Genomic Structure and Functional Characterization of NBPhox (PMX2B), a Homeodomain Protein Specific to Catecholaminergic Cells That Is Involved in Second Messenger-Mediated Transcriptional Activation

doi:10.1006/geno.1999.5845

Genomics

Volume 59, Issue 1, 1 July 1999, Pages 40-50

https://doi.org/10.1006/geno.1999.5845 Get rights and content

Abstract

Homeodomain proteins play essential roles in basic processes during embryogenesis and development. NBPhox, a vertebrate paired-like homeodomain protein specific to catecholaminergic cells, may have a fundamental role in determining and maintaining the catecholaminergic phenotype. Here we describe the human and mouse genomic structures and the functional characterization of NBPhox. The genomic structure of NBPhox is highly conserved at the nucleotide level between human and mouse and is also quite similar to that of other paired-like homeodomain proteins, Arix/Phox2a, CRX, OTX1, and OTX2. The human NBPhox gene (HGMW-approved symbol PMX2B) maps to chromosomal region 5p12–p13. When the NBPhox expression plasmid was introduced into PC12h cells, the transcription from the promoter of the dopamine β-hydroxylase (DBH) gene was slightly stimulated. However, NBPhox substantially enhances second messenger-mediated activation of the DBH promoter by forskolin and/or phorbol ester. Furthermore, we found that NBPhox can also enhance second messenger-mediated activation of the c-fos promoter and several enhancers, including cyclic AMP-response element, the binding site for activator protein 1, and serum-response element. Our findings provide strong evidence that a homeodomain protein is involved in the activation of several genetic regulatory elements responsive to second messenger-mediated signals. These data suggest that this family of proteins may be involved in determining and maintaining a cell-specific phenotype through regulation of certain genes responsive to second messenger-mediated signals.

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Association analysis of the PHOX2B gene with Hirschsprung disease in the Han Chinese population of Southeastern China
2009, Journal of Pediatric Surgery
Hirschsprung disease (HSCR, OMIM 142623) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. The PHOX2B gene is involved in neurogenesis and disruption of Phox2b in mice results in a HSCR-like phenotype. The first association study of the PHOX2B gene with HSCR derived from Chinese population in Hong Kong; here, we address the question of whether PHOX2B acts as a predisposing factor in HSCR pathogenesis in Chinese population in mainland.
To investigate the contribution of PHOX2B to the HSCR phenotype, polymerase chain reaction amplification and direct sequencing were used to screen PHOX2B coding regions and intron/exon boundaries for mutations and polymorphisms in 102 patients with HSCR and 96 ethnically matched controls, in Han Chinese populations of Southeastern China.
In this study, we genotyped 4 single nucleotide polymorphisms (SNPs) (including 1 novel SNP) located within the PHOX2B gene. Statistically significant differences were found for c.701 A > G and IVS2 + 100 A > G, and the log-additive model was accepted as the best inheritance model (odds ratio [OR], 1.79; 95% confidence interval [CI], 1.11-2.87) for IVS2 + 100 A > G. We also showed that the haplotype-A G A N composed of 4 SNPs exhibited significant association with the disease (P = .03); this haplotype was more frequently observed in cases than in controls (OR, 2.31; 95% CI, 1.11-4.82).
Our study provided further evidence that the PHOX2B gene is involved in the susceptibility to HSCR in the Han Chinese population. Our findings are in accordance with the involvement of PHOX2B in the signaling pathways governing the development of enteric neurons.
PHOX2B in respiratory control: Lessons from congenital central hypoventilation syndrome and its mouse models
2009, Respiratory Physiology and Neurobiology
Phox2b is a master regulator of visceral reflex circuits. Its role in the control of respiration has been highlighted by the identification of heterozygous PHOX2B mutations as the cause of Central Congenital Hypoventilation Syndrome (CCHS), a rare disease defined by the lack of CO₂ responsiveness and of breathing automaticity in sleep. Phox2b^27Ala/+ mice that bear a frequent CCHS-causing mutation do not respond to hypercapnia and die in the first hour after birth from central apnoea. They are therefore a reliable animal model for CCHS. Neurons of the retrotrapezoïd nucleus/parafacial respiratory group (RTN/pFRG) were found severely depleted in these mice and no other neuronal loss could be identified. Physiological experiments show that RTN/pFRG neurons are crucial to driving proper breathing at birth and are necessary for central chemoreception and the generation of a normal respiratory rhythm. To date, the reason for the selective vulnerability of RTN/pFRG neurons to PHOX2B protein dysfunction remains unexplained.
PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome
2005, American Journal of Human Genetics
The Phox2b gene is necessary for autonomic nervous-system development. Phox2b^−/− mice die in utero with absent autonomic nervous system circuits, since autonomic nervous system neurons either fail to form or degenerate. We first identified the Phox2b human ortholog, PHOX2B, as the gene underlying congenital central hypoventilation syndrome (CCHS, or Ondine curse), with an autosomal dominant mode of inheritance and de novo mutation at the first generation. We have subsequently shown that heterozygous mutations of PHOX2B may account for several combined or isolated disorders of autonomic nervous-system development—namely, tumors of the sympathetic nervous system (TSNS), such as neuroblastoma and late-onset central hypoventilation syndrome. Here, we report the clinical and molecular assessments of a cohort of 188 probands with CCHS, either isolated or associated with Hirschsprung disease and/or TSNS. The mutation-detection rate was 92.6% (174/188) in our series, and the most prevalent mutation was an in-frame duplication leading to an expansion of +5 to +13 alanines in the 20-alanine stretch at the carboxy terminal of the protein. Such findings suggest PHOX2B mutation screening as a simple and reliable tool for the diagnosis of CCHS, independent of the clinically variable phenotype. In addition, somatic mosaicism was detected in 4.5% of parents. Most interestingly, analysis of genotype-phenotype interactions strongly supports the contention that patients with CCHS who develop malignant TSNS will harbor either a missense or a frameshift heterozygous mutation of the PHOX2B gene. These data further highlight the link between congenital malformations and tumor predisposition when a master gene in development is mutated.
Molecular cloning and characterization of the promoter region of the human Phox2b gene
2004, Molecular Brain Research
The closely related homeodomain transcription factors, Phox2a and Phox2b, are restrictively expressed in central and peripheral noradrenergic (NA) neurons in an overlapping but distinct manner, and critically regulate the differentiation and neurotransmitter identity of NA neurons. The structure and function of the human Phox2a (hPhox2a) promoter has recently been reported. Towards the long-term goal of delineating the regulatory cascade of NA neuron differentiation, we isolated a human Phox2b (hPhox2b) genomic clone encompassing approximately 7.8 kb of the 5′ upstream promoter region, the entire exon–intron structure and 4.5 kb of the 3′ flanking region. Two transcription start sites are identified to reside 115 and 110 nucleotides upstream of the start codon, based on both primer extension and 5′-rapid amplification of the cDNA ends analyses. In addition, transient transfection assays indicate that 1.1 kb or longer upstream sequences of the hPhox2b gene may confer cell type-specific gene expression in certain, but not all cell lines. The promoter activity of the hPhox2b gene is modestly transactivated by forced co-expression of Phox2b and the hPhox2b gene promoter contains a high-affinity binding site at −320 to −295 bp. This study provides a frame to further elucidate the molecular mechanisms underlying the regulation of Phox2a and Phox2b gene expression and its relation to NA differentiation.
Germline Mutations of the Paired-Like Homeobox 2B (PHOX2B) Gene in Neuroblastoma
2004, American Journal of Human Genetics
Neuroblastoma (NB) is a frequent pediatric tumor for which recurrent somatic rearrangements are known. Germline mutations of predisposing gene(s) are suspected on the basis of rare familial cases and the association of NB with other genetically determined congenital malformations of neural crest–derived cells—namely, Hirschsprung disease (HSCR) and/or congenital central hypoventilation syndrome (CCHS). We recently identified the paired–like homeobox 2B (PHOX2B) gene as the major disease-causing gene in isolated and syndromic CCHS, which prompted us to regard it as a candidate gene in NB. Here, we report on germline mutations of PHOX2B in both a familial case of NB and a patient with the HSCR-NB association. PHOX2B, therefore, stands as the first gene for which germline mutations predispose to NB.
Expression of Phox2 transcription factors and induction of the dopaminergic phenotype in primary sensory neurons
2002, Molecular and Cellular Neuroscience
Cell-type-specific transcription factors may regulate phenotypic diversity by conferring selective responsiveness to relatively nonspecific environmental cues. To test this hypothesis, we examined whether the homeodomain transcription factors Phox2a/2b play a role in activity-dependent expression of the dopaminergic phenotype using petrosal ganglion (PG) sensory neurons as a model. The timing of Phox2a/2b expression is precisely correlated with the ability of PG neurons to express the dopamine-synthesizing enzyme, tyrosine hydroxylase (TH), in response to depolarizing stimuli. Phox2a/2b expression is highest at embryonic day 16.5, when virtually all PG neurons exhibit activity-dependent TH induction, and subsequently falls in parallel with the loss of activity-dependent TH induction. Expression is maintained, however, in all dopaminergic neurons. Physiologic stimulation of PG neurons in vivo induces TH expression exclusively in Phox2a/2b⁺ cells. Our data suggest that constitutive expression of Phox2a/2b defines the potential of neurons to become dopaminergic in response to membrane depolarization during a critical window of phenotypic plasticity.

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^☆: Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession Nos. AB015671 and AB015672.

¹: These authors contributed equally to this work.

²: To whom correspondence should be addressed at Pharmaceutical Frontier Research Laboratories, Japan Tobacco, Inc., 13-2, Fukuura 1-chome, Kanazawa-ku, Yokohama, Kanagawa 236, Japan. Telephone: 81-45-786-7693. Fax.: 81-45-786-7692. E-mail: [email protected].

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Regular ArticleGenomic Structure and Functional Characterization of NBPhox (PMX2B), a Homeodomain Protein Specific to Catecholaminergic Cells That Is Involved in Second Messenger-Mediated Transcriptional Activation☆

Abstract

Trends Genet.

Curr. Opin. Neurobiol.

Cell

Cell

Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Genomics

Genomics

Neuron

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Cell. Biol.

Brain Res. Mol. Brain Res.

Epilepsy and brain abnormalities in mice lacking the Otx1 gene

Nat. Genet.

Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation

Development

Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription

Mol. Cell. Biol.

Patterning of the Drosophila embryo by a homeodomain-deleted Ftz polypeptide

Nature

Pax genes and organogenesis

BioEssays

Regular Article
Genomic Structure and Functional Characterization of NBPhox (PMX2B), a Homeodomain Protein Specific to Catecholaminergic Cells That Is Involved in Second Messenger-Mediated Transcriptional Activation☆