Elaine J. Lewis

The homeodomain protein Arix interacts synergistically with cyclic AMP to regulate expression of neurotransmitter biosynthetic genes.

Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine β-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.

AP1 PROTEINS MEDIATE THE CAMP RESPONSE OF THE DOPAMINE BETA -HYDROXYLASE GENE

Neurotransmitter biosynthesis is regulated by environmental stimuli, which transmit intracellular signals via second messengers and protein kinase pathways. For the catecholamine biosynthetic enzymes, dopamine β-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 β-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 β-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 β-hydroxylase promoter construct in PC12 and CATH.a cells. In addition to the role of c-Fos in regulating dopamine β-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 β-hydroxylase promoter to elicit activation of gene expression.

Paired-Like Homeodomain Proteins Phox2a/Arix and Phox2b/NBPhox Have Similar Genetic Organization and Independently Regulate Dopamine β-Hydroxylase Gene Transcription

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.

Transcription Factor AP‐2 Regulates Expression of the Dopamine β‐Hydroxylase Gene

Abstract: Expression of the gene encoding the neurotransmitter biosynthetic enzyme dopamine β‐hydroxylase (DBH) is regulated in a tissue‐specific pattern, and transcription is influenced by environmental stimuli. Using the promoter proximal region of the rat DBH gene and nuclear extracts from SHSY‐5Y neuroblastoma cells, a DNA‐protein complex was identified that is competitive with oligonucleotides containing the recognition site of transcription factor AP‐2. DNase footprint analysis identified an AP‐2 binding site between −136 and −115 of the DBH promoter. Mutation of that AP‐2 site results in a sevenfold reduction of basal reporter gene expression, but second messenger‐stimulated activity is retained. Cotransfection of an AP‐2 expression vector and a DBH promoter‐reporter construct into cultured cells results in a sixfold stimulation of reporter gene expression, demonstrating the ability of AP‐2 to trans‐activate the DBH promoter. These results identify a new regulatory element on the rat DBH gene and suggest that the AP‐2 site plays a role in maintaining basal levels of DBH transcription.

A Mutation in the ATP7B Copper Transporter Causes Reduced Dopamine β-Hydroxylase and Norepinephrine in Mouse Adrenal

The copper-transporting ATPases Atp7A and Atp7B play a major role in controlling intracellular copper levels. In addition, they are believed to deliver copper to the copper-requiring proteins destined for the secretory vesicles. One cuproprotein, dopamine β-hydroxylase (DBH) functions in the biosynthesis of norepinephrine and epinephrine, neurohormones in endocrine and nervous tissue. To evaluate the consequences of loss of Atp7B on the function of DBH, the level of proteins in adrenal gland were compared between normal mice and mice containing a null mutation in the ATP7B gene. The levels of DBH, as well as another vesicular protein, chromogranin A, are reduced in the ATP7B−/− mice. In addition to the lower level of enzyme, the products of DBH catalytic activity, norepinephrine and epinephrine, are also decreased. Although these changes are a consequence of ATP7B gene function, Atp7B mRNA is not normally expressed in the adrenal gland. Instead, Atp7A mRNA is present. The levels of copper and DBH RNA within adrenals of the ATP7B−/− mice are not different from the wild type. The results of these experiments suggest that copper-requiring enzymes are affected by a loss of ATP7B even in tissue not normally expressing this protein. Therefore the multisystemic effects observed in Wilson disease, the human disorder characterized by mutation in ATP7B, may be a secondary consequence of the major accumulation of copper in liver.

A Negative Regulatory Element in the Rat Dopamine β-Hydroxylase Gene Contributes to the Cell Type Specificity of Expression

Abstract: Dopamine β‐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.

Phox2 and dHAND transcription factors select shared and unique target genes in the noradrenergic cell type.

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 β-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.