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Title: 5’heterogeneity of glucocorticoid receptor messenger RNA : associations with tissue-specific and constitutive regulation
Author: Munn, H. L.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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The rat GR gene includes a complex 5’ promoter region encoding at least eleven untranslated alternate exons 1, eight of which lie in a 3kb CpG island close to exon 2. The subject for this investigation has been the association between alternate exons 1 and the tissue-specific and constitutive regulation of GR. Cell-specific alternate exon 1 expression was analysed by RT-PCR in various rodent cell lines. All CpG island exons examined (14, 15, 16, 17, 110, and 111) were expressed in all cell lines tested, while expression of exon 11, which lies >30kb upstream, was restricted to cells of the immune system. Promoter activity was analysed by transient transfection in B103 (rat neural), C6 (rat glioma) and H4IIE (rat liver) cell lines, using constructs forming both a 3’ and 5’ deletion series of the CpG island region fused to a luciferase reporter gene. ‘P2’ contains the entire promoter and is the most active construct in all cell lines tested. Transfections in B103 cells using the 5’ deletion series identified 2 regions important for high promoter activity. Transfections using the 3’ deletion series, which assesses individual exon 1-associated promoter activity, demonstrated that only ‘PI7’ showed high tissue-specific activity restricted to the neural cell lines B103 and C6. Further transfections with constructs based on PI7 identified a 134bp region required for high promoter activity in neutral cells, which does not show properties associated with a classical enhancer. DNaseI footprinting studies of the 134bp region identified protein binding at a putative AP2 site. However, gel mobility shift assays showed evidence of Sp1, but not AP2 binding. With regard to tissue-specific regulation of GR, only exon 17 showed associated neural/specific promoter activity. Constitutive expression of GR is ensured by a redundant promoter mechanism of ubiquitously expressed CpG island exons 1, which combine to generate both robustness to mutations and high overall promoter activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available