Microscopic observations that whole chromosomes and individual loci occupy specific regions within a cell (nucleus) imply that certain DNA sequences are spatially more proximal to each other than others. With the development of 3C methods, this has been shown to transcend to the molecular level with specific regions of DNA detected colocalizing more frequently than others. The 3C methods have enabled unprecedented insight into the local and global organization of genomes based on the frequency of contacts detected between DNA segments.
43 This has put great emphasis on the ‘interactions’ detected between distant regions of DNA (both inter- and intra-chromosomal) and what the fluctuation of the contact frequency may imply.
The detection of an interaction between two genomic sequences can be a result of three main processes: 1) the thermodynamic movement of the DNA polymer and molecular crowding within the cell or nucleus results in the random contact between two chromosome regions; 2) two chromosome regions come into close spatial proximity as part of defining structural features such as centromere and telomere clustering, and the formation of the nucleolus; and 3) two chromosome regions come into close spatial proximity through an active (e.g. ATP driven) cellular process that influences functional output of the genome (i.e. transcription, replication and DNA repair). Experimental controls and statistical methods are used to distinguish between the different types of interactions with the greatest interest in the interactions that contribute to cellular function, the genotype to phenotype translation. Clear examples of functional interactions detected as a result of DNA loop formation between regulatory sequences and promoters of the genes they regulate are beginning to emerge and the role of the interactions is being unravelled. However, detailed examples are few and the understanding of the organization of the genome at the global level and its involvement in transcription remain vague. Furthermore, the extent and importance of changes in genome organization over time in response to the environment remains largely unexplored.
1.7 G
OALS OF THE THESISThe extensive investigation of the spatial organization of bacterial nucleoids and eukaryotic nuclear chromosomes has clearly demonstrated that they are highly organized. The establishment of the spatial organization of genomes appears to be a balance between the forces and processes that compact and compartmentalize the chromosomes (e.g. DNA supercoiling, DNA binding proteins, macromolecular crowding, and entropy-driven depletion attraction) and the requirement for the DNA to be accessible to essential cellular processes such as DNA replication and transcription. Despite the significant advances in techniques for the investigation of spatial genome organization our understanding of the functional significance of specific three-dimensional genome conformations is limited. Furthermore, how changes in the spatial organization of genomes through time participate in the
44 regulation of cellular processes such as transcription, and consequently in a cell’s ability to adapt, have been minimally characterized.
To gain further insight into the role that the spatial organization of genomes has on the genotype - phenotype translation will require the integrated study of chromosome organization at the molecular level and cellular processes. Further, most studies of the spatial genome organization using proximity-based ligation methods have been performed on asynchronous cells and multicellular organisms. Consequently, due to the dynamic nature of the chromatin fibre, this may cloud our interpretation of the relationship between genome structure and function. Therefore, the utilization of synchronous cell cultures will likely provide a more detailed understanding of the role spatial genome organization plays in functional cellular processes. In an attempt to further our understanding of the role that the spatial organization of genomes and changes therein have on the genotype - phenotype translation, I have addressed three main questions relating to genome structure and function in space and through time.
1) Do changes in the spatial organization of the Escherichia coli nucleoid in response to nutrient deprivation have a functional role in adapting to the stress?
Following an induced amino acid starvation by the treatment with serine hydroxamate there is an observable expansion of the E. coli nucleoid and changes in gene transcription. Combining GCC with microarray data on gene transcript levels, I investigated the potential involvement for specific DNA-DNA contacts in the modulation of specific gene transcript levels enabling the cell to cope with the changing environment. I further investigated the role of DNA replication and the classical NAPs in the spatial organization of the nucleoid.
2) Are there changes in spatial genome organization throughout the cell cycle of Schizosaccharomyces pombe and do these changes facilitate the establishment of cell cycle specific transcription profiles?
S. pombe cells were synchronized at three phases of the cell cycle: G1, G2 and M phases and GCC and RNA-seq were performed on these cells. I determined whether there were detectable differences in genome organization between the three phases of the cell cycle, which included an in vivo molecular level analysis of metaphase chromosomes. I investigated the functional role of the changes in
45 genome organization observed in the establishment and maintenance of cell cycle specific transcription profiles.
3) Can mt-nDNA interactions that were detected in the Budding yeast also be found in the Fission yeast and do they serve functional roles?
It had been demonstrated in S. cerevisiae that Interactions between mitochondrial genes (i.e. COX1 and Q0182, a dubious mitochondrial ORF) and nuclear encoded loci (i.e. MSY1 and, RSM7, respectively), are dependent upon a functional electron transport chain and mitochondrial encoded reverse transcriptase machinery (Rodley et al., 2012). I investigated whether the levels of the nuclear encoded MSY1 and RSM7 gene transcripts changed when the interaction frequency of the respective interactions was reduced by the knockout of mitochondrial reverse transcriptase activity.
I characterized the relationship between mt-nDNA interactions and cellular function throughout the S. pombe cell cycle. I mapped mt-nDNA interactions using GCC for S. pombe cells synchronized at the G1, G2, and M phases of the cell cycle. The enrichment for particular protein products and transcriptional levels of nuclear encoded genes that were associated with the detected mt-nDNA interactions were assessed. In addition, the role for mt-nDNA interactions in the regulation of nuclear DNA replication was also investigated.
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