Multiple Protein Factors Interact with the cis-Regulatory Elements of the Proximal Promoter in a Cell-Specific Manner and Reg\ ulate Transcription of the Dopamine b-Hydroxylase Gene

Abstract

The dopamine beta-hydroxylase (DBH) gene is expressed selectively in noradrenergic and adrenergic neurons and neuroendocrine cells in the nervous system. A cAMP response element (CRE) residing at -181 to -174 bp 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 area with a composite promoter structure. Using the DBH-expressing human neuroblastoma SK-N-BE(2)C and DBH-negative HeLa cell lines as model systems, we report here that this CRE/YY1/AP1 area interacts with multiple nuclear proteins, including CRE-binding protein (CREB) and transcription factor YY1 in a cell-specific manner. In support of the notion that multiple proteins bind to the CRE/YY1/AP1 area, DNase I foot-printing analysis has demonstrated that nuclear extracts protect an extended region (from -186 to -150 bp) relative to that protected by the purified CREB (from -186 to -171 bp). Site-directed mutational analysis has revealed differential roles of potential cis-regulatory motifs in regulation 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 identified by DNase I footprint analysis in the upstream 260 bp promotor 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.