In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer

Abstract

DBA/2J mouse contains two resin gene loci (Ren1(d) and Ren2(d)). Ren2(d) but not Ren1(d) is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on in vitro studies, we have postulated that a negative regulatory element (NRE) in the resin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Ren1(d) or Ren2(d) promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Ren1(d) fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2(d) promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Ren1(d) construct with Ren1(d)-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Ren1(d) fragment-CAT construct or Ren2(d) fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human c-myc NRE oligonucleotides which share homology with Ren1(d)-NRE competed effectively with these oligonucleotides for the regulation of Ren1(d) gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes.