Identification ad characterization of a novel phorbol ester-responsive DNA sequence in the 5'-flanking region of the human dopamine β-hydroxylase gene

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), enhances transcription of many eukaryotic genes, including that for dopamine β- hydroxylase (DBH). In the present study, we report identification and characterization of a novel sequence motif residing in the 5'-flanking region of the human DBH gene, which mediates transcriptional induction by TPA. Deletional analyses indicated the promoter region between -223 and -187 base pairs to be critical whereas this region does not contain any putative regulatory motifs with significant sequence homology to the AP-1 motif, extensive deletional and site-directed mutational analyses indicated that a sequence between -210 and -199 base pairs, 5'-ATCCGCCTGTCT-3', may represent a novel TPA-response element (TRE). In addition, alteration of the YY1- binding site decreased TPA-mediated induction of the DBH promoter activity, suggesting that contiguous cis-regulatory element(s) cooperate with this novel sequence motif. Furthermore, insertional mutation analyses between the YY1-binding site and the cyclic AMP-responsive element indicated that the stereospecificity of these motifs is important for intact transcriptional induction by TPA. Taken together, these data suggest that transcriptional up- regulation of the human DBH gene in response to TPA requires coordination of a novel TRE (human DBH TRE, hDTRE), cyclic AMP-responsive element, and the YYl-binding site.