Glucocorticoids (GCs) exert diverse effects on multiple cell types and tissues. The variability in GC sensitivity can give rise to disease states hence the importance of GC sensitivity modulators. GCs act through the glucocorticoid receptor (GR), a ligand-activated nuclear hormone receptor (NR), which interacts with the DNA to regulate gene transcription depending on the chromatin structure. GR itself modulates chromatin through epigenetic modification of histone residues. In the present study, novel modulators of GC sensitivity, altering GR-mediated gene expression through dynamic or epigenetic regulatory mechanisms, are identified and explored. Metastasis-related methyltransferase1 (Merm1/WBSCR22), is a histone methyltransferase, previously shown to methylate histone H3 Lysine 9 (H3K9), a repressive methyl mark, to inhibit target gene transcription. Our GR reporter transient transfections assays showed that Merm1 potentiated GR transactivation through its methyltransferase and SAM domains. Merm1 knockdown significantly impaired both GR transactivation, and transrepression of endogenous genes, including GILZ. The ChIP assay analysis confirmed that both GR and Merm1 bound the GILZ promoter and Merm1 regulated ligand-induced GR recruitment. Merm1 regulated tri-methylation of H3K4 (H3K4me3) and di-methylation of H3K79 (H3K79me2). At the GILZ locus, GR induced H3K4me3 and inhibited H3K79me2. Merm1 regulated both of these and also maintained basal H3K79me2. The GR-induced H3K4me3 followed by loss of H3K79me2 showed that these events were driven by H3K4 methylation. In conclusion, Merm1 regulates chromatin structure to affect GR recruitment, and mediates GR actions of transcription by histone methylation. In the second part of the thesis, the biological consequence of temporal dynamics of GC delivery to target cell gene expression and apoptosis has been investigated. For this purpose a flow-through culture system was designed and modified for pulsatile and continuous delivery of GC to HeLa cells and primary T cells. Pulsatile cortisol caused a significant reduction in cell survival compared to continuous exposure of the same cumulative dose in HeLa population. This was due to increased apoptosis. Transcription factor (TF) binding site analysis of the microarray data identified CCAAT-displacement protein (CDP) as a common TF binding site in the differentially regulated target genes. Mouse mammary tumour virus (MMTV) gene is regulated by CDP and is also GC responsive. MMTV-Luc was also differentially regulated between pulsatile and continuous cortisol. In primary T cells, GILZ and FKBP5 genes were more highly induced with continuous than with the same equivalent concentration given in pulses. In conclusion, cortisol oscillations exert important effects on target cell gene expression, and phenotype. In summary, GC sensitivity is modulated via different mechanisms. Our data illustrate a novel regulatory mechanism whereby GR activity is altered through histone modifications and chromatin remodelling. In addition, GC oscillations provide frequency modulation to GR-mediate gene expression with a resulting differential pattern of gene transcription and cellular response.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553483 |
| Date | January 2012 |
| Creators | Jangani, Maryam |
| Contributors | Donn, Rachelle; Ray, David; Bruce, Ian |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/novel-modulators-of-glucocorticoid-sensitivity(6c463259-031b-4514-ac93-98585cc939b0).html |
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