In order to equally segregate their genetic material into daughter cells during mitosis, it is essential that chromosomes undergo major restructuring to facilitate compaction. However, the process of transforming diffuse, entangled interphase chromatin into discrete, highly organised chromosomal structures is extremely complex, and currently not completely understood. The complexes involved in chromatin compaction and sister chromatid decatenation in preparation for mitosis include condensins I and II. Mutations in condensin subunits have been identified in human tumours, reflecting the importance of accurate cell division in the prevention of aneuploidy and tumour formation. Most mutations described in TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic Mutations in Cancer) are missense, and therefore likely to only partially affect condensin function. Most functional genetic studies of condensin, however, have used loss of function systems, which typically cause severe chromosome segregation defects and cell death. Mice carrying global hypomorphic mutations within the kleisin subunit of the condensin II complex develop T cell lymphomas. The Caph2nes/nes mouse model is therefore a good system for understanding how condensin dysfunction can influence tumourigenesis. However, little is known about which cellular processes are affected in mutant cells before transformation. I therefore set out to use the Caph2nes/nes mouse model to study the consequences of the condensin II deficiency on cell cycle regulation in several different hematopoietic lineages. The Caph2nes/nes mice are viable and fertile, with no obvious abnormalities other than the thymus, which is drastically reduced in size. Previous studies reported greater than a hundred-fold reduction in the number of CD4+ CD8+ thymocytes. I set out to understand why the alteration of a ubiquitously expressed protein which functions in a fundamental cellular process would result in such a cell-type specific block in development. To achieve this, I investigated the possibility that condensin II is involved in interphase processes as well as in mitosis. In addition, I studied the aspects of T cell development that may make this lineage particularly vulnerable to condensin II deficiency. Finally, I carried out a preliminary investigation into the biochemical properties of the condensin complexes. During my PhD., I found strong evidence to suggest that the Caph2nes/nes T cell-specific phenotype arises due to abnormal cell division. However, I was unable to find any evidence to support the hypothesis that the phenotype is a consequence of abnormal interphase processes. Upon systematic analysis of several stages of hematopoietic differentiation, I found that at a specific stage of T cell development, the mutation results in an increased proportion of cells with abnormal ploidy, followed by a drastic reduction in cell numbers. Erythroid cells revealed a similar increase in the frequency of hyperdiploid cells, but no reduction in cell numbers. B cells and hematopoietic precursors did not reveal an increase in hyperdiploidy, or a reduction in cell numbers in wildtype relative to mutant. Subsequently, I found preliminary evidence to suggest that the T cell-specificity may be due to more rapid progression of CD4+ CD8+ T cells from S phase to M phase, relative to other hematopoietic stages. Finally, a preliminary investigation into the biochemical properties of the condensin complex revealed apparent imbalances in the expression of condensin subunits in T, B and erythroid cells. The sedimentation profile of CAP-H2 from whole-thymus extract did not exclude the possibility that condensin subunits might be forming heavier-weight complexes with non-SMC proteins. Further work must be carried out to determine whether this sedimentation pattern is unique to T cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:721199 |
| Date | January 2016 |
| Creators | Woodward, Jessica Christina |
| Contributors | Bickmore, Wendy ; Wood, Andrew |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/22921 |
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