Sequencing and characterization of the human thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene promoter

The thiazide-sensitive Na-Cl cotransporter SLC12A3 displays expression restricted to distal convoluted tubule cells where it catalyzes the uptake of sodium and chloride through the apical membrane, We sequenced 1959 bp of the 5 ' flanking region of human SLC12A3, located the area of transcription initiation, and used deletion constructs of the flanking region to determine areas that affect reporter gene expression in two cell lines, MDCT and CHO. Amplification of the 5 ' end of SLC12A3 cDNA from an adapter-ligated human kidney cDNA library demonstrated that transcription initiation is confined to an area from -18 to -6 bp upstream of the translation start codon. Maximum promoter activity (9.815 +/- 0.864 times control) was observed in MDCT cells using a promoter containing 1019 bp of the 5 ' flanking region, A promoter containing only 134 bp of the 5 ' flanking region upstream of the translation initiation codon maintained reporter gene expression at levels equal to 75% of that maximally observed (7.375 +/- 0.533 times control). Sequence analysis of this minimal promoter responsible for most of the SLC12A3 promoter activity revealed a TATA element, two Sp binding sites, a potential E box, and a potential binding site for NF-1/CTF or NY-I/CP-I. This promoter, and all other promoter constructs from SLC12A3, displayed repressor activity in CHO cells. A construct containing sequence 94 bp upstream of the initiation codon with two potential Sp binding sites was required for this repression, Protein-DNA interactions between the 182 bp region immediately upstream of the start codon and the nuclear proteins from rat kidney cortex and HeLa cells were examined to further clarify the role of the putative binding sites for SLC12A3 expression. Physiological studies investigating the effects of osmolarity, pH, and mineralocorticoid steroid on promoter activity demonstrated that the promoter activity was repressed by acidification, whereas no effects of increased osmolarity or deoxycorticosterone acetate addition were observed. (C) 2001 Academic Press.