Abstract
Dopamine beta-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.
Highlights
Cell-specific transcription is accomplished by the precise and transient interactions of numerous and complex molecular factors
It has been shown that ectopically expressed dHAND induces digit duplication in transgenic mice, and similar effects were observed with a dHAND mutant lacking the basic domain, but this mutant was not able to activate transcription in reporter assays when expressed with E12 or to bind to DNA in electrophoretic mobility shift assay (EMSA) [21]
To investigate the ability of dHAND to interact with Arix at the DBH promoter region, NT2 cells were transiently transfected with the DBH-(Ϫ232/ϩ10)luciferase reporter plasmid plus various expression plasmids
Summary
Cell-specific transcription is accomplished by the precise and transient interactions of numerous and complex molecular factors. DHAND has been shown to associate with the bHLH proteins E12 [6] and eHAND [18] and GATA4 and p300 [19]; and the cardiospecific enhancer adenylosuccinate synthetase was the first target gene for dHAND [20] All of these studies demonstrated the dependence of dHAND binding to E-boxes within the promoter region to activate gene expression. It has been shown that ectopically expressed dHAND induces digit duplication in transgenic mice, and similar effects were observed with a dHAND mutant lacking the basic domain, but this mutant was not able to activate transcription in reporter assays when expressed with E12 or to bind to DNA in electrophoretic mobility shift assay (EMSA) [21] These latter results suggest that dHAND could activate transcription independent of direct DNA binding, but other proteins are required to recruit dHAND to the promoter region. Whereas the action of Arix is dependent upon the presence of its DNA recognition sites, the action of dHAND is independent of DNA binding
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