ROLE OF PROXIMAL PROMOTER ELEMENTS IN REGULATION OF RENIN GENE-TRANSCRIPTION

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to in duce neoplasia in renal renin-expressing cells, express high levels of renin mRNA from the endogenous Ren-1(c) gene. We have used these cell s to characterize the role of the Ren-lc proximal promoter (+6 to -117 ) in the regulation of renin gene transcription. It was found that 4.1 kilobases (kb) of Ren-1(c) 5'-flanking sequence, in combination with the proximal promoter, are required for strong activation (similar to 2 orders of magnitude over the basal level of the promoter alone) of t he 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 combina tion with the promoter. The resulting transcripts initiate at the norm al renin start site. Electrophoretic mobility shift assays identified a sequence at approximately position -60 in the promoter region that b inds nuclear proteins specific for renin-expressing As4.1 cells. Mutat ions in this sequence, which disrupt binding of nuclear protein(s), co mpletely abolish activation of transcription by the 4.1-kb fragment. A ctivation of transcription by the 241-base pair enhancer was still obs erved, although it was diminished in magnitude (60-fold over the mutat ed promoter alone). We present a model derived from the current data t hat suggests that regulation of renin expression is achieved through c ooperation of transcription factors binding at the proximal promoter e lement and a distal enhancer element to abrogate or override the effec ts of an intervening negative regulatory region.