A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:630335 |
| Date | January 2014 |
| Creators | Corless, Samuel |
| Contributors | Van Heyningen, Veronica; Gilbert, Nicholas |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/9623 |
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