Search

The homeodomain transcription factors Arix/Phox2a and NBPhox/Phox2b play a role in the specification of the noradrenergic phenotype of central and peripheral neurons. To better understand the functions of these two factors, we have compared the genetic organization, chromosomal location, and transcriptional regulatory properties of Arix and NBPhox. The gene structure is very similar, with each gene containing three exons and two introns, extending a total of approximately 5 kb. Arix and NBPhox are unlinked in human and mouse genomes. NBPhox is located on human Chromosome 4p12 and mouse Chromosome 5, while Arix is located on human Chromosome 11q13 and mouse Chromosome 7. Both proteins bind to three sites in the promoter proximal region of the rat dopamine beta-hydroxylase gene (DBH). In vitro, Arix and NBPhox form DNA-independent multimers and exhibit cooperative binding to the DB1 regulatory element, which contains two homeodomain recognition sites. Both proteins regulate transcription from the rat DBH promoter, and transcription is synergistically increased in the presence of the protein kinase A catalytic subunit (PKA) plus either Arix or NBPhox. The transcription factors exhibit similar concentration-dependent efficacies, and when they are coexpressed, transcription is stimulated to a value approximately equal to that seen with either factor alone. The N-terminal segment of Arix is essential for transcriptional regulatory activity, and this region bears 50% identity with NBPhox, suggesting a similar mechanism of transcriptional activation of the DBH gene. We conclude from this study that Arix and NBPhox exhibit indistinguishable and independent transcriptional regulatory properties on the DBH promoter.

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present 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 within the network of the general transcription machinery.

Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH) is regulated by cell type-specific transcription factors, including the homeoprotein Arix, and second messengers, including cyclic AMP. The cis-acting regulatory sites of the DBH gene which respond to Arix and cAMP lie adjacent to each other, between bases -180 and -150, in a regulatory element named DB1. Neither Arix nor cyclic AMP analogs alone effectively stimulate transcription from the DBH promoter in non-neuronal cell cultures. However, when Arix is present together with cAMP, transcription is substantially activated. Synergistic transcription from the DBH promoter can also be elicited by cotransfection of Arix with an expression vector encoding the catalytic subunit of protein kinase A. Nuclear extracts from PC12 cells display a cAMP-induced complex binding to the DB1 element, and antisera to transcription factors CREB, CREM, Fos, and Jun indicate that these proteins, or closely related family members, interact with DB1. A dominant negative construct of CREB inhibits the response of the DBH promoter to protein kinase A. These results demonstrate a synergistic interaction between a homeodomain protein and the cAMP signal transduction system and suggest that similar interactions may regulate the tissue-specific expression of neuroendocrine genes.

The interaction between cell-cell contact and cyclic AMP-mediated control of the rat tyrosine hydroxylase (TH) gene was investigated in subclones of the PC12 rat pheochromocytoma cell line. Increasing cell culture density and elevation of intracellular cyclic AMP levels with forskolin both cause augmentation of TH RNA levels. However, the extent of increase in TH RNA following forskolin treatment is less in cultures grown at high density than those at low density, suggesting that there may be an interaction in the mechanism by which these two treatments modulate TH RNA levels. The role of cis-acting sequences in the TH gene in the induction of TH RNA by cyclic AMP and cell density was determined by the use of plasmid constructs containing the 5′-flanking sequences of the TH gene directing the transcription of the reporter gene, chloroamphenicol acetyltransferase (CAT). Using transient transfection assays in PC12 cells, we have mapped the site of cyclic AMP regulation of the TH gene to a region between −60 and −41. Stable transformants of PC12 cells which express p5TH CAT ( −773 +27 ) were isolated and the activity of CAT following treatment of cells with forskolin and growth at different cell densities was evaluated. CAT activity does not differ between cells grown at low or high density. Forskolin induces CAT activity 2–4 fold, but the extent of induction does not vary with changes in cell culture density. We conclude from these experiments that the intracellular mechanism by which increased cell-cell contact modulates TH RNA levels is not through interaction with the same genomic elements as those which regulate gene expression by cyclic AMP.

The noradrenergic cell type is characterized by the expression of proteins involved in the biosynthesis, transport, and secretion of noradrenaline and is dependent on the sequential and combinatorial expression of numerous transcription factors, including Phox2a, Phox2b, dHAND, GATA2, GATA3, and MASH1. Phox2a and Phox2b transactivate the promoter of the gene encoding the noradrenergic biosynthetic enzyme, dopamine beta-hydroxylase (DBH), and dHAND potentiates the activity of Phox2a. In this study, we use chromatin immunoprecipitation assays to identify target genes of the Phox2 proteins and dHAND. All three proteins are bound to the DBH and PHOX2B promoter regions in SH-SY5Y neuroblastoma cells. The interaction between Phox2a and dHAND is analyzed by fluorescent anisotropy, which demonstrates that dHAND causes an eightfold increase in the affinity of Phox2a for its recognition sites on the DBH promoter region. The Phox2 proteins are not found on the genes encoding other noradrenergic enzymatic or transport proteins but are reciprocally bound to each other's promoters in SH-SY5Y cells. Together with Phox2a and Phox2b, dHAND is bound to the PHOX2B promoter and is also associated with the GATA2 and eHAND genes in the absence of the Phox2 proteins. These results demonstrate the direct interactions of the Phox2 and dHAND transcription factors within a noradrenergic cell type. The Phox2 proteins were found to share all target genes, whereas dHAND binds to genes independently of Phox2a.

Neurotransmitter biosynthesis is regulated by environmental stimuli, which transmit intracellular signals via second messengers and protein kinase pathways. For the catecholamine biosynthetic enzymes, dopamine beta-hydroxylase and tyrosine hydroxylase, regulation of gene expression by cyclic AMP, diacyl glycerol, and Ca2+ leads to increased neurotransmitter biosynthesis. In this report, we demonstrate that the cAMP-mediated regulation of transcription from the dopamine beta-hydroxylase promoter is mediated by the AP1 proteins c-Fos, c-Jun, and JunD. Following treatment of cultured cells with cAMP, protein complexes bound to the dopamine beta-hydroxylase AP1/cAMP response element element change from consisting of c-Jun and JunD to include c-Fos, c-Jun, and JunD. The homeodomain protein Arix is also a component of this DNA-protein complex, binding to the adjacent homeodomain recognition sites. Transfection of a dominant negative JunD expression plasmid inhibits cAMP-mediated expression of the dopamine beta-hydroxylase promoter construct in PC12 and CATH.a cells. In addition to the role of c-Fos in regulating dopamine beta-hydroxylase gene expression in response to cAMP, a second pathway, involving Rap1/B-Raf is involved. These experiments illustrate an unusual divergence of cAMP-dependent protein kinase signaling through multiple pathways that then reconverge on a single element in the dopamine beta-hydroxylase promoter to elicit activation of gene expression.

The recently described homeodomain protein ARIX is expressed specifically in noradreneric cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine {beta}-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.

In order to characterize the specificity of expression of the neurotransmitter biosynthetic gene dopamine beta-hydroxylase (DBH), the identification of proteins that interact with the DB1 enhancer was initiated. A homeobox-containing cDNA was isolated from a PC12 expression cDNA library screened with the DB1 enhancer. The homeodomain is a member of the paired-like class, and is encoded by several nonidentical cDNAs. The cDNAs contain the same sequence in the homeodomain and 3′ coding and noncoding sequences, but diverge in sequence 5′ to the homeodomain. This family of homeobox-containing cDNAs is named Arix. Arix mRNA transcripts are found only in noradrenergic, DBH-positive tissues, and in cell lines derived from those tissue. The DB1 enhancer contains two binding sites for the Arix homeodomain, and both sites contribute to basal activity of the DBH promoter. When introduced into tissue culture, Arix regulates the transcriptional activity from the DBH promoter, and also from the promoter of the tyrosine hydroxylase gene, encoding the initial enzyme of the catecholamine biosynthetic pathway. The pattern of expression of the Arix transcripts, the presence of the homeodomain, and the transcriptional regulatory properties suggest that this family of proteins may be involved in the specificity of expression of the catecholamine biosynthetic genes.

Dopamine beta-hydroxylase (DBH) catalyzes the synthesis of the neurohormone norepinephrine and is expressed only in noradrenergic and adrenergic cells of the nervous and endocrine systems. We have previously described a positive-acting genetic regulatory element of the DBH that contributes to the restricted expression of this gene. In the study described here, we identify and characterize a negative-acting regulatory element within the 5'-flanking region of the rat DBH gene. This negative regulatory element, which is located between -282 and -232, represses transcription from the DBH promoter in cell lines of noncatecholaminergic origin and binds to nuclear factors found in extracts from both the catecholaminergic cell line SHSY-5Y and the noncatecholaminergic cell lines JEG-3 and C6. The negative regulatory region will reduce transcription from a heterologous promoter in two noncatecholaminergic cell lines. These experiments demonstrate that the selective expression of the DBH gene is controlled by both positive- and negative-acting genetic regulatory elements.

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H1 histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED50 values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.