Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for tripler DNA formation
Sd. Auerbach et al., Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for tripler DNA formation, BIOCHEM J, 347, 2000, pp. 105-114
The mRNA for the epithelial Na+ channel gamma subunit (gamma ENaC) is regul
ated developmentally in the lung, colon and distal nephron and in response
to Na+ deprivation and systemic corticosteroids in the distal colon. Becaus
e such regulation is likely to be at the level of gene transcription, we ex
amined the function of the promoter and other 5' Banking elements of the hu
man gamma ENaC gene. The proximal 5' flanking region contains two GC boxes
but does not contain a TATA box, A 450 bp human gamma ENaC fragment (-459 t
o + 40) directed the expression of luciferase in H441 cells and primer exte
nsion analysis in transfected cells confirmed the correct initiation of hum
an gamma ENaC-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 synt
hesized and tested in a gel mobility-shift assay. Incubation of this radiol
abelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells res
ulted in the formation of four specific and distinct DNA-protein complexes.
On the basis of antibody 'supershift' assays, one of these factors corresp
onds to Spl, whereas the other three correspond to Sp3. Further upstream, a
n approx. 300 nt (-1143 to -839) polypurinepolypyrimidine tract (PPy tract)
containing internal mirror repeats was identified. When contained in a sup
ercoiled plasmid, the approx. 1200nt 5' flanking region was sensitive to S1
endonuclease, which was consistent with the formation of an intramolecular
tripler DNA ('H-DNA') structure with an unpaired single strand. High-resol
ution mapping with S1 endonuclease and sequencing of S1-generated clones co
nfirmed that all SI-sensitive sites were within the PPy tract. Finally, a n
egative regulatory element was identified between -1525 and -1296 that func
tioned in lung, colon and collecting duct cell lines.