protein 1 SSC = Saline sodium citrate TBE = Tris-borate EDTA TBS = Tris-buffered saline T = Thymine TIFF = Tagged Image File Format TRITC = Tetramethylrhodamine isothiocyanate TRF1 = Telomere repeat binding factor 1 tRNA = Transfer RNA UV = Ultraviolet VSMC = Vascular smooth muscle cells v/v = Volume/ volume WS = Werner’s Syndrome w/v = Weight/ volume X-EDMD = X-linked Emery-Dreifuss Muscular Dystrophy viii Table of Contents Abstract .............................................................................................................................. i Declaration ....................................................................................................................... iii Acknowledgements .......................................................................................................... iv Abbreviations .................................................................................................................... v Table of Contents ............................................................................................................. ix Table of Figures ............................................................................................................. xviii Table of Tables .............................................................................................................. xxiii 1. Introduction ..............................................................
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