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Previous studies demonstrate that the mouse renin gene is regulated by a complex enhancer of transcription located 2.6 kilobases upstream of the transcription start site which is under both positive and negative influence. We demonstrate herein that a positive regulatory element (Eb) is repeated 10 bp upstream (Ec), and both are required for baseline activity of the enhancer. The Eb and Ec core sequences are identical to the consensus sequence for the nuclear hormone receptor superfamily of transcription factors, and transcriptional activity of constructs containing the enhancer is increased after treatment with retinoic acid. Maximal induction requires both Eb and Ec. Expression of endogenous renin and a renin-promoter controlled transgene in As4.1 cells, and kidney renin mRNA in C57BL/6J mice was induced after retinoid treatment. Gel mobility supershift analysis revealed the binding of RAR␣ and RXR␣ to oligonucleotides containing both Eb and Ec. Reverse transcriptase-polymerase chain reaction analysis revealed that As4.1 cells express both receptor isoforms, along with RAR␥, but do not express RAR␤, RXR␤, or RXR␥. Co-transfection of an expression vector encoding wild-type RAR␣ increased enhancer activity, whereas a dominant negative mutant of RAR␣ significantly attenuated retinoic acid-induced activity of the enhancer. These results demonstrate the importance of the Eb and Ec motifs in controlling baseline activity of the renin enhancer, and suggest the potential importance of retinoids in regulating renin expression.The renin-angiotensin system is a critical regulator of arterial pressure and electrolyte homeostasis and is required for continued development of the kidney after birth. The cleavage of angiotensinogen by renin is thought to be the rate-limiting step in the biosynthesis of angiotensin II and is tightly regulated. Transcription of the renin gene, storage and processing of renin in juxtaglomerular cell secretory granules, and secretion of renin into the systemic circulation, each dictate the level of angiotensin II produced. Although the regulation of the renin gene has been studied for many years, the molecular mechanisms controlling its cell-specific expression and regulation in response to physiological cues remains incomplete.Recent studies have identified an enhancer of transcription located upstream of the renin gene which can markedly induce transcription of renin promoter reporter constructs when transfected into As4.1 cells, a renin expressing tumor cell line isolated from the kidney thought to be derived from juxtaglomerular cells (1, 2). This enhancer, located ϳ2.6 kb 1 upstream of the mouse renin gene is partially homologous to a sequence located ϳ12 kb upstream of the human renin gene (3). We previously used mouse/human chimeric enhancers spanning the conserved and nonconserved region to identify important sequences controlling expression (4). Those studies revealed that a 40-bp segment (m40) in the promoter proximal region of the mouse renin enhancer was required for maximal activity....

Abstract-A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4.1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences. (Circ Res. 1999;85:479-488.)Key Words: transfection Ⅲ transcription factor Ⅲ kidney Ⅲ juxtaglomerular cell Ⅲ electrophoretic mobility shift assay R enin (REN) is thought to be the rate-limiting enzyme controlling the generation of angiotensin II, the effector hormone of the REN-angiotensin system. The main source of circulating REN is juxtaglomerular cells of the kidney, although REN expression has been reported in a number of extrarenal tissues. Expression of REN mRNA and its release from the kidney is thought to be controlled at the transcriptional, posttranscriptional, and posttranslational level and is regulated by physiological cues such as arterial pressure, plasma sodium, and sympathetic nerve activity. 1 Although the REN-angiotensin system has been studied for decades, the molecular mechanisms controlling the regulation of REN gene expression and REN release remain unclear (reviewed in Reference 2).Initial clues for the existence of a major regulatory element(s) controlling REN gene expression came from a number of transgenic studies that implicated...

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