5' heterogeneity of glucocorticoid receptor mRNA: associations with tissue-specific, constitutive and auto-regulation
Munn, Helen L.
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Glucocorticoids are steroid hormones that exert profound effects on virtually all tissues. The principal determinant of cellular sensitivity to glucocorticoids is the level of glucocorticoid receptor (GR). Despite near-ubiquitous expression, GR levels vary widely between tissues. A complete lack of GR is lethal at birth and even mild dysregulation has significant pathophysiological consequences. 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 region close to exon 2. The subject for this investigation has been the association between the alternate exons 1 of the GR promoter and the tissue-specific, constitutive and auto-regulation of GR.Cell-specific alternate exon 1 expression was analysed by RT-PCR in various rodent cell lines. All 'CpG island' exons 1 examined (I4, I5, 16, I7, 110 andln) were expressed in all cell lines tested, while expression of exon 11, which lies outside the CpG island region, ~30.2kb upstream of exon 2, 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 was the most active construct in all cell lines tested. 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 Pl7 identified a 134bp region required for high promoter activity in neural cells, which was orientation-specific and did not show properties associated with a classical enhancer. This directionality was mirrored in DNasel protection studies in which a footprint was identified on only one strand of the 134bp fragment. While gel mobility shift assays showed evidence of Spl binding over the region, the physiological significance of this remains unclear. It may be the case that this fragment represents a region at which directional transcription is initiated. Transfections in B103 cells using the 5' deletion series identified two regions important for high promoter activity. Further analysis showed that the activity of these regions was position-dependent within the context of the whole promoter. It appears, therefore, that alternate GR exons 1 are not associated with individual promoters that are selectively regulated in different cell types. Rather, tissue-specific levels of GR arise through a complex arrangement of interacting regulatory elements over the whole promoter region.The role of individual exon 1 splice donor sites was investigated by site-directed mutagenesis of splice donor sites at exons U, lio and In within P2. Following transient transfection in B103 cells, none of the mutant constructs showed a loss in promoter activity relative to intact P2. RT-PCR identified longer transcripts associated with the mutant exons, reflecting the use of downstream 'cryptic' splice donor sites. There appears to be redundancy between alternate exons 1, which can therefore be seen to form a robust promoter mechanism that ensures constitutive GR expression. DNasel hypersensitive site mapping in H4IIE cells showed that a ~3kb fragment, encompassing the entire CpG island region, is bounded by hypersensitive sites and shows general sensitivity to nuclease digestion, consistent with an extended region of transcriptionally active chromatin.The role of individual exons 1 in the autoregulation of GR was investigated by transient transfection of the 3' and 5' deletion series of the GR promoter into B103 cells treated with the synthetic glucocorticoid, dexamethasone. Results showed that the mechanism of autoregulation does not operate selectively on individual exons 1, but instead down-regulates the transcriptional activity of the whole GR CpG island region.Results were therefore consistent with a model of GR transcriptional regulation in which exons 1 are not associated with individual promoters that are selectively regulated by tissue-specific or auto-regulatory signals; rather, regulation occurs through position-dependent interactions of elements arranged over the whole CpG island promoter 'region'. However, while selection of alternate exon 1 transcription initiation sites may not be of primary importance to the tissue-specific or auto¬ regulation of GR, the multiple exon 1 structure does ensure that promoter activity is especially robust to mutations and thus ensures the constitutive expression of this vital housekeeping gene.