The homeodomain protein Arix promotes protein kinase A-dependent activation of the dopamine beta-hydroxylase promoter through multiple elements and interaction with the coactivator cAMP-response element-binding protein-binding protein

The differentiation and maintenance of a neurotransmitter phenotype is guid ed by the interaction of exogenous cues with intrinsic genetic machinery. F or the noradrenergic phenotype, these influences combine to activate the ex pression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). in this study, we evaluate the molecu lar mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual h omeodomain binding sites in the rat DBH promoter region and find that all a re essential for both basal and cAMP-dependent protein kinase A (PKA)-stimu lated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core r ecognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PHA in SHSY-5Y neuroblastoma and HepG2 he patoma cells. Intracellular Arix-Arix interactions are evident and may cont ribute to the interdependence of homeodomain binding sites. Analysis of fun ctional domains of Arix reveals an N-terminal activation domain and a C-ter minal repression domain. The N terminus of Arix contains an amino acid moth similar to a region in Brachyury and Pax9 transcription factors. The N ter minal activation domain of Arix interacts with the transcriptional co-activ ator, cAMP-response element-binding protein-binding protein, which potentia tes transcription from the DBH promoter in a PEA-dependent manner, The pres ent study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators wi thin the network of the general transcription machinery.