Characterization of putative transcription factor motifs within human <i>SLC12A3</i> gene and the effect of sVpr on the association of MR to its binding regions.

(A) Human DCT cells were exposed to aldosterone or vehicle, with or without sVpr, and were processed for the ChIP assay, using anti-FLAG antibody, targeting FLAG-MR. Fifteen primer pairs (S2 Table), located around the transcription start site of the SLC12A3 gene, encoding NCC, were used for qRT-PCR. The fold-enrichment of the specific FLAG-MR-binding DNA sequences to that of the normal rabbit anti serum control was compared among four treatment groups: Control, aldosterone, sVpr, and aldosterone plus sVpr. As shown in the column graphs, the regions of highest Vpr-specific binding (determined by the ratio of aldosterone response to Vpr response) were amplicons P-9 and P-10. *P P B) CV-1 cells were transfected with WT or mutated SLC12A3 constructs. The construct pTSC-Luc contains one putative MR response element (MRE) motif (5’-CAATCAAATGGTGTTCTGC-3’, amplicon P-9) and one putative SP1 motif (5’- CCCTCCCTGGACACC-3’, amplicon P-10). Amplicons P-9 and P-10 exerted the largest suppressive effects by Vpr on transcriptional activity. Putative MRE and SP1 motifs flanking the SLC12A3 transcription start site were mutated with random sequences (S2 Table) to eliminate the putative regulatory motif sequences. Each reporter construct contained a fragment of the SLC12A3 gene. CV-1 cells were transfected with reporter constructs as shown and exposed to aldosterone and/or sVpr. sVpr attenuated aldosterone-stimulated reporter activity with the intact P-9 and P-10 constructs but had no effect with single or dual mutants of P-9 and P-10 amplicons. ***P < 0.001 vs control, ANOVA, followed by Bonferroni correction.