POSITIVE AND NEGATIVE ELEMENTS CONTRIBUTE TO THE CELL-SPECIFIC EXPRESSION OF THE RAT DOPAMINE-BETA-HYDROXYLASE GENE

Dopamine beta-hydroxylase catalyzes the final step in noradrenaline sy nthesis and is expressed exclusively in noradrenergic and adrenergic c ells. In order to identify elements within the dopamine beta-hydroxyla se (DBH) gene which contribute to the regulation of tissue-specific ex pression, we have analyzed the expression of the rat DBH promoter by t ransient transfection in both DBH-expressing and non-expressing cell l ines. We have found that 1 kilobase of the DBH promoter can direct exp ression of the luciferase reporter gene in the DBH-expressing PC12, CA TH.a, and SK-N-SH cell lines, but not in the non-DBH-expressing C6 gli oma or CA77 cell lines. This activity was localized to a region betwee n -133 and -173 upstream of the transcription start site. This element , however, also directed expression in non-DBH-expressing cell lines, but was inhibited when sequences between -212 and -388 were included. This inhibitory region contains sequences homologous to a silencer ele ment recently identified in the human DBH gene, and shares homology wi th other previously identified silencer elements. Gel retardation expe riments demonstrate that the rat DBH inhibitory region and the silence r elements found in the rat sodium type II channel and SCG10 genes bin d a similar factor. The region between -133 and -173, which contains a consensus cyclic AMP response element (CRE), was also found to be res ponsive to cAMP in both DBH-expressing and non-expressing cells. Inclu sion of sequences between -173 and -190 diminished the cAMP induction in PC12 cells, and nearly abolished the induction in C6 and CA77 cells , suggesting the presence of an additional negative element which inhi bits cAMP induction in non-DBH expressing cells. DNA binding assays us ing antibodies to CRE binding protein-related transcription factors id entified ATF-1 binding to the rat DBH-CRE, and further suggest that in hibition of cAMP regulation may be due to inhibition of ATF-1 binding by an additional factor, which binds to the DBH promoter immediately u pstream of the CRE. These results demonstrate the importance of both p ositive and negative regulatory elements in the regulation of tissue-s pecific expression of the rat DBH gene.