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.