AbstractMammalian nuclear function depends on the complex interaction of genetic and epi-genetic elements coordinated in space and time. Structure and function overlap to sucha degree that they are usually considered as being inextricably linked. In this work Icombine a experimental approach with a computational one in order to answer two mainquestions in the field of mammalian chromosome organization.In the first section of this thesis, I attempted to answer the question to what extent doeschromatin from different chromosome territories share the same space inside the nucleus?This is a relatively open question in the field of chromosome territories. It is well-knownand accepted that interphase chromosomes are spatially constrained inside the nucleus andthat they occupy their own territory, however, the degree of spatial interaction betweenneighbouring chromosomes is still under debate. Using labelling methods that directlyincorporate halogenated DNA precursors into newly replicated DNA without the needfor immuno-detection or in situ hybridization, we show that neighbouring chromosometerritories colocalise at very low levels. We also found that the native structure of DNAfoci is partially responsible for constraining the interaction of chromosome territoriesas disruption of the innate architecture of DNA foci by treatment with TSA resulted inincreased colocalisation signal between adjacent chromosomes territories.The second major question I attempted to answer concerned the correlation betweennuclear function and the banding pattern observed in human mitotic chromosomes. Hu-man mitotic chromosomes display characteristic patterns of light and dark bands whenvisualized under the light microscope using specific chemical dyes such as Giemsa. De-spite the long standing use of the Giemsa banding pattern in human genetics for identi-fying chromosome abnormalities and mapping genes, little is known about the molecularmechanisms that generate the Giemsa banding pattern or its biological relevance. Therecent availability of many genetic and epigenetic features mapped to the human genomepermit a high-resolution investigation of the molecular correlates of Giemsa banding.Here I investigate the relationship of more than 50 genomic and epigenomic features withlight (R) and dark (G) bands. My results confirm many classical results, such as the lowgene density of the most darkly staining G bands and their late replication time, usinggenome-wide data. Surprisingly, I found that for virtually all features investigated, Rbands show intermediate properties between the lightest and darkest G bands, suggest-ing that many R bands contain G-like sequences within them. To identify R bands thatshow properties of G bands, I employed an unsupervised learning approach to classify Rbands on their genomic and epigenomic properties and show that the smallest R bandsshow a tendency to have characteristics typical of G bands. I revisit the evidence sup-porting the boundaries of G and R bands in the current cytogenomic map and concludethat inaccurate placement of weakly supported band boundaries can explain the interme-diate pattern of R bands. Finally, I propose an approach based on aggregating data frommultiple genomic and epigenomic features to improve the positioning of band boundariesin the human cytogenomic map. My results suggest that contiguous domains showing ahigh degree of uniformity in the ratio of heterochromatin and euchromatin sub-domainsdefine the Giemsa banding pattern in human chromosomes.
|Date of Award||1 Aug 2013|
|Supervisor||Dean Jackson (Supervisor) & Casey Bergman (Supervisor)|
- Chromosome Biology
- Nuclear Architecture
- Human Genome