An E-box motif conveys inhibitory activity on the atrial natriuretic peptide gene

Atrial natriuretic peptide (ANP) is a potent diuretic, natriuretic, and vasorelaxant hormone that is expressed early in ventricular hypertrophy. Expression of human ANP is controlled by a series of regulatory elements located in the 5' flanking sequence of its gene. We generated a series of 5' deletion mutations extending from -2600 to -1150 relative to the transcription start site and linked them to a chloramphenicol acetyltransferase reporter gene. Using transient transfection analysis, we have identified a negative regulatory element between -1206 and -1152 relative to the start site. Each of a series of 5' deletion mutants, when introduced into fibroblast cultures, expressed the reporter function at a level that was significantly less (< 20%) than that seen with the -1152 reporter construct, whereas comparably transfected atrial cardiocytes demonstrated no change in reporter activity, implying that the repressor function is specific to cell type. The critical region (from -1206 to -1152) associates with a soluble protein present in cardiac fibroblast extracts in a sequence-specific fashion. Deoxyribonuclease I footprint analysis demonstrated the presence of several protected regions, including one that overlies an E-box motif (CAACTG), an element that in other systems has been implicated in promoting differentiation in the myocyte lineage. Site-directed mutagenesis of the E-box motif suppressed both the protein-binding and inhibitory activities of the 54-bp fragment. In summary, we have found a region in the 5' flanking sequence of the human ANP gene that represses transcriptional activity in nonmyocardial cells. This element may play an important role in the restriction of ANP gene expression to cardiac myocytes.