Promoter analysis of the gene encoding the beta-subunit of the rat amiloride-sensitive epithelial sodium channel.
The amiloride-sensitive epithelial Na(+) channel (ENaC), found in the apical membrane of Na(+)-absorptive epithelia, is made up of three differentially regulated subunits: alpha, beta, and gamma. We undertook a study of the 5'-end of the gene encoding the beta-ENaC subunit in the rat. 5'-Rapid amplification of cDNA ends and RNase protection assays indicated multiple transcription start sites over a 50-bp region. Sequencing 1.3 kb of the 5'-flanking DNA revealed putative binding sites for PEA3, Sp1, activator protein (AP)-1 and Oct-1 but neither a TATA box nor consensus sites for steroid hormone receptor binding. Transient transfections of reporter constructs driven by beta-ENaC 5'-flanking DNA in the representative epithelial cell lines Madin-Darby canine kidney, MLE-15, and Caco-2 revealed a negative element present between positions -424 and -311 that affected basal transcription rates. Gel shift assays showed protein-DNA binding activity of an AP-1 consensus site in this region; however, mutation of the AP-1 site did not abrogate the repressive activity of the region in transient transfections. Deletion of two clusters of Sp1 consensus binding sites between -1 and -51 bp and between -169 and -211 bp indicated that the proximal cluster was essential to basal promoter activity in transfected cell lines. In a comparison of these data with those in published studies on alpha- and gamma-ENaC promoters, the beta- and gamma-subunit promoters appear to be more similar to each other than to the alpha-promoter.
Bremner
Harry Robert
HR
Canadian Institutes of Health Research Group in Lung Development, Lung Biology Programme, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Freywald
Tanya
T
O'Brodovich
Hugh M
HM
Otulakowski
Gail
G
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics; D004285:Dogs; D053503:Epithelial Sodium Channels; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics; D013237:Stereoisomerism; D018808:Transcription Factor AP-1 / Q000235:genetics; D014158:Transcription, Genetic
D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1