GC- and E-box motifs as regulatory elements in the proximal promoter region of the neuronal nicotinic receptor α7 subunit gene

The alpha 7 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 similar to 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 alpha 7 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-l) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, alpha 7 promoter activity increased by up to B-fold when alpha 7 constructs and an Egr-l expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-l activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-l sites was removed. Taken together, these studies indicate that the alpha 7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.