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Title: Regulation of GR recruitment to the chromatin template during basal glucocorticoid pulsatility and stress
Author: Pooley, John Robert
ISNI:       0000 0004 2725 4780
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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The secretion of glucocorticoids from the adrenal gland is necessary for appropriate metabolism, immune regulation and responses to stress exposure. Glucocorticoid secretion occurs with defined patterns sensitive to physiological or pathological state, and communicates detailed messages to the cellular interior. These signals are conveyed in part by the genomic actions of the glucocorticoid receptor (GR), a ligand-activated transcription factor modifying the transcriptome by interacting with DNA. Little attention has been afforded to how GR-mediated genomic signalling interacts with the defined pattern of glucocorticoid secretion in vivo, or the varied physiological contexts in which glucocorticoid secretion occurs. In the first part of the thesis the dynamics of GR interaction with the pattern of hormone presentation is explored. Well recognised circadian glucocorticoid secretion comprises high amplitude pulses of adrenal secretory activity with an ultradian frequency (approximately one pulse/hr). The importance of the ultradian component of this secretion pattern, conserved through mammalian evolution has been previously suggested by detailed studies revealing an influence of physiological and pathophysiological state on rhythm components. By scrutiny of other systems possessing ultradian rhythms, we hypothesised a functional significance to the ultradian pattern of glucocorticoid release. The emerging hypothesis that ultradian secretion is important for appropriate gene regulation was examined. Utilising chromatin immunoprecipitation to define GR occupancy of endogenous gene regulatory regions, we describe a dynamic interaction of the GR with DNA binding sites in cell lines and animal tissues. GR responded individually to pulses of glucocorticoid exposure, and interacted with the genome in a manner defined temporally by ligand availability. The effect of this behaviour on the output of known glucocorticoid-regulated genes is discussed, incorporating the work of colleagues and collaborators using DNA binding ELISA and quantitative RT-PCR. The transcriptional program facilitated by this mode of interaction can be described as 'gene pulsing'. The second part of the thesis addresses context-dependence III glucocorticoid signalling .. Context -dependant behavioural and physiological responses to glucocorticoid likely anse through context-dependent molecular events. Access of transcription factors to DNA of higher organisms is controlled by the chromatin environment and by the availability of other transcription factors, which can either permit or hinder binding site accessibility. We hypothesised that the population of accessible binding sites for GR genome-wide is not absolute or fixed in any given cell type. Rather, changes in the chromatin landscape could arise from signalling components activated by stimulus exposure. Such changes may permit focused targeting of GR to DNA in a manner appropriate to the context of glucocorticoid exposure. Utilising the rodent response to stress as a context different from a baseline quiescent state, we designed experiments to test whether an equivalent elevation in plasma corticosterone produces the same genome-wide pattern of GR binding regardless of context (null hypothesis). Our approach depends upon high throughput sequencing technology to define regions of enriched GR binding determined by chromatin immunoprecipitation analysis (ChIP-seq). The thesis presents the experimental approach incorporating available preliminary data but does not address the hypothesis directly at this stage. Our results necessitate a revision of the classical view of glucocorticoid receptor function at the genome and hint at how the transcriptional program generated by GR's episodic association with the genome might be disrupted in human disease. A review of the clinical use of glucocorticoids, and the current approach to novel glucocorticoid therapeutics may be warranted.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available