GC- and E-box motifs as regulatory elements in the proximal promoter region of the neuronal nicotinic receptor alpha7 subunit gene.
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.
Department of Neurochemistry, Universidad Miguel Hernández, 03550 San Juan, Alicante, Spain.
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Carrasco-Serrano
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Campos-Caro
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Viniegra
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Ballesta
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Criado
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D019439:Chromaffin Cells; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000032:analysis; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011978:Receptors, Nicotinic; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors