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Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites.

ABSTRACT: Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons and neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with the HD-binding site residing within a composite promoter of the human DBH gene, designated domain IV, in a cell-specific manner and directly controls noradrenergic-specific DBH promoter activity. In this report, we demonstrate that three additional protein-binding sites (i.e., domains I, II, and III) between domain IV and the TATA box are critical for intact DBH promoter activity in noradrenergic cells and that they activate DBH transcription in a highly concerted manner. Transient transfection assays of mutant DBH reporter constructs indicated that domain I was active in every cell line tested, whereas domain III was preferentially active in DBH-positive cells. Remarkably, mutation of domain II was associated with inactivation of DBH promoter activity exclusively in DBH-positive cell lines, defining it as another noradrenergic-specific promoter element. The cell-specific profile of the promoter function of these sequence motifs was further supported by in vitro DNA-binding studies and Southwestern analysis. Furthermore, competition and antibody supershift assays show that transcription factors Sp1 and AP2 are the cognate nuclear factors interacting with domains I and III, respectively. Parallel evidence indicates that domain II is another Phox2a-binding site, demonstrating at least two binding sites for this factor in the upstream DBH promoter. Strikingly, four tandem copies of domain II increased the promoter activity of a minimal DBH promoter by 100- to 200-fold in DBH-positive cell lines without compromising cell specificity. Cotransfection of Phox2a-expression vector dramatically increased the activity of the multiple domain II promoter only in DBH-negative cell lines, confirming that domain II is responsive to Phox2a. Collectively, this study emphasizes a critical role of Phox2a as well as its functional synergism with other transcription factors (e.g., CREB, AP2, and Sp1) in transcriptional activation of the DBH gene.

SUBMITTER: Kim HS

PROVIDER: S-EPMC6792837 | biostudies-literature | 1998 Oct

REPOSITORIES: biostudies-literature

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Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites.

Kim H S HS Seo H H Yang C C Brunet J F JF Kim K S KS

The Journal of neuroscience : the official journal of the Society for Neuroscience 19981001 20

Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons and neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with the HD-binding site residing within a composite promoter of the human DBH gene, designated domain IV, in a cell-specific manner and directly controls noradrenergic-specific DBH promoter activity. In this report, ...[more]

PMID: 9763470

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Project description:IntroductionDisulfiram has been claimed to be useful in cocaine addiction therapy, its efficacy being attributed to dopamine-beta-hydroxylase (DBH) inhibition. Our previous results indicate that disulfiram and the selective DBH inhibitor nepicastat increase extracellular dopamine (DA) in the rat medial prefrontal cortex (mPFC), and markedly potentiated cocaine-induced increase. Concomitantly, in rats with cocaine self-administration history, cocaine-seeking behavior induced by drug priming was prevented, probably through overstimulation of D1 receptors due to the DA increase. The present research was aimed at studying the neurochemical mechanisms originating the enhanced DA release.MethodsNoradrenergic system ablation was attained by intracerebroventricular (i.c.v.) administration of the neurotoxin anti-DBH-saporin (aDBH-sap). DA, noradrenaline (NA), and DOPAC were assessed by HPLC after ex vivo tissue extraction or in vivo microdialysis. Control and denervated rats were subjected to microdialysis in the mPFC and caudate nucleus to evaluate the effect of nepicastat-cocaine combination on extracellular DA levels and their regulation by α2-adrenoceptors.ResultsFifteen days after neurotoxin or its vehicle administration, tissue and extracellular NA were reduced to less than 2% the control value, while extracellular DA was increased by approximately 100%. In control rats, nepicastat given alone and in combination with cocaine increased extracellular DA by about 250% and 1100%, respectively. In denervated rats, nepicastat slightly affected extracellular DA, while in combination with cocaine increased extracellular DA by 250%. No differences were found in the caudate nucleus. Clonidine almost totally reversed the extracellular DA elevation produced by nepicastat-cocaine combination, while it was ineffective in denervated rats.ConclusionsThis research shows that the increase of extracellular DA produced by nepicastat alone or in combination with cocaine was prevented by noradrenergic denervation. The results indicate that nepicastat enhances DA release from noradrenergic terminals supposedly by removing NA from α2-autoreceptors. In addition to the inhibition of DA uptake, the latter mechanism may explain the synergistic effect of cocaine on nepicastat-induced DA release.

Project description:Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency phenotypes, but did not reveal an impact of the rs11115 variant on DBH expression in mice.

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Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites.

Publications

Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites.

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