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Kwangsoo Kim - One of the best experts on this subject based on the ideXlab platform.

  • Trim11 increases expression of dopamine β-hydroxylase gene by interacting with Phox2b
    Biochemical and biophysical research communications, 2008
    Co-Authors: Seok Jong Hong, Han Chae, Thomas Lardaro, Sunghoi Hong, Kwangsoo Kim
    Abstract:

    The homeodomain transcription factor Phox2b is one of the key determinants involved in the development of noradrenergic (NA) neurons in both the central nervous system (CNS) and the peripheral nervous system (PNS). Using yeast two-hybrid screening, we isolated a Phox2b interacting protein, Trim11, which belongs to TRIM (Tripartite motif) or RBCC proteins family, and contains a RING domain, B-boxes, a coiled-coil domain, and the B30.2/SPRY domain. Protein-protein interaction assays showed that Phox2b was able to physically interact with Trim11. The B30.2/SPRY domain of Trim11 was required for the interaction with Phox2b. Expression of Phox2b and Trim11 was detected in the sympathetic ganglia (SG) of mouse embryos. Forced expression of Trim11 with Phox2b further increased mRNA levels of dopamine beta-hydroxylase (DBH) gene in primary avian neural crest stem cell (NCSC) culture. This study suggests a potential role for Trim11 in the specification of NA phenotype by interaction with Phox2b.

  • the camp pathway in combination with bmp2 regulates PHOX2A transcription via camp response element binding sites
    Journal of Biological Chemistry, 2006
    Co-Authors: Chutamas Benjanirut, Seok Jong Hong, Kwangsoo Kim, Maryline Paris, Wenhorng Wang, Ronald L Hullinger, Ourania M Andrisani
    Abstract:

    Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor PHOX2A, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on PHOX2A transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPHOX2A-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous PHOX2A expression, the 7.5-kb hPHOX2A reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPHOX2A fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPHOX2A promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous PHOX2A transcription and prolonged CREB phosphorylation, indicating PHOX2A chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine PHOX2A promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to PHOX2A-luciferase reporters. We conclude these CREs are functional, with CREB directly activating PHOX2A transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on PHOX2A transcription.

  • age associated changes in mrna levels of phox2 norepinephrine transporter and dopamine β hydroxylase in the locus coeruleus and adrenal glands of rats
    Journal of Neurochemistry, 2005
    Co-Authors: Mengyang Zhu, Weiping Wang, Abiye H Iyo, Gregory A Ordway, Kwangsoo Kim
    Abstract:

    Age-related changes in the gene expression of the transcription factors, PHOX2A and 2b, and two marker proteins, norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), of noradrenergic neurons were characterized in the locus coeruleus (LC) and adrenal glands using in situ hybridization. Analysis of changes was performed in rats that were 1–23 months of age. Compared to 1-month-old rats, there was a 62% increase of PHOX2A messenger RNA (mRNA) in the LC of 3-month-old rats, and a decline of 37% in 23-month-old rats. In contrast, levels of Phox2b mRNA in the LC remained unchanged in 3-month-old rats, but declined to a 30% reduction in 23-month-old rats. Interestingly, mRNA levels of NET in the LC decreased with increasing age to a reduction of 29%, 30% and 43% in 3-, 8- and 23-month-old rats, respectively. Similarly, DBH mRNA in the LC declined with increasing age to a 56% reduction in 23-month-old rats. mRNA levels of PHOX2A, Phox2b, NET and DBH in the adrenal medulla of 23-month-old rats were significantly lower than those of 1-month-old rats. Semi-quantitative reverse transcription assays of the same genes yielded data similar to in situ hybridization experiments, with β-actin mRNA levels being unchanged across the ages. Taken together, these data reveal that reduced Phox2 mRNAs in the LC and adrenal medulla of aging rats are accompanied by a coincidental decline in mRNA levels of NET and DBH and suggest a possible relationship between Phox2 genes and the marker genes in noradrenergic neurons after birth.

  • genetically engineered dopamine β hydroxylase gene promoters with better phox2 binding sites drive significantly enhanced transgene expression in a noradrenergic cell specific manner
    Molecular Therapy, 2005
    Co-Authors: Dong Youn Hwang, Michelle M Hwang, Hansoo Kim, Kwangsoo Kim
    Abstract:

    A continuously growing body of evidence suggests that dysregulation of noradrenergic (NA) neurons is implicated in the etiology and pathophysiology of various human diseases such as depression, drug addiction, and autonomic dysfunction. An efficient NA neuron-specific promoter is potentially valuable to investigate the precise role of NA neurons in normal as well as in diseased brain and to treat the associated disorders by gene therapy. In this study, we tested a novel strategy to modify genetically the promoter of the human dopamine β-hydroxylase (hDBH) gene to overcome its inherent weakness while maintaining its cell-type specificity. We optimized the nucleotide sequence motifs of PHOX2-binding sites (PRS2 and PRS3) residing within the hDBH promoter. Optimization of both PRS2 and PRS3 motifs significantly increased their binding affinities to PHOX2A, leading to a dramatic increase in the promoter strength (>20-fold). More importantly, these modifications do not alter the level of transgene expression in non-NA cells either in vitro or in vivo, demonstrating tight cell-type specificity. This work shows that a cellular gene promoter can be genetically modified to strengthen its promoter activity without losing cell-type specificity by optimizing critical cis-regulatory elements. Our genetically engineered promoter may be useful for cell-type-specific gene targeting as well as for generating in vivo animal models with altered gene expression in a specific cell type.

  • molecular cloning and characterization of the promoter region of the human phox2b gene
    Molecular Brain Research, 2004
    Co-Authors: Seok Jong Hong, Han Chae, Kwangsoo Kim
    Abstract:

    Abstract 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.

Diego Fornasari - One of the best experts on this subject based on the ideXlab platform.

  • PHOX2A and phox2b are differentially regulated during retinoic acid driven differentiation of sk n be 2 c neuroblastoma cell line
    Experimental Cell Research, 2016
    Co-Authors: Simona Di Lascio, Elena Saba, Debora Belperio, Andrea Raimondi, Helen Lucchetti, Diego Fornasari, Roberta Benfante
    Abstract:

    PHOX2B and its paralogue gene PHOX2A are two homeodomain proteins in the network regulating the development of autonomic ganglia that have been associated with the pathogenesis of neuroblastoma (NB), because of their over-expression in different NB cell lines and tumour samples. We used the SK-N-BE(2)C cell line to show that all-trans retinoic acid (ATRA), a drug that is widely used to inhibit growth and induce differentiation in NBs, regulates both PHOX2A and PHOX2B expression, albeit by means of different mechanisms: it up-regulates PHOX2A and down-regulates PHOX2B. Both mechanisms act at transcriptional level, but prolonged ATRA treatment selectively degrades the PHOX2A protein, whereas the corresponding mRNA remains up-regulated. Further, we show that PHOX2A is capable of modulating PHOX2B expression, but this mechanism is not involved in the PHOX2B down-regulation induced by retinoic acid. Our findings demonstrate that PHOX2A expression is finely controlled during retinoic acid differentiation and this, together with PHOX2B down-regulation, reinforces the idea that they may be useful biomarkers for NB staging, prognosis and treatment decision making.

  • Alanine expansions associated with congenital central hypoventilation syndrome impair PHOX2B homeodomain-mediated dimerization and nuclear import
    'American Society for Biochemistry & Molecular Biology (ASBMB)', 2016
    Co-Authors: Simona Di Lascio, Debora Belperio, Roberta Benfante, Diego Fornasari
    Abstract:

    Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to congenital central hypoventilation syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA binding, transcriptional activity, and nuclear localization and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism but also involves a dominant negative effect and/or toxic gain of function for PHOX2B mutations. Because PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study, we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import, and our data clearly show that the expanded C terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity

  • transcriptional dysregulation and impairment of phox2b auto regulatory mechanism induced by polyalanine expansion mutations associated with congenital central hypoventilation syndrome
    Neurobiology of Disease, 2013
    Co-Authors: Simona Di Lascio, T. Bachetti, Elena Saba, Roberta Benfante, Isabella Ceccherini, Diego Fornasari
    Abstract:

    The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a polyalanine repeat expansion mutation involving a polyalanine tract of twenty residues in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. As one of the targets of PHOX2B is the PHOX2B gene itself, we tested the transcriptional activity of wild-type and mutant proteins on the PHOX2B gene promoter, and found that the transactivation ability of proteins with polyalanine expansions decreased as a function of the length of the expansion, whereas DNA binding was severely affected only in the case of the mutant with the longest polyalanine tract (+13 alanine). Co-transfection experiments using equimolar amounts of PHOX2B wild-type and mutant proteins in order to simulate a heterozygous state in vitro and four different PHOX2B target gene regulatory regions (PHOX2B, PHOX2A, DBH, TLX2) clearly showed that the polyalanine expanded proteins alter the transcriptional activity of wild-type protein in a promoter-specific manner, without any clear correlation with the length of the expansion. Moreover, although reduced transactivation may be caused by retention of the wild-type protein in the cytoplasm or in nuclear aggregates, this mechanism can only be partially responsible for the pathogenesis of CCHS because of the reduction in cytoplasmic and nuclear accumulation when the +13 alanine mutant is co-expressed with wild-type protein, and the fact that the shortest polyalanine expansions do not form visible cytoplasmic aggregates. Deletion of the C-terminal of PHOX2B leads to a protein that correctly localizes in the nucleus but impairs PHOX2B wild-type transcriptional activity, thus suggesting that protein mislocalization is not the only mechanism leading to CCHS. The results of this study provide novel in vitro experimental evidence of a transcriptional dominant-negative effect of PHOX2B polyalanine mutant proteins on wild-type protein on two different PHOX2B target genes.

  • The E3 ubiquitin ligase TRIM11 mediates the degradation of congenital central hypoventilation syndrome-associated polyalanine-expanded PHOX2B
    Journal of Molecular Medicine, 2012
    Co-Authors: Sara Parodi, T. Bachetti, Simona Di Lascio, Diego Fornasari, Eleonora Zanni, Paola Bocca, Ignazia Prigione, Maria Pennuto, Isabella Ceccherini
    Abstract:

    Expansions of a polyalanine (polyA) stretch in the coding region of the PHOX2B gene cause congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by the absence of adequate control of autonomic breathing. Expansion of polyA in PHOX2B leads to protein misfolding and accumulation into inclusions. The mechanisms that regulate mutant protein degradation and turnover have been poorly elucidated. Here, we investigate the regulation of degradation of wild-type and polyA-expanded PHOX2B. We show that expanded PHOX2B is targeted for degradation through the ubiquitin–proteasome system, resulting in lowered levels of the mutant protein relative to its wild-type counterpart. Moreover, we show that mutant PHOX2B forms ubiquitin-positive inclusions, which sequester wild-type PHOX2B. This sequestration correlates with reduced transcriptional activity of endogenous wild-type protein in neuroblastoma cells. Finally, we show that the E3 ubiquitin ligase TRIM11 plays a critical role in the clearance of mutant PHOX2B through the proteasome. Importantly, clearance of mutant PHOX2B by TRIM11 correlates with a rescue of PHOX2B transcriptional activity. We propose that CCHS is partially caused by a dominant-negative effect of expanded PHOX2B due to the retention of the wild-type protein in pathogenic aggregates. Our results demonstrate that TRIM11 is a novel modifier of mutant PHOX2B toxicity and represents a potential therapeutic target for CCHS.

  • phox2b mediated regulation of alk expression in vitro identification of a functional relationship between two genes involved in neuroblastoma
    PLOS ONE, 2010
    Co-Authors: T. Bachetti, Simona Di Lascio, Daniela Di Paolo, Valentina Mirisola, Chiara Brignole, Marta Bellotti, Irene Caffa, Chiara Ferraris, M Fiore, Diego Fornasari
    Abstract:

    Background Neuroblastoma (NB) is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and the transcription factor Paired-like Homeobox 2B (PHOX2B), in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis. Methodology/Principal Findings Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target. Conclusions/Significance These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.

Jeanfrancois Brunet - One of the best experts on this subject based on the ideXlab platform.

  • autonomic neurocristopathy associated mutations in phox2b dysregulate sox10 expression
    Journal of Clinical Investigation, 2012
    Co-Authors: Mayumi Nagashimada, Jeanne Amiel, Jeanfrancois Brunet, Hiroshi Ohta, Kazuki Nakao, Toshihiro Uesaka, Teruhiko Wakayama, Hideki Enomoto
    Abstract:

    The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine β-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

  • forced expression of phox2 homeodomain transcription factors induces a branchio visceromotor axonal phenotype
    Developmental Biology, 2007
    Co-Authors: Marierose Hirsch, Jeanfrancois Brunet, Joel C Glover, Heloise D Dufour, Christo Goridis
    Abstract:

    What causes motor neurons to project into the periphery is not well understood. We here show that forced expression of the homeodomain protein Phox2b, shown previously to be necessary and sufficient for branchio-visceromotor neuron development, and of its paralogue PHOX2A imposes a branchiomotor-like axonal phenotype in the spinal cord. Many Phox2-transfected neurons, whose axons would normally stay within the confines of the neural tube, now project into the periphery. Once outside the neural tube, a fraction of the ectopic axons join the spinal accessory nerve, a branchiomotor nerve which, as shown here, does not develop in the absence of Phox2b. Explant studies show that the axons of Phox2-transfected neurons need attractive cues to leave the neural tube and that their outgrowth is promoted by tissues, to which branchio-visceromotor fibers normally grow. Hence, Phox2 expression is a key step in determining the peripheral axonal phenotype and thus the decision to stay within the neural tube or to project out of it.

  • essential role of gata transcription factors in sympathetic neuron development
    Development, 2004
    Co-Authors: Konstantina Tsarovina, Alexandre Pattyn, Jeanfrancois Brunet, Jutta Stubbusch, F Muller, Jacqueline Van Der Wees, Christoph Schneider, Hermann Rohrer
    Abstract:

    Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, Phox2b, Hand2 and PHOX2A. Here, we have studied the function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, Phox2b, Hand2 and PHOX2A expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of Phox2b, PHOX2A, Hand2 and Mash1. Together with the lack of Gata2/3 expression in Phox2b knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development.

  • coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of cns progenitors
    Genes & Development, 2003
    Co-Authors: Alexandre Pattyn, Jeanfrancois Brunet, Omar Abdel Samad, Filippo M Rijli, Anna Vallstedt, Jose M Dias, Robb Krumlauf, Johan Ericson
    Abstract:

    Neural progenitor cells often produce distinct types of neurons in a specific order, but the determinants that control the sequential generation of distinct neuronal subclasses in the vertebrate CNS remain poorly defined. We examined the sequential generation of visceral motor neurons and serotonergic neurons from a common pool of neural progenitors located in the ventral hindbrain. We found that the temporal specification of these neurons varies along the anterior-posterior axis of the hindbrain, and that the timing of their generation critically depends on the integrated activities of Nkx- and Hox-class homeodomain proteins. A primary function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. These findings assign new roles for Nkx, Hox, and Phox2 proteins in the control of temporal neuronal fate determination, and link spatial and temporal patterning of CNS neuronal fates.

  • paired like homeodomain proteins PHOX2A and phox2b are responsible for noradrenergic cell specific transcription of the dopamine β hydroxylase gene
    Journal of Neurochemistry, 2002
    Co-Authors: Chunying Yang, Jeanfrancois Brunet, Hyemyung Seo, Heesun Kim, Chunhyung Kim, Kwangsoo Kim
    Abstract:

    Abstract: Recently, a murine paired-like homeobox gene, PHOX2A, has been identified whose product is critical for the development of several major noradrenergic neuron populations, including the locus coeruleus. In noradrenergic neurons, dopamine β-hydroxylase (DBH) is a hallmark protein and catalyzes the conversion of dopamine to noradrenaline. Our previous studies have shown that a composite promoter (domain IV), residing at −185 to −150 bp upstream of the transcription start site, is critical for DBH transcription and is comprised of multiple cis-acting elements, including a cyclic AMP response element, a YY1 binding site, and two core motifs of the homeodomain (HD)-binding site. Here, we show that the HD-binding site residing within domain IV is a noradrenergic-specific cis-acting element. In contrast, the cyclic AMP response element is active in all cell lines tested. We provide evidence that PHOX2A is expressed only in DBH-positive cell lines and interacts with the HD-binding site. Forced expression of PHOX2A robustly activates DBH promoter activity in DBH-negative cell lines (>10-fold), but increased it only marginally (<50%) in DBH-positive cell lines. Furthermore, another protein factor with an identical HD, Phox2b, also activates DBH transcription with an efficiency comparable to that of PHOX2A. In contrast, neither PHOX2A nor Phox2b was able to transactivate tyrosine hydroxylase transcription, indicating that these transcription factors differentially activate catecholamine-synthesizing gene transcription. Together with the PHOX2A knockout experiment, the studies described here make PHOX2A and Phox2b the first strong candidate transcription factors for determining a neurotransmitter phenotype in vertebrates.

Hermann Rohrer - One of the best experts on this subject based on the ideXlab platform.

  • neuroblastoma phox2b variants stimulate proliferation and dedifferentiation of immature sympathetic neurons
    The Journal of Neuroscience, 2010
    Co-Authors: Tobias Reiff, Konstantina Tsarovina, Afsaneh Majdazari, Mirko Schmidt, Isabel Del Pino, Hermann Rohrer
    Abstract:

    Neuroblastoma is a pediatric tumor that is thought to arise from autonomic precursors in the neural crest. Mutations in the PHOX2B gene have been observed in familial and sporadic forms of neuroblastoma and represent the first defined genetic predisposition for neuroblastoma. Here, we address the mechanisms that may underlie this predisposition, comparing the function of wild-type and mutant Phox2b proteins ectopically expressed in proliferating, embryonic sympathetic neurons. Phox2b displays a strong antiproliferative effect, which is lost in all Phox2b neuroblastoma variants analyzed. In contrast, an increase in sympathetic neuron proliferation is elicited by Phox2b variants with mutations in the homeodomain when endogenous Phox2b levels are lowered by siRNA-mediated knockdown to mimic the situation of heterozygous PHOX2B mutations in neuroblastoma. The increased proliferation is blocked by Hand2 knockdown and the antiproliferative Phox2b effects are rescued by Hand2 overexpression, implying Hand2 in Phox2b-mediated proliferation control. A Phox2b variant with a nonsense mutation in the homeodomain elicits, in addition, a decreased expression of characteristic marker genes. Together, these results suggest that PHOX2B mutations predispose to neuroblastoma by increasing proliferation and promoting dedifferentiation of cells in the sympathoadrenergic lineage.

  • the bhlh transcription factor hand2 is essential for noradrenergic differentiation of sympathetic neurons
    Development, 2006
    Co-Authors: Marsha E Lucas, F Muller, Roland Rudiger, Paul D Henion, Hermann Rohrer
    Abstract:

    The basic helix-loop-helix transcription factor Hand2 , together with Ascl1, PHOX2A, Phox2b and Gata2/Gata3 , is induced by bone morphogenetic proteins in neural crest-derived precursor cells during sympathetic neuron generation. Hand2 overexpression experiments and the analysis of its function at the Dbh promotor implicated Hand2 in the control of noradrenergic gene expression. Using the zebrafish hand2 deletion mutant hands off , we have now investigated the physiological role of hand2 in the development of sympathetic ganglia. In hands off mutant embryos, sympathetic precursor cells aggregate to form normal sympathetic ganglion primordia characterized by the expression of phox2b, PHOX2A and the achaete-scute family member zash1a/ascl1 . The expression of the noradrenergic marker genes th and dbh is strongly reduced, as well as the transcription factors gata2 and tfap2a ( Ap-2 α). By contrast, generic neuronal differentiation seems to be unaffected, as the expression of elavl3 (HuC) is not reduced in hands off sympathetic ganglia. These results demonstrate in vivo an essential and selective function of hand2 for the noradrenergic differentiation of sympathetic neurons, and implicates tfap2a and gata2 as downstream effectors.

  • essential role of gata transcription factors in sympathetic neuron development
    Development, 2004
    Co-Authors: Konstantina Tsarovina, Alexandre Pattyn, Jeanfrancois Brunet, Jutta Stubbusch, F Muller, Jacqueline Van Der Wees, Christoph Schneider, Hermann Rohrer
    Abstract:

    Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, Phox2b, Hand2 and PHOX2A. Here, we have studied the function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, Phox2b, Hand2 and PHOX2A expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of Phox2b, PHOX2A, Hand2 and Mash1. Together with the lack of Gata2/3 expression in Phox2b knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development.

  • Interaction of Mash1 and Phox2b in sympathetic neuron development.
    Molecular and cellular neurosciences, 2004
    Co-Authors: Matthias Stanke, Jutta Stubbusch, Hermann Rohrer
    Abstract:

    The transcription factors Mash1 and Phox2b are both essential for sympathetic neuron development. To understand in more detail their function and interaction, Phox2b and Mash1 were ectopically expressed in vivo, in peripheral nerve precursors. Here, we demonstrate that the Phox2b-induced generation of ectopic noradrenergic neurons in chick peripheral nerve involves the induction of Cash1, the chick homolog of Mash1. All Phox2-induced neurons coexpress the noradrenergic marker genes TH and DBH. Conversely, Mash1 induces neuronal differentiation characterized by the expression of generic neuronal genes SCG10, Hu and NF160; however, only a subpopulation of these neurons also displays an autonomic, noradrenergic phenotype. This context-dependent action of Mash1 implicates autonomic codeterminants, required for noradrenergic differentiation in response to Mash1. In contrast, Phox2b coordinates generic and noradrenergic gene expression, recruiting Mash1/Cash1, which may have a major function in the control of pan-neuronal gene expression during noradrenergic neuron development.

  • bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons
    Neuron, 1999
    Co-Authors: Carolin Schneider, Helmut Wicht, Jana Enderich, Michael Wegner, Hermann Rohrer
    Abstract:

    Abstract Bone morphogenetic proteins (BMPs) induce autonomic neurogenesis in neural crest cultures and stimulate sympathetic neuron development when overexpressed in vivo. We demonstrate that inhibition of BMPs in the chick embryo by the BMP antagonist Noggin prevents sympathetic neuron generation. In Noggin-treated embryos, the noradrenergic marker genes tyrosine hydroxylase ( TH ) and dopamine-β-hydroxylase ( DBH ), panneuronal neurofilament 160 ( NF160 ) and SCG10 genes, and the transcriptional regulators Phox2b and PHOX2A are not expressed in sympathetic ganglia. Whereas initial ganglion development is not affected, the expression of the basic helix–loop–helix transcription factor Cash-1 is strongly reduced. These results demonstrate that BMPs are essential for sympathetic neuron development and establish Cash-1 and Phox2 genes as downstream effectors of BMPs in this lineage.

Ourania M Andrisani - One of the best experts on this subject based on the ideXlab platform.

  • time dependent activation of PHOX2A by the cyclic amp pathway modulates onset and duration of p27kip1 transcription
    Molecular and Cellular Biology, 2009
    Co-Authors: Min Hwa Shin, Wenhorng Wang, Nirmala Mavila, Sasha Vega Alvarez, Mark C Hall, Ourania M Andrisani
    Abstract:

    In noradrenergic progenitors, PHOX2A mediates cell cycle exit and neuronal differentiation by inducing p27(Kip1) transcription in response to activation of the cyclic AMP (cAMP) pathway. The mechanism of cAMP-mediated activation of PHOX2A is unknown. We identified a cluster of phosphoserine-proline sites in PHOX2A by mass spectrometry. Ser206 appeared to be the most prominent phosphorylation site. A phospho-Ser206 PHOX2A antibody detected dephosphorylation of PHOX2A that was dependent on activation of the cAMP pathway, which occurred prior to neuronal differentiation of noradrenergic CAD cells. Employing serine-to-alanine and serine-to-aspartic acid PHOX2A substitution mutants expressed in inducible CAD cell lines, we demonstrated that the transcriptional activity of PHOX2A is regulated by two sequential cAMP-dependent events: first, cAMP signaling promotes dephosphorylation of PHOX2A in at least one site, Ser206, thereby allowing PHOX2A to bind DNA and initiate p27(Kip1) transcription; second, following dephosphorylation of the phosphoserine cluster (Ser202 and Ser208), PHOX2A becomes phosphorylated by protein kinase A (PKA) on Ser153, which prevents association of PHOX2A with DNA and terminates p27(Kip1) transcription. This represents a novel mechanism by which the same stimulus, cAMP signaling, first activates PHOX2A by dephosphorylation of Ser206 and then, after a built-in delay, inactivates PHOX2A via PKA-dependent phosphorylation of Ser153, thereby modulating onset and duration of p27(Kip1) transcription.

  • homeodomain transcription factor PHOX2A via cyclic amp mediated activation induces p27kip1 transcription coordinating neural progenitor cell cycle exit and differentiation
    Molecular and Cellular Biology, 2006
    Co-Authors: Maryline Paris, Wenhorng Wang, Min Hwa Shin, David S Franklin, Ourania M Andrisani
    Abstract:

    Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27(Kip1) is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27(Kip1) transcription have not been identified. We demonstrate the homeodomain transcription factor PHOX2A, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27(Kip1) transcription. PHOX2A transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27(Kip1) transcription, G(1) arrest, and neuronal differentiation. Small interfering RNA silencing of either PHOX2A or p27(Kip1) suppresses p27(Kip1) transcription and neuronal differentiation, suggesting a causal link between p27(Kip1) expression and differentiation. Conversely, ectopic PHOX2A expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27(Kip1) transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed PHOX2A activated by cAMP signaling binds homeodomain cis-acting elements of the p27(Kip1) promoter in vivo and mediates p27(Kip1)-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link PHOX2A activation with p27(Kip1) transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.

  • the camp pathway in combination with bmp2 regulates PHOX2A transcription via camp response element binding sites
    Journal of Biological Chemistry, 2006
    Co-Authors: Chutamas Benjanirut, Seok Jong Hong, Kwangsoo Kim, Maryline Paris, Wenhorng Wang, Ronald L Hullinger, Ourania M Andrisani
    Abstract:

    Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor PHOX2A, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on PHOX2A transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPHOX2A-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous PHOX2A expression, the 7.5-kb hPHOX2A reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPHOX2A fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPHOX2A promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous PHOX2A transcription and prolonged CREB phosphorylation, indicating PHOX2A chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine PHOX2A promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to PHOX2A-luciferase reporters. We conclude these CREs are functional, with CREB directly activating PHOX2A transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on PHOX2A transcription.

  • the camp pathway regulates both transcription and activity of the paired homeobox transcription factor PHOX2A required for development of neural crest derived and central nervous system derived catecholaminergic neurons
    Journal of Biological Chemistry, 2005
    Co-Authors: Sigeng Chen, Maryline Paris, Ronald L Hullinger, Ourania M Andrisani
    Abstract:

    Abstract Pluripotent neural crest (NC) cells differentiate to diverse lineages, including the neuronal, sympathoadrenal lineage. In primary NC cultures, bone morphogenetic protein 2 (BMP2) requires moderate activation of cAMP signaling for induction of the sympathoadrenal lineage. However, the mechanism by which cAMP signaling synergizes with BMP2 to induce the sympathodrenal lineage is unknown. Herein, we demonstrate that moderate activation of cAMP signaling induces both transcription and activity of proneural transcription factor PHOX2A. In NC cultures inhibition of cAMP-response element-binding protein (CREB)-mediated transcription by expression of dominant-negative CREB suppresses PHOX2A transcription and sympathoadrenal lineage development. Interestingly, the constitutively active CREBDIEDML, despite inducing PHOX2A transcription, is insufficient for sympathoadrenal lineage development, requiring activation of the cAMP pathway. Because CREBDIEDML-mediates cAMP-dependent transcription without requiring activation by the cAMP-dependent protein kinase A (PKA), these results identify PKA activation as necessary in sympathoadrenal lineage development. Treatment of NC cultures with the PKA inhibitor H89 or 1-10 nm okadaic acid (OA), a serine/threonine PP2A-like phosphatase inhibitor, suppresses sympathoadrenal lineage development. Likewise, OA treatment of the CNS-derived catecholaminergic CAD cell line inhibits cAMP-mediated neuronal differentiation. Specifically, OA inhibits cAMP-mediated PHOX2A dephosphorylation, cAMP-dependent PHOX2A DNA binding in vitro, and cAMP- and PHOX2A-dependent dopamine-β-hydroxylase-luciferase reporter expression. Together, these results support cAMP-dependent PHOX2A dephosphorylation is required for its activation. We conclude that moderate activation of cAMP signaling has dual inputs in catecholaminergic, sympathoadrenal lineage development; that is, regulation of both PHOX2A transcription and activity. These results provide the first mechanistic understanding of how moderate activation of the cAMP pathway in synergy with BMP2 promotes sympathoadrenal lineage development.

  • differential expression of sympathoadrenal lineage determining genes and phenotypic markers in cultured primary neural crest cells
    In Vitro Cellular & Developmental Biology – Animal, 2001
    Co-Authors: Matthew L Bilodeau, Ronald L Hullinger, Theresa Boulineau, John D M Greulich, Ourania M Andrisani
    Abstract:

    Bone morphogenetic protein-2 (BMP-2) promotes the development of primary neural crest cells grown in tissue culture to the sympathoadrenal (SA) lineage. Independent studies have characterized the expression patterns of SA-lineage genes in developing chicken embryo; however, studies using cultured primary neural crest cells have characterized only the expression patterns of the catecholaminergic markers, tyrosine hydroxylase (TH) and catecholamines (CAs). To further explore the molecular mechanisms that control SA-cell development using the in vitro model system, it is crucial to define the expression patterns of both the catecholaminergic markers and the genes regulating SA-lineage determination. Accordingly, we defined, in the absence and presence of BMP-2, the temporal expression patterns of TH and CA, the SA lineage-determining genes ASH-1, PHOX2A, and Phox2b, the GATA-2 gene, and the pan-neuronal SCG10 gene. Comparison of these data with the reported temporal and spatial patterns of expression in vivo demonstrate that the inductive steps of SA-lineage determination, including the specification of neurotransmitter identity and neuronal fate, are recapitulated in the neural-crest culture system.