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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ItemSequence structure and evolution of the mouse Y centromere( 2013)The centromere is a fundamental structure required for the faithful segregation of eukaryotic chromosomes during cell division. Despite this function being highly conserved, centromere sequences evolve rapidly, and are frequently diverged between even closely related species. While the centromere sequences of many model multicellular eukaryotes are now well characterised, one centromere sequence, namely that of the mouse Y chromosome, has remained unidentified. This lack of sequence information has left a significant gap in our knowledge of centromere biology and chromosome evolution in this important mammalian model organism. In mouse, the centromere sequence of all chromosomes except the Y chromosome consists of a highly conserved, tandemly repeated minor satellite DNA. Why the Y centromere should lack minor satellite DNA is unknown, but this observation suggests that the Y centromere DNA has evolved in relative isolation from the minor satellite sequence. Here, a bioinformatics approach has been used to identify a putative Y centromere DNA that has led to the complete molecular characterisation of the C57BL/6J mouse Y centromere. This newly identified satellite DNA (Ymin) is composed of a complex, highly diverged minor satellite-like sequence that is organised as a 2.3 kb higher-order repeat (HOR) unit. The homogeneous HOR units are tandemly repeated across the core of the Y centromere array and are flanked by diverged multimeric and monomeric Ymin repeats. This sequence architecture is more reminiscent of the organisation of the human centromeres and is quite distinct from the minor satellite sequence organisation found at all other mouse centromeres. The complete characterisation of the C57BL/6J (M.m. molossinus) mouse Y centromere DNA has been facilitated by the entire 90 kb Ymin array being contained within a single, fully sequenced BAC clone; RP24-110P17. The sequence conservation of the Ymin DNA was also investigated in other mouse strains. This led to the characterisation of a new, 1.6 kb HOR unit that is specific for the Y centromere of the subspecies M.m. domesticus. A comparative sequence analysis between the canonical HOR units from M.m. molossinus and M.m. domesticus mice indicates the Y centromere DNA is diverging at an accelerated rate, with major turnover of the HOR arrays driving rapid divergence of sequence and higher-order structure at the mouse Y centromere. A comparative sequence analysis of the human and chimpanzee centromeres indicates a similar rapid divergence for the primate Y centromere relative to other primate centromeres. Together, these data suggest that accelerated divergence of the Y centromere DNA may be a general feature of mammalian Y chromosome evolution. Somewhat paradoxically, an analysis of inbred mice transmitting the same Y chromosome over several hundred inbred generations provided no evidence for a change in Y centromere array length. This is despite considerable evidence for unequal sister chromatid exchange, which drives the expansion and contraction of tandem repeat arrays, being pervasive in the evolution of the mouse Y centromere DNA. The identification and molecular characterisation of the mouse Y centromere sequence fills a significant and long standing void in centromere biology and chromosome evolution. The diverged nature of the Y centromere DNA and its unique organisational architecture is consistent with this sequence evolving independently from the centromeres of all other mouse chromosomes.
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ItemCharacterisation of the centromere protein FAM44A in human and mouse cells( 2012)The centromere is responsible for ensuring correct segregation of newly replicated sister chromatids into daughter cells. This structure is found in all eukaryotes ranging from single cell to complex multicellular organisms. Any errors in chromosome segregation, including mutations in proteins that have a role in the assembly of the spindle microtubule attachment site, known as the kinetochore, can result in daughter cells with an abnormal chromosomal number, or aneuploidy. In humans, changes in chromosome number significantly contribute to medical conditions such as spontaneous abortions, infertility and birth disorders (Hassold and Hunt, 2001), and is commonly linked with cancer via changes in copy numbers of oncogenes and tumour-suppressor genes. A novel centromere protein, FAM44A, was identified by the screening of a panel of patient sera with autoimmune antibodies that localise to the centromere. These sera were chosen for their presence of uncommon fragment sizes as visualised by Western blot. One such serum sample was subsequently used to probe a HeLa cDNA phage expression library. The 330 kDa FAM44A protein was identified, and contains the following chromatin domains; AT-hook motif, Cps15 domain, histone deacetylase interaction domain, and proline rich domain. The main aim of this study was to localise and functionally characterise the cell cycle roles of the FAM44A protein in mammalian cells. The cellular localisation of FAM44A using a FAM44A-specific antibody demonstrated that this protein is a centromeric and is present during all the mitotic stages. The functional study used RNAi-mediated down-regulation of FAM44A transcripts, which demonstrated a clear mitotic progression defect where accumulation of the cells at different mitotic stages was observed at 48 and 72 hours post siRNA knockdown. Further, several mitotic defects were observed to include, poor chromosome alignment during metaphase, lagging anaphases and chromatin bridges and an increase in the number of cells with micronuclei. These mitotic defects indicate that this protein plays an important role in the correct segregation of chromosomes during mitosis. Interestingly, further analysis showed most of these aberrant chromosomes and micronuclei were acentric, which suggest that FAM44A could be involved DNA repair, chromatin modification or remodelling processes. The identification and characterisation of FAM44A in this study contributes to the growing list of novel centromere proteins discovered in recent years. From the interesting mitotic phenotype observed, we can anticipate that further in-depth characterisation will fully define FAM44A as an important component of the centromere and chromatin that has multiple functions in the regulation of the cell cycle and chromosome segregation.
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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.
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ItemFunctional analysis of a novel mammalian zinc-finger centromere protein, ZNF397( 2010)The centromere is responsible for ensuring correct segregation of newly replicated sister chromatids into daughter cells. This fundamental role is conserved in eukaryotic organisms from yeast to humans. An error in chromosome segregation, including mutations in proteins that have a role in the assembly of the kinetochore, can result in daughter cells with an abnormal chromosomal number (aneuploidy). In humans, aneuploidy is a major contributor to birth defects, spontaneous abortions and infertility (Hassold and Hunt 2001), and is often associated with cancer due to the loss of tumour-suppressor genes or gain of oncogenes. A novel centromere protein, ZNF397, has recently been identified using a centromere positive autoimmune serum from a patient with watermelon stomach disease. ZNF397 protein belongs to the classical C2H2 group of the zinc-finger protein superfamily, which is one of the largest families in the human proteome. It contains two conserved domains; a leucine-rich SCAN domain and nine C2H2 zinc fingers. Bioinformatic analysis showed that ZNF397 is conserved in placental mammals. To date, only a few proteins containing zinc-finger domains have been identified at the centromeric or pericentric loci in various eukaryotic organisms. Stable human cell lines expressing a green fluorescent protein-ZNF397 fusion demonstrated that ZNF397 was centromeric and it co-localised with constitutive centromere protein CENP-A during interphase to early prophase of human cells. Deletion and domain-swap constructs indicated that the SCAN domain was necessary, but not sufficient, for centromere localisation. ZNF397 also localised to an active neocentromere, not the inactive α-satellite centromere on a pseudo-dicentric neocentromere chromosome four. Knockout studies in mice have revealed that ZNF397 was not essential for chromosome segregation, development or reproduction of the mice. The presence of ZNF397 in interphase cells but not on mitotic chromosomes suggests that it is not a constitutive structural protein and that it is unlikely to be directly involved in microtubule capture, mitotic segregation, or cytokinesis. An attractive hypothesis is that ZNF397 plays a role in the regulation of transcription at the centromere. Two possible mechanisms of action for ZNF397 are proposed; 1) ZNF397 could be directly involved in transcription of centromeric repeats or 2) ZNF397 could repress genes by recruiting them to the centromeric heterochromatin environment.