Paediatrics (RCH) - Theses
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ItemThe role of centromere defects in cancer formation and progression( 2016)The centromere plays a crucial role in genome inheritance — ensuring proper segregation of sister chromatids into each daughter cell. In cancer, especially in later stages of solid tumours, cells are often presented with extensive chromosomal abnormalities. However, the involvement of defective centromeres in the disease formation and progression has not been carefully studied. Hence, my PhD project aimed to investigate the role of defective centromeres in cancer progression using the NCI-60 panel of cancer cell lines. The spectrum of centromere-related abnormalities were first characterised with pan-centromeric FISH probes and then with the addition of CENP-A immunofluorescence. For HOP-92 and SN12C, cell lines showing high prevalence of functional dicentric chromosomes, expansion of single cell clones from the initial heterogeneous population was carried out to further study the involvement of dicentric chromosomes in cancer genomic instability. Neocentromere formation sites in cancer cell lines were investigated using cell lines with high prevalence of neocentric chromosomes. A neocentromere was found in T-47D which marked the first report of a neocentromere discovered in a long-established and widely used cell line, and also the first neocentromere to be reported in breast cancer. Separately, an antibody screen to identify antibodies recognising components of an active centromere in methanol-acetic acid stored cells was performed because thus far, antibodies that are widely used for functional centromere detection mainly worked on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. I found a commercially available rabbit monoclonal anti-CENP-C that worked on fixed samples and in addition, I combined three methods (FISH, immunofluorescence and mFISH) to obtain more information from the same metaphase spread. I then proceeded to test anti-CENP-C in my method, CENP-IF-cenFISH-mFISH, on dicentric- and neocentric-containing cancer cell lines and proposed other utilities for this method which I have developed.
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ItemHuman centromeric and neocentromeric chromatin( 2000-09)Human centromeres contain large arrays of α-satellite DNA that are thought to provide centromere function. These arrays show size and sequence variations. However, the lower limit of the sizes of these DNA arrays in normal centromeres is unknown. Using a set of chromosome-specific α-satellite probes for each of the human chromosomes, interphase Fluorescence In Situ Hybridisation (FISH) was performed in a population screening study. This study demonstrated that extreme reduction of chromosome-specific α-satellite is unusually common in chromosome 21 (screened with the αRI probe), with a prevalence of 3.70%, compared to <=.12 % for each of chromosomes 13 and 17, and 0 % for the other chromosomes. No analphoid centromere was identified in over 17,000 morphologically normal chromosomes studied. All the low-alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins including CENtromere Protein-A (CENP-A), CENtromere Protein-B (CENP-B), CENtromere Protein-C (CENP-C), and CENtromere Protein-E (CENP-E). Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual αRI as well as other non-αRI α-satellite DNA suggesting that centromere function may be provided by (i) the residual αRI DNA, (ii) other non-αRI a-satellite sequences, (iii) a combination of i and ii, or (iv) an activated neocentromere DNA. (For complete abstract open document)
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ItemHuman centromere organisation and function( 2012)Centromeres are essential for correct chromosome segregation during cell division. Whilst centromeric function is conserved throughout eukaryotes, the profile of centromeric DNA, although generally repetitive, is not, and neocentromeres have been reported at various euchromatic sites devoid of satellite DNA. The lack of DNA sequence conservation has led to the notion that centromere identity is maintained by epigenetic mechanisms. The repetitive nature of canonical centromeres has made studying the organization of the centromere difficult, although the discovery of neocentromeres has provided a useful model for studying the function and organization of the centromere domain, and several studies have used neocentromeres to investigate several proteins required for successful centromere formation and function. One such neocentromere model is the 10q25 neocentromere of Mardel(10), which has previously been used to map the binding domains of several centromere-associated proteins. This study utilized the 10q25 neocentromere model to map the binding domains of three global proteins reported to be enriched at canonical centromeres. The linker histone, histone H1 and the chromatin assembly protein, HMGA1 are enriched at the 10q25 domain following neocentromere formation, suggesting a possible role for each protein at active centromeres. The transcriptional regulator, CTCF, was present at the 10q25 domain both prior to and after neocentromere formation. However, neocentromere formation altered the binding profile of CTCF at the 10q25 domain, with an additional binding cluster observed overlapping the core CENP-A containing domain on the Mardel(10) neocentromere, suggesting a role for CTCF at active centromeres unrelated to the DNA sequence. This study also looked at the role CTCF may be playing at the centromere. Depletion of CTCF resulted in a decrease in transcription at canonical mouse centromeres and at the 10q25 neocentromere, and also resulted in an increase in mitotic segregation defects. Whilst it is evident that the presence of CTCF is important at the centromere for correct centromeric function, the exact mechanism by which CTCF acts at the centromere is still uncertain. Neocentromere-derived mini-chromosomes (NC-MiCs) were developed by telomere associated chromosome truncation (TACT) of the arms of the Mardel(10) marker chromosome. These NC-MiCs provide a useful model for studying the effects large-scale chromosomal rearrangements have on the size and organization of chromatin domains as they can be compared to the progenitor Mardel(10). This study used NC-MiC6, a 1.4Mb mini-chromosome derived from Mardel(10) and still containing the entire Mardel(10) CENP-A binding domain, to investigate the effect of severe chromosomal alterations on the size and organization of the centromere by analysis of the binding domain of CENP-A on NC-MiC6. The CENP-A binding domain on NC-MiC6 was reduced to one-third the size of the Mardel(10) CENP-A binding domain, despite the chromosome size being reduced by 98%. This reduction in size did not dramatically alter the stability of the chromosome, and no correlation was found between the chromosome size and the CENP-A domain size for neocentromeres. Whilst the ratio between chromosome size and centromere size is unlikely to be the trigger for the shrinkage and reorganization of the chromatin domain observed in the 10q25 CENP-A binding domain, the disruption of the chromatin scaffold matrix (S/MAR) or the flanking pericentric heterochromatin in the formation of NC-MiC6 may be responsible for this alteration. Whilst the evidence presented in this study provide further insights into the organization and function of the centromere, our knowledge is still far from complete and further studies are required before we will fully understand this complex and essential domain. Neocentromeres devoid of α-satellite DNA provide a useful model to obtain this information as they can be used to generate a linear ‘road map’ of protein binding at the neocentromere. This information can be used to further understand the organization of the centromere, and can be used in conjunction with three dimensional studies to understand the higher-order chromatin organization of the centromere, thus shedding some light on this complex domain.