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Chromosome Territory Position and Active Relocation in Normal and Hutchinson-Gilford Progeria Fibroblasts

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Chromosome Territory Position and Active Relocation in Normal and Hutchinson-Gilford Progeria Fibroblasts Chromosome Territory Position and Active Relocation in Normal and Hutchinson-Gilford Progeria Fibroblasts A thesis submitted for the degree of Doctor of Philosophy by Ishita Shailesh Mehta School of Health Sciences and Social Care Brunel University September 2009 Abstract: Radial chromosome positioning in interphase nuclei is non-random and can alter according to developmental, differentiation, proliferation or disease status. The aim of this thesis is to understand how chromosome re-positioning is elicited and to identify the nuclear structures that assist this re-localisation event. By positioning all human chromosomes in primary fibroblasts that have left the proliferative cell cycle, the study within this thesis has demonstrated that in cells made quiescent by reversible growth arrest, chromosome positioning is altered considerably. Upon removal of serum from the culture medium, chromosome re- positioning took less than 15 minutes, required energy and was inhibited by drugs affecting the polymerization of myosin and actin. The nuclear distribution of nuclear myosin 1β was dramatically different in quiescent cells as compared to proliferating cells. If the expression of nuclear myosin 1β was suppressed using interference RNA procedures the movement of chromosomes after 15 minutes in low serum was inhibited. When high serum was restored to the serum starved cultures chromosome repositioning was only evident after 24-36 hours that coincided with a return to a proliferating distribution of nuclear myosin 1β. Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature ageing syndrome due to a mutation in LMNA gene that codes for lamin A protein – a critical component of the nuclear lamina and the nuclear matrix. The mutation causes this protein to be accumulated in a farnesylated form (also known as progerin) within the nuclei of HGPS patient cells. Interestingly, the positions of chromosome 10, 13 and 18 territories, in proliferating HGPS fibroblasts, are different from proliferating control cells and in fact resemble that of normal quiescent fibroblasts. Unlike control ii fibroblasts, the repositioning of chromosome territories in response to serum withdrawal is dysfunctional in HGPS patient cells and interestingly this defect coincides with atypical distribution of nuclear myosin 1β. Moreover, the effect of accumulation of progerin is not only restricted to the nuclear lamina, but has more widespread effects with the presence and distribution of some nucleolar proteins also adversely affected in progeria cells. Treatment of HGPS patient cells with a drug FTI-277 that inhibits farnesylation of the lamin A protein, also restores the chromosome territory dynamics and the nucleolar antigen localisation within the interphase nuclei, thus reaffirming that functional lamin A protein is indeed vital for maintenance of interphase chromosome territory organisation, chromosome behaviour and regulation of nucleolar proteins. In summary, the studies within this thesis have demonstrated that chromosome repositioning within interphase nuclei is rapid upon withdrawal of serum, but repositioning after readdition of serum requires a longer time and passage of cells through mitosis upon re-stimulation of serum starved cells. Importantly, the nuclear lamina and the nuclear motors comprising nuclear myosin 1β appear to be vital for this global reorganisation of interphase chromosome territories. iii Table of Contents: Abstract: ................................................................................................................ ii Table of Contents: ..................................................................................................iv Declaration: ........................................................................................................... x Acknowledgements: ...............................................................................................xi Abbreviations: .................................................................................................... xiii List of Figures: .................................................................................................... xxi Chapter 1: Introduction ...................................................................................... 1 1.1. The eukaryotic cell cycle: ................................................................................ 2 1.1.1. Proliferative cell division cycle ............................................................ 2 1.1.2. Non-proliferative stages in eukaryotic cells life-span: ........................... 3 1.2. Nuclear architecture: ...................................................................................... 9 1.2.1. Nuclear envelope, the lamina and lamin proteins: ................................... 11 1.2.2. The Nucleolus: ....................................................................................... 35 1.2.3. Nuclear motors: ...................................................................................... 40 1.3. Genome organisation: ................................................................................... 49 1.3.1. Features regulating positioning of genomic regions in interphase nuclei: 50 1.3.2. Models of chromosome organisation within interphase nuclei: ................ 55 1.3.3. Cell cycle and interphase chromosome positioning: ................................ 59 1.3.4. Interaction of the genome with other components of the nuclear architecture: ......................................................................................................................... 61 iv 1.3.5. Functional significance of genome organisation in interphase nuclei: ...... 64 1.3.6. Genome organisation and disease: .......................................................... 67 1.4.Overview: ...................................................................................................... 69 Chapter 2: Chromosome territory organisation in quiescent fibroblasts ........ 72 2.1. Introduction: ................................................................................................. 73 2.2. Methods and Materials: ................................................................................ 76 2.2.1. Cell Culture: ........................................................................................... 76 2.2.2. Two-dimensional in situ hybridisation (2D FISH): .................................. 78 2.2.3. Three dimensional fluorescence in situ hybridization (3D-FISH): ........... 83 2.2.4. Indirect immunofluorescence: ................................................................. 86 2.3. Results: ......................................................................................................... 87 2.3.1. Positions of chromosomes 10, 18 and X in normal proliferating human dermal fibroblasts: ............................................................................................ 87 2.3.2. Positions of all human chromosomes in normal quiescent human dermal fibroblasts: ....................................................................................................... 91 2.3.2. Chromosome 10 occupies differential locations in normal proliferating, quiescent and senescent human dermal fibroblasts: ........................................... 98 2.3.3. Positions of chromosomes 10 and X in normal human dermal fibroblasts made quiescent by serum starvation, topoinhibition and stress: ........................100 2.3.4. Positions of chromosomes 10 and X in serum-starved senescent cells resembles that of normal senescent cells: .........................................................103 2.4. Discussion: ..................................................................................................107 v Chapter 3: Repositioning of chromosome territories - a nuclear motor dependent process .............................................................................................111 3.1. Introduction: ................................................................................................112 3.2. Methods and Materials: ...............................................................................116 3.2.1. Cell culture: ...........................................................................................116 3.2.2. Treatment with inhibitors: ......................................................................116 3.2.3. siRNA transfection for MYO1C: ...........................................................119 3.2.4. 2D-FISH, 3D-FISH and indirect immunofluorescence: ..........................121 3.3. Results: ........................................................................................................122 3.3.1. Alteration in chromosome territory position – a rapid response to serum withdrawal: .....................................................................................................122 3.3.2. Restoration of proliferative chromosome position following re-stimulation of quiescent cells: ............................................................................................128 3.3.3. Rapid chromosome movement following serum starvation is an energy dependent process: ..........................................................................................132 3.3.4. Effect of microtubule polymerisation inhibition on movement of whole chromosome territories: ...................................................................................136 3.3.5. Rapid chromosome movement following serum starvation requires nuclear
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