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Volume 271, Number 37, Issue of September 13, 1996 pp. 22499-22505
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of Proximal Promoter Elements in Regulation of Renin Gene Transcription

(Received for publication, April 10, 1996, and in revised form, June 21, 1996)

Nenad Petrovic , Thomas A. Black , John R. Fabian , Colleen Kane , Craig A. Jones , John A. Loudon , J. Pablo Abonia , Curt D. Sigmund Dagger and Kenneth W. Gross

From the Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263 and the Dagger  Cardiovascular Diseases Division, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin-expressing cells, express high levels of renin mRNA from the endogenous Ren-1c gene. We have used these cells to characterize the role of the Ren-1c proximal promoter (+6 to -117) in the regulation of renin gene transcription. It was found that 4.1 kilobases (kb) of Ren-1c 5'-flanking sequence, in combination with the proximal promoter, are required for strong activation (~2 orders of magnitude over the basal level of the promoter alone) of the chloramphenicol acetyltransferase reporter in transfection assays. Within the 4.1-kb fragment, a 241-base pair region was identified that retains full activity in an orientation-independent manner in combination with the promoter. The resulting transcripts initiate at the normal renin start site. Electrophoretic mobility shift assays identified a sequence at approximately position -60 in the promoter region that binds nuclear proteins specific for renin-expressing As4.1 cells. Mutations in this sequence, which disrupt binding of nuclear protein(s), completely abolish activation of transcription by the 4.1-kb fragment. Activation of transcription by the 241-base pair enhancer was still observed, although it was diminished in magnitude (60-fold over the mutated promoter alone). We present a model derived from the current data that suggests that regulation of renin expression is achieved through cooperation of transcription factors binding at the proximal promoter element and a distal enhancer element to abrogate or override the effects of an intervening negative regulatory region.


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