MULTIPLE PROTEIN FACTORS INTERACT WITH THE CIS-REGULATORY ELEMENTS OFTHE PROXIMAL PROMOTER IN A CELL-SPECIFIC MANNER AND REGULATE TRANSCRIPTION OF THE DOPAMINE-BETA-HYDROXYLASE GENE

The dopamine beta-hydroxylase (DBH) gene is expressed selectively in n oradrenergic and adrenergic neurons and neuroendocrine cells in the ne rvous system. A cAMP response element (CRE) residing at -181 to -174 b p from the transcription start site of the human DBH gene seems to be essential for DBH transcription, Potential cis-regulatory motifs such as AP1 and YY1 occur proximal to and overlap this CRE, endowing the ar ea with a composite promoter structure, Using the DBH-expressing human neuroblastoma SK-N-BE(2)C and DBH-negative HeLa cell lines as model s ystems, we report here that this CRE/YY1/AP1 area interacts with multi ple nuclear proteins, including CRE-binding protein (CREB) and transcr iption factor YY1 in a cell-specific manner. In support of the notion that multiple proteins bind to the CRE/YY1/AP1 area, DNase 1 footprint ing analysis has demonstrated that nuclear extracts protect an extende d region (from -186 to -150 bp) relative to that protected by the puri fied CREB (from -186 to -171 bp). Site-directed mutational analysis ha s revealed differential roles of potential cis-regulatory motifs in re gulation of DBH transcription. Strikingly, the YY1 element positively regulated basal DBH transcription while simultaneously regulating cAMP -mediated induction negatively, which is a novel mechanism of promoter function, Furthermore, three additional DNA-binding sites have been:i dentified by DNase 1 footprint analysis in the upstream 260 bp promote r region of the human DBH gene, of which two sites are cell-specific. These results support a model whereby multiple proteins bind to the 5' -proximal area in a cell-specific manner and coordinately regulate the cell type-specific transcriptional activation of the DBH gene.