Common fragile sites are a set of genomic locations with a propensity to form lesions, breaks and gaps on mitotic chromosomes upon induction of replication stress. While the exact reasons for their fragility are unknown, CFS display instability in a cell-type specific manner, suggesting a substantial contribution from an epigenetic component. CFSs also overlap with sites of increased breakage and deletions in tumour cells, as well as evolutionary breakpoints, implying that their features shape genome stability in vivo. Previously, factors such as delays in replication timing, low origin density and transcription of long genes have been implicated in instability at CFS locations but comprehensive molecular studies are lacking. Chromatin structure, an important factor that fits the profile of cell-type specific contributor, has also not been investigated yet. Throughout their efforts to determine the factors that lead to the appearance of CFS lesions, investigators have focused on a single component at a time, potentially missing out complex interactions between cellular processes that could underlie fragility. Additional difficulties come from the cell-type specificity of CFS breakage: it indicates that only cell type-matched data would be informative, limiting the scope for studies using publicly available data. To perform a comprehensive study defining the role of different factors in determining CFS fragility, I explored replication timing, transcriptional landscapes and chromatin environment across a number of CFSs in two cell types exhibiting differential CFS breakage. Initially, I characterised the patterns of CFS fragility in the two cell types on both the cytogenetic and the molecular level. I then used a FISH-based technique to investigate the process of mitotic compaction at active CFS sites and found that the cytogenetically fragile core of these sites sits within larger regions which display a tendency to mis-fold in mitosis. The aberrant compaction of these regions could be observed on cytogenetically normal metaphase chromosomes, suggesting that finer scale abnormalities in chromosome structure underlie the cytogenetically visible breaks at fragile sites. I also investigated the links between transcription of long genes and CFS fragility using two approaches: I quantified levels of expression across all fragile sites using RNA-seq and modified transcription at a single active CFS using the CRISPR genome engineering methodology. My results indicate a complex interplay between transcription and CFS fragility: no simple linear correlation can be observed, but an increase of transcriptional levels at the active CFS led to a corresponding increase in fragility. To investigate the influence of the cell type specific replication programme and replication stress on CFS instability, I mapped replication timing genome-wide in unperturbed cells and under conditions of replication stress in both cell types. I found that replication stress induces bi-directional changes in replication timing throughout the genome as well as at CFS regions. Surprisingly, the genomic regions showing the most extreme replication timing alterations under replication stress do not overlap with CFS, implying that CFS instability is not fully explained by replication delays as previously suggested. Instead, I observed a range of replication-stress induced timing changes across CFS regions: while some CFSs appear under-replicated, others display switches to both earlier and later replication as well as differential recruitment of both early and late origins, implying that dis-regulation of replication timing and origin firing, rather than simply delays, underlie the sensitivity to CFS regions to replication stress. Finally, I investigated large-scale chromatin states at two active CFSs throughout S phase and into G2, the cell cycle stages most relevant stage for CFS breakage. I found that changes in large-scale chromatin architecture accompany the replication timing shifts triggered by replication stress, raising the possibility that such alterations contribute to instability. In conclusion, I assessed the influence of multiple relevant factors on CFS fragility. I found that bi-directional replication timing changes and alterations in interphase chromatin structure are likely to play a role, converging to promote mitotic folding problems which ultimately result in the well-described cytogenetic lesions on metaphase chromosomes and genomic instability.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:738877 |
| Date | January 2017 |
| Creators | Boteva, Lora |
| Contributors | Gilbert, Nicholas ; Jackson, Andrew |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28803 |
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