Transient expression analyses of DNA extending 2.4 kb upstream of the human renin gene

Abstract

The influence on homologous and heterologous promoter activity of DNA extending 2.4 kb upstream of the human renin gene ( REN) was examined by transient expression assay in JEG-3 cells, using the gene for chloramphenicol acetyl transferase (CAT) as reporter, and cotransfection with pCH110 to control for transfection efficiency. Analyses of constituent subfragments of the region 5′ of residue −144, using the herpes simplex virus thymidine kinase ( tk) promoter to drive transcription, provided no evidence for negative regulatory influences within the −2400 to −144 DNA. That distal 5′-flanking DNA may have little influence on promoter activity is further supported by a sharp decline in nucleotide homology between human, rat and mouse renin genes further upstream than human residue −604. Constructs containing renin DNA to residue + 13, i.e., which retained the REN promoter, all displayed very low CAT activities, consistent with negative cis-acting control within the −149 to + 13 region. This finding contrasts with results of similar studies for mouse, in which renin gene control was suggested to be mediated primarily via cell-specific trans-acting activator(s) acting on yet-to-be identified enhancer(s). Mouse renin genes have, however, a common DNA insertion that could have disrupted the negative element in this region, and which might contain enhancer target(s) for trans-acting factor(s). In conclusion, the present study involving JEG-3 cells has demonstrated that distal human renin 5′-flanking DNA has little cis-acting influence on promoter activity, whereas DNA located within 100 base pairs of the renin promoter may have a negative regulatory effect. Control of the human renin gene may involve conserved cis-acting sequences in the proximal 0.6 kb of 5′-flanking DNA, although strong conservation is also seen in sequences extending into the first intron. Since trans-acting factors that might be present only in cells that express renin could be important in renin gene control, similar studies as these are required in renin-synthesizing cell lines before any conclusions can be drawn concerning the cis-acting influences on renin promoter activity.