Positive and negative elements contribute to the cell-specific expression of the rat dopamine beta-hydroxylase gene.

Abstract

Dopamine beta-hydroxylase catalyzes the final step in noradrenaline synthesis and is expressed exclusively in noradrenergic and adrenergic cells. In order to identify elements within the dopamine beta-hydroxylase (DBH) gene which contribute to the regulation of tissue-specific expression, we have analyzed the expression of the rat DBH promoter by transient transfection in both DBH-expressing and non-expressing cell lines. We have found that 1 kilobase of the DBH promoter can direct expression of the luciferase reporter gene in the DBH-expressing PC12, CATH.a, and SK-N-SH cell lines, but not in the non-DBH-expressing C6 glioma or CA77 cell lines. This activity was localized to a region between -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 element recently identified in the human DBH gene, and shares homology with other previously identified silencer elements. Gel retardation experiments demonstrate that the rat DBH inhibitory region and the silencer elements found in the rat sodium type II channel and SCG10 genes bind a similar factor. The region between -133 and -173, which contains a consensus cyclic AMP response element (CRE), was also found to be responsive to cAMP in both DBH-expressing and non-expressing cells. Inclusion 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 inhibits cAMP induction in non-DBH expressing cells. DNA binding assays using antibodies to CRE binding protein-related transcription factors identified ATF-1 binding to the rat DBH-CRE, and further suggest that inhibition of cAMP regulation may be due to inhibition of ATF-1 binding by an additional factor, which binds to the DBH promoter immediately upstream of the CRE. These results demonstrate the importance of both positive and negative regulatory elements in the regulation of tissue-specific expression of the rat DBH gene.