Identification of a short cis-acting element in the human vasopressin type 2 receptor gene which confers high-level expression of a reporter gene specifically in collecting duct cells.
In the kidney, water reabsorption is mainly regulated by the binding of arginine vasopressin to vasopressin type 2 (V2) receptors. These receptors are expressed selectively in principal cells of the collecting ducts. To identify molecular mechanisms responsible for the cell-specific expression of the V2 receptor, we have analyzed the proximal promoter of the corresponding gene. We report the identification of a 33-bp enhancer [collecting duct tissue-specific element 1 (CSE1)] that induced high levels of expression of the luciferase reporter gene in three collecting duct cell lines, but not in other renal cell lines. In gel shift assays, CSE1 bound a DNA-binding protein expressed selectively in collecting duct cell lines, and a 7-bp mutation, which abolished the activity of CSE1 in transient transfection experiments, also abolished the binding of this protein. Furthermore, decoy experiments performed using CSE1 showed that this sequence was involved not only in the expression of a construct containing 4.2 kb of the V2 receptor proximal promoter, but also in the expression of the endogenous V2 receptor gene. CSE1 appears to act mostly by counteracting the inhibitory effects of a strong ubiquitous repressor element that we called CIE1. Collectively, these results identify the first functional collecting duct-specific cis-acting element.
Calmont
A
A
The University of Paris VI, France.
Reichwald
K
K
Ronco
P
P
Rossert
J
J
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018938:Caco-2 Cells; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007672:Kidney Cortex; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D017483:Receptors, Vasopressin / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D017483:Receptors, Vasopressin; D008156:Luciferases