Identification of a novel region in the proximal promoter of the mouse renin gene critical for expression

Abstract

An enhancer at −2.6 kb and a HOX·PBX-binding site at −60 bp have been demonstrated to be critical to expression of the mouse renin gene ( Ren-1c) in As4.1 cells. In this report, we show that a region (−197 to −70) immediately 5′ to the HOX·PBX-binding site is also critical for Ren-1cexpression. Deletion of this region in a construct containing 4.1 kb of the Ren-1c5′-flanking sequence resulted in a 99% reduction in Ren-1cpromoter activity in As4.1 cells, suggesting the pivotal role for the region in the regulation of the mouse renin gene. Electrophoretic mobility shift and supershift assays have identified two nuclear factor I-binding sites and a Sp1/Sp3-binding site within the distal portion of the region (−197 to −103). Mutation of these three sites caused a 90% decrease in Ren-1cpromoter activity. Mutational analysis and electrophoretic mobility shift assays have also identified three additional transcription factor-binding sites within the region from −103 to −69, each of which contributes to high-level expression of Ren-1cin As4.1 cells. Finally, we have shown that the Ren-1cenhancer is the target for endothelin-1 (ET-1)-induced inhibition of Ren-1cexpression and the transcription factor-binding sites in the proximal promoter are required for the maximal ET-1 inhibitory effect.