FIG. 3.

Characterization of the human WNK1 renal promoter (rP). (A) Northern blot analysis of WNK1 expression in cultured cells. Fifteen micrograms of poly(A)⁺ RNA from MDCK cells, HEK cells, and CHO cells was hybridized with human WNK1 exon 1 (Ex 1) probe (specific for the full-length transcript) (P1), human exon 5-6 probe (specific for both kinase domain-containing and -defective isoforms), or human probe specific for transcripts ending at the second polyadenylation signal (polyA probe). Hybridization with the exon 1 probe revealed two bands, except for MDCK cells. These two bands result from the use of two polyadenylation sites, as shown when the blot was hybridized with the probe specific for the second poly(A) signal: only the larger band is observed. A shorter band appeared for the MDCK cells when exon 5-6 was probed, corresponding to the renal kinase-defective isoform. (B) Endogenous expression of WNK1 isoforms in cultured HEK 293 cells. QRT-PCR was used to quantify WNK1 transcripts. The full-length isoform (under P1 control) was amplified with primers binding to the 5′ sequence of exon 1, both kinase domain-containing isoforms (under P1 and P2 transcriptional control) were amplified with primers binding to exons 2 and 3, and the kidney-specific kinase-defective isoform was amplified with primers binding to exons 4a and 5. Relative expression of the different WNK1 isoforms is shown. (C) Functional analysis of the proximal 5′ flanking region of exon 4a. Reporter constructs containing the indicated lengths of the 5′ rP relative to the transcription initiation site (+1) are indicated on the left. Luciferase activity normalized with respect to that for pGL3-Basic is shown, after the transfection of CHO, HEK 293, or MDCK cells. Histograms represent means, and bars indicate the minima and maxima for at least three experiments. (D) Comparison of human (h.) and mouse (m.) minimal promoter sequences. Sequences were aligned with BLAST 2. I, sequence identity; -, gaps. Lines indicate consensus transcription factor binding sites identified with the TESS program, and the bent arrow indicates the transcription initiation site mapped by 5′RACE-PCR.