FLUORESCENCE ACTIVATED CELL SORTING OF TRANSIENTLY TRANSFECTED As4.1 CELLS SHOWS RENIN ENHANCER DIRECTS ON/OFF SWITCHING OF RENIN PROMOTER IN VITRO

Abstract

1. The proximal promoter of the renin gene is weak and its activity is influenced by a strong, far-upstream enhancer. This and the ability of renin expression in renal afferent arteriolar cells to be 'recruited' under chronic stimulation is consistent with the on/off switching (variegation) model of gene expression. If true, this would provide an example in which variegation controls a physiologically regulable gene. 2. The present study tested the hypothesis that renin promoter activity may accord with the variegation model, at least in individual juxtaglomerular (mouse As4.1) cells in vitro. 3. As4.1 cells were transiently transfected with constructs containing the mouse renin (Ren-1c) enhancer adjacent to the Ren-1c promoter and a linked reporter gene encoding enhanced green fluorescent protein (EGFP). The EGFP signal from individual cells was monitored by fluorescence activated cell sorting. 4. In the presence of the renin enhancer there was 10-fold higher EGFP expression in transfected cells compared with cells transfected with EGFP constructs containing the promoter alone. There was, moreover, an 8-fold increase in the number of EGFP expressing cells. However, EGFP expression in individual transfected cells was similar in the presence or absence of the enhancer. 5. Results from the in vitro system used suggest that the Ren-1c enhancer does not regulate the rate of promoter activity, but rather increases the probability of achieving an active transcriptional state. Limitations of these findings are discussed.