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

Abstract

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 DNAbinding 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.Cell-specific transcription is accomplished by the precise and transient interactions of numerous and complex molecular factors. Basic helix-loop-helix (bHLH) 1 proteins are transcription factors that play an important role in the development and differentiation of specific cell types such as neurons (1), appendages (2), and myocytes (3, 4). These bHLH proteins have been shown to bind to defined consensus sites, E-boxes (CANNTG), through the basic domain and require dimerization partners (via helices) to activate transcription. These proteins have been divided into several classes based on structure, function, and tissue specificity. Class I bHLH proteins (E12 and E47) are expressed ubiquitously in all tissues and can function as homodimers, but prefer to form heterodimers with other bHLH proteins (5). Class II proteins (dHAND, NeuroD, and MyoD) are expressed in specific tissues and are believed to be functionally active when complexed with a class I protein (5, 6). dHAND/HAND2 (deciduum, heart, autonomic nervous system, and neural crest cell derivatives) is a 30-kDa member of the class II bHLH protein family (7,8) and has been detected in Xenopus, zebrafish, chicks, mice, and humans (9). dHAND is required for cardiac (7), vascular (10), and craniofacial...

Hand2 elevates cardiomyocyte production during zebrafish heart development and regeneration

“…Mice in which the Hand2 gene was replaced with a DNA binding-deficient form of Hand2 exhibited relatively normal hearts at E11.5 (Liu et al, 2009), in contrast to the severe ventricular hypoplasia seen in Hand2 mutants by E10.5 (Srivastava et al, 1997). It is presumed that the DNA binding-deficient form of Hand2 can influence transcription through dimerization with other bHLH factors, as well as through interactions with larger protein complexes (Rychlik et al, 2003;Xu et al, 2003). To test whether the early role of hand2 during cardiomyocyte production is dependent on DNA binding or dimerization, we evaluated whether previously characterized DNA binding-deficient and dimerization-deficient versions of Hand2 can expand cmlc2 expression.…”

The bHLH transcription factorhand2is essential for noradrenergic differentiation of sympathetic neurons

“…As the number of ascl1-, phox2b-and phox2a-expressing sympathetic precursor cells is initially not affected, a function of Hand2 in the control of th and dbh expression is the most probable mechanism of action, and indirect effects on cell migration can be excluded. A direct action is supported by the interaction and synergistic function of Phox2a and Hand2 at the Dbh promotor (Xu et al, 2003;Rychlik et al, 2003). Although evidence for the function of Hand2 at the Th promotor is still lacking, it is tempting to speculate that Hand2 and Phox2a act together to induce both Dbh and Th in this lineage.…”

“…A function of Hand2 in the control of Dbh expression has been suggested by the finding that Hand2 cooperates with Phox2a in activating transcription from Dbh promotor reporter constructs (Xu et al, 2003;Rychlik et al, 2003;McFadden et al, 2002;Howard, 2005;Firulli, 2003). There is also in vivo evidence implicating a role for Hand2 in the maintenance of Th and Dbh expression in autonomic neurons (Müller and Rohrer, 2002).…”

“…However, as Phox2a/b are also expressed in non-noradrenergic neurons in both PNS and CNS Pattyn et al, 2000a;Pattyn et al, 2000b;Pattyn et al, 1997), it is evident that the action of Phox2 on the expression of noradrenergic marker genes requires additional co-regulators that are differentially expressed or modified between noradrenergic and non-noradrenergic neurons. Indeed, a number of additional transcriptional regulators have been implicated in the control of Th and Dbh in sympathetic precursor cells, including Creb (Swanson et al, 1997;Adachi and Lewis, 2002), Ap-2␣ (Barallo-Gimeno et al, 2004;Holzschuh et al, 2003;Knight et al, 2003;O'Brien et al, 2004) (also known as Tfap2a), Gata2/3 (Lim et al, 2000;Tsarovina et al, 2004) and Hand2 (also known as dHand) (Howard et al, 1999;Howard et al, 2000;Xu et al, 2003;Rychlik et al, 2003).…”

Essential role of Gata transcription factors in sympathetic neuron development

“…The basic helix-loop-helix (bHLH) transcription factor Hand2 (previously known as dHand) has recently been identified as a noradrenergic co-determinant, due to its ability to elicit noradrenergic differentiation in neural crest (Howard et al, 2000) and parasympathetic precursors (Müller and Rohrer, 2002), and due to its expression in noradrenergic sympathetic but not in parasympathetic ciliary neurons (Müller and Rohrer, 2002). The effects on the expression of the noradrenergic marker gene dopamine-β-hydroxylase (Dbh) can be explained by a direct interaction with Phox2a to stimulate transcription from the Dbh promotor (Xu et al, 2003;Rychlik et al, 2003). Finally, members of the Gata family of transcription factors have been implicated in the control of noradrenergic differentiation (Groves et al, 1995;Lim et al, 2000).…”