Noradrenergic-Specific Transcription of the Dopamine β-Hydroxylase Gene Requires Synergy of MultipleCis-Acting Elements Including at Least Two Phox2a-Binding Sites

摘要: Dopamine β-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic adrenergic neurons neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with HD-binding site residing within composite promoter human DBH gene, designated domain IV, cell-specific manner directly controls noradrenergic-specific activity. In report, we demonstrate three additional protein-binding sites (i.e., domains I, II, III) between IV TATA box are critical for intact activity cells they activate transcription highly concerted manner. Transient transfection assays mutant reporter constructs indicated I was active every cell line tested, whereas III preferentially DBH-positive Remarkably, mutation II associated inactivation exclusively lines, defining it as another element. The profile function these sequence motifs further supported by vitro DNA-binding studies Southwestern analysis. Furthermore, competition antibody supershift show factors Sp1 AP2 cognate nuclear interacting III, respectively. Parallel evidence indicates Phox2a-binding site, demonstrating at least two binding factor upstream promoter. Strikingly, four tandem copies increased minimal 100- 200-fold lines without compromising specificity. Cotransfection Phox2a-expression vector dramatically multiple only DBH-negative confirming responsive Phox2a. Collectively, study emphasizes role Phox2a well its functional synergism other (e.g., CREB, AP2, Sp1) transcriptional activation gene.