Human amiloride-sensitive epithelial Na+ channel γ subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation

Abstract

The mRNA for the epithelial Na+ channel γ subunit (γENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na+ deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5ʹ flanking elements of the human γENaC gene. The proximal 5ʹ flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human γENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human γENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody ‘supershift’ assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5ʹ flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA (‘H-DNA’) structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.