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J. Biol. Chem., Vol. 278, Issue 49, 49652-49660, December 5, 2003

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The Interaction between dHAND and Arix at the Dopamine -Hydroxylase Promoter Region Is Independent of Direct dHAND Binding to DNA^*

Jennifer L. Rychlik, Vincent Gerbasi, and Elaine J. Lewis

From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239

Dopamine -hydroxylase (DBH) catalyzes the production of norepinephrine, and its expression defines the noradrenergic phenotype. Transcription factors dHAND, a basic helix-loop-helix protein, and Arix/Phox2a, a homeoprotein, have been demonstrated to play a role in the differentiation and maintenance of catecholaminergic neurons. Three Arix regulatory sites have been identified in the DBH promoter proximal region, but there is no such evidence for dHAND. Cotransfection with a DBH promoter-luciferase reporter construct plus dHAND or dHAND-E12 expression plasmids did not alter luciferase activity, whereas transfection with Arix resulted in a 2.5-fold stimulation of luciferase activity. However, a 5.5-fold increase was observed when Arix and dHAND were combined, and an 8-fold level of expression was observed when Arix was transfected with a dHAND mutant lacking the basic DNA-binding domain. When the homeodomain sites in the DBH promoter proximal region were mutated, all activity was lost, demonstrating dependence upon Arix-DNA interaction for transcriptional activation. In electrophoretic mobility shift assays, the addition of dHAND decreased the amount of Arix needed to elicit a mobility shift with the DBH homeodomain sites, and the dHAND basic mutant potentiated Arix binding in a manner similar to wild-type dHAND. The dHAND-Arix complex was dissociated upon the addition of an unlabeled competitor containing a homeodomain, but not upon the addition of a competitor containing E-boxes. Arix coprecipitated with antisera directed against recombinant dHAND, demonstrating direct protein-protein interactions. These results indicate that the activation of the DBH promoter by Arix is potentiated by dHAND via a mechanism independent of a direct interaction of dHAND with DNA.

Received for publication, August 5, 2003 , and in revised form, September 18, 2003.

^* This work was supported by Grant GM38696 from the National Institutes of Health and by a grant from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, L224, 3181 SW Sam Jackson Park Rd., Oregon Health and Science University, Portland, OR 97239. Tel.: 503-494-6699; Fax: 503-494-8393; E-mail: rychlikj{at}ohsu.edu.

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