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J Biol Chem, Vol. 273, Issue 32, 20021-20028, August 7, 1998

GC- and E-box Motifs as Regulatory Elements in the Proximal Promoter Region of the Neuronal Nicotinic Receptor 7 Subunit Gene

Carmen Carrasco-Serrano^§, Antonio Campos-Caro^§, Salvador Viniegra^§, Juan J. Ballesta^§**, and Manuel Criado^§

From the Departments of  Neurochemistry and ^** Pharmacology, and ^§ Instituto de Neurociencias, Universidad Miguel Hernández, 03550 San Juan, Alicante, Spain

The 7 subunit is a component of -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 ~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 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-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, 7 promoter activity increased by up to 5-fold when 7 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 7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.

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