Abstract
The alpha7 subunit is a component of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in bovine adrenomedullary chromaffin cells. The proximal promoter of the gene coding for this subunit contains several GC-boxes and one E-box. Deletion analysis and transient transfections showed that a 120-base pair region (-77 to +43) including all of these elements gave rise to approximately 70 and 95% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of the different elements indicated that both GC and E motifs contribute to the activity of the alpha7 gene in a very prominent way. Using electrophoretic mobility shift assays, the upstream stimulatory factor (USF) was shown to be a component of the complexes that interacted with the E-box when nuclear extracts from chromaffin and SHSY-5Y cells were used. Binding of the early growth response gene transcription factor (Egr-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, alpha7 promoter activity increased by up to 5-fold when alpha7 constructs and an Egr-1 expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-1 activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-1 sites was removed. Taken together, these studies indicate that the alpha7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.
Highlights
Nicotinic acetylcholine receptors1 are members of a supergene family of neurotransmitter-gated ion channels [1, 2]
␣7 subunit expression is restricted to adrenergic cells by a mechanism that appears to be mediated by the immediate early gene transcription factor Egr-1
These constructs were introduced into SHSY-5Y and chromaffin cells, a neuroblastoma cell line and a primary cell culture, respectively, that express the ␣7 subunit endogenously [18, 30]
Summary
Plasmid Constructions—The isolation and characterization of the 5Ј-flanking sequence of the bovine ␣7 subunit gene has been previously described [23]. All ␣7 promoter-LUC gene fusions were made in the pGL2-Basic vector (Promega, Madison, WI), introducing in its polylinker, upstream of the luciferase gene, the suitable ␣7 promoter fragments. Deletion analysis was performed by partially digesting an ApaI–HindIII fragment (from Ϫ339 to ϩ43 in the ␣7 sequence) with BstUI, which leaves blunt ends at GCGC sequences, and subcloning the partial digests into pBluescript (Stratagene, Heidelberg, Germany) vector cut with HincII–HindIII. Sequence analysis allowed selection of the appropriate fragments, which were cloned into pGL2-Basic and further transfected. The basic strategy for site-directed mutagenesis of the different elements at the Ϫ77 to Ϫ15 region of the ␣7 promoter (see Fig. 2) consisted of the following steps. The basic strategy for site-directed mutagenesis of the different elements at the Ϫ77 to Ϫ15 region of the ␣7 promoter (see Fig. 2) consisted of the following steps. (a) PCR
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