Glucocorticoids are widely used as a clinical treatment mainly due to their potent anti-inflammatory abilities, however some patients develop glucocorticoid resistance and high levels of steroid are required to maintain effective treatment. Use of prolonged high glucocorticoid levels is associated with several severe side effects. ARID1a mutations are frequently identified across numerous cancers and have been linked to glucocorticoid resistance. ARID1a is a subunit of the SWI/SNF complex important for binding the glucocorticoid receptor (GR) and for interactions of the complex with DNA. Chromatin remodelling by the SWI/SNF complex is a vital component of genomic GR signalling. Chromatin is dynamically opened and closed at GR binding sites in target genes to regulate transcription. Loss of the full length ARID1a is thought to impact upon GR dependent gene regulation. In this study transcriptional profiling using next-generation RNA sequencing enables the assessment of GR dependent gene transcription in the absence of the full length ARID1a and Chromatin immunoprecipitation is used to assess differences in GR and RNA Polymerase II binding. In the first part of this thesis we compare two cell lines, SKOV3 and HeLa, which contain a truncated or full length ARID1a protein respectively. Here we show a limited response to glucocorticoids in SKOV3 cells and reveal greatly diminished GR binding at a regulatory site known to require remodelling. In the second part, ARID1a knock-down and overexpression of the ARIDla C-terminal are assessed. In these chapters we identify novel 'roles of GR with ARID1a in cell cycle regulation. This thesis highlights several molecular mechanisms through which the loss of ARID1a can impair GR regulation and disproves previous hypotheses that ARID1a is essential for large-scale regulation of robustly glucocorticoid responsive genes. This research may form the foundations for further research leading to a better understanding of how to approach treatment of cancers with ARID1a mutations.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:702466 |
| Date | January 2016 |
| Creators | Stubbs, Felicity Ellen |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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