AN E-BOX MOTIF CONVEYS INHIBITORY ACTIVITY ON THE ATRIAL-NATRIURETIC-PEPTIDE GENE

Atrial natriuretic peptide (ANP) is a potent diuretic, natriuretic, an d vasorelaxant hormone that is expressed early in ventricular hypertro phy. Expression of human ANP is controlled by a series of regulatory e lements located in the 5' flanking sequence of its gene. We generated a series of 5' deletion mutations extending from -2600 to -1150 relati ve to the transcription start site and linked them to a chloramphenico l acetyltransferase reporter gene. Using transient transfection analys is, we have identified a negative regulatory element between -1206 and -1152 relative to the start site. Each of a series of 5' deletion mut ants, when introduced into fibroblast cultures, expressed the reporter function at a level that was significantly less (< 20%) than that see n with the -1152 reporter construct, whereas comparably transfected at rial cardiocytes demonstrated no change in reporter activity, implying that the repressor function is specific to cell type. The critical re gion (from -1206 to -1152) associates with a soluble protein present i n cardiac fibroblast extracts in a sequence-specific fashion. Deoxyrib onuclease I footprint analysis demonstrated the presence of several pr otected regions, including one that overlies an E-box motif (CAACTG), an element that in other systems has been implicated in promoting diff erentiation in the myocyte lineage. Site-directed mutagenesis of the E -box motif suppressed both the protein-binding and inhibitory activiti es of the 54-bp fragment. In summary, we have found a region in the 5' flanking sequence of the human ANP gene that represses transcriptiona l activity in nonmyocardial cells. This element may play an important role in the restriction of ANP gene expression to cardiac myocytes.