Paediatrics (RCH) - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemUnderstanding the role of condensin II in chromosome structure( 2015)The condensin complex plays a key role in organising mitotic chromosomes. In vertebrates, there are two condensin complexes that have independent and cooperative roles in folding mitotic chromosomes. Condensin is an ancient complex that is present in every species from bacteria to human and is increasingly being linked to disease. This project will analyse two poorly studied areas of condensin biology: 1) The of phospho-regulation of condensin in mitosis, and 2) the role of condensin in interphase. In the first study, I dissect the role of a Cdk1 phosphorylation site on the condensin II subunit CAP-D3 in chicken DT40 cells. I show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis and results in overloading of condensin II onto chromosomes. This is a very rare example of a mutation that directly causes chromosome hypercondensation. My results imply phosphorylation of CAP-D3 acts to limit condensin II binding onto mitotic chromosomes and demonstrates that the ratio of condensin I:II is a critical determinant in shaping mitotic chromosomes. In the second study, I analyse the role of condensins in interphase chromosome organisation using conditional knockouts in condensin subunits. Using 3D FISH analyses, I show that condensins not only function during mitosis to fold chromosomes but also during interphase. These findings have important implications as the three-dimensional organisation of the genome plays a critical role in organising gene expression.
-
ItemCondensin: a dynamic organizer of vertebrate chromosome structure: analysis of condensin binding sites and function in vertebrates( 2013)Condensin is essential for the packaging of chromatin into condensed chromosomes in all eukaryotes. It is a multi-subunit protein complex consisting of two core subunits SMC2 and 4 and three auxiliary components CAP-H, CAP-D2, CAP-G for condensin I and CAP-H2, CAP-D3, CAP-G2 for condensin II. Condensin I associates with chromosomes after the nuclear envelope breaks down, and is required for the formation of higher order structure of condensed chromosomes, whereas, condensin II binds to DNA throughout the cell cycle and initiates prophase chromosome condensation. While its role in chromosome condensation is well-established, where condensin I binds in the vertebrate genome is a key outstanding question. This thesis illustrates the first genome-wide map of condensin I in vertebrate cells, which sheds light on the role of condensin I in chromosome condensation and will possibly assist to understand its increasing link to diseases. The genome-wide map suggests that condensin I is enriched at promoters of actively transcribed RNA polymerase II-driven genes, in addition to tRNA and rRNA genes. Furthermore, condensin I is enriched in discrete regions along the central axis, and high concentration of condensin I binding in centromeres and telomeres were observed which is thought to be required for the structural integrity of chromosomes for proper chromosome segregation during cell division. Importantly, this genomic localization pattern of condensin in vertebrates is also seen in bacteria and yeast, suggesting a conserved condensin binding in three kingdoms of life. Another key finding from this study is that condensin I regulates the gene expression through bookmarking. Although how condensin binds to these regions and regulates the genes is enigmatic, this finding is opening a window of new direction in the understanding of condensin and chromosome structure and their link to diseases.
-
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.