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Seok Jong Hong

Publications and source records attributed to Seok Jong Hong.

8 recordsLinked to original sources
PubMed2006

GATA-3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB.

The zinc finger transcription factor GATA-3 is a master regulator of type 2 T-helper cell development. Interestingly, in GATA-3-/- mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA-3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA-3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA-3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH promoter residing at -61 to -39 bp. Surprisingly, this domain does not contain GATA-3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site-directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA-3. Furthermore, protein-protein interaction assays showed that GATA-3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA-3 may regulate TH gene transcription via a novel and distinct protein-protein interaction, and directly contributes to NA phenotype specification.

Animals↗
PubMed2005

The cAMP pathway in combination with BMP2 regulates Phox2a transcription via cAMP response element binding sites.

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.

Animals↗
PubMed2005

Molecular consequences of PHOX2B missense, frameshift and alanine expansion mutations leading to autonomic dysfunction.

Heterozygous mutations of the PHOX2B gene account for a broad variety of disorders of the autonomic nervous system, either isolated or combined, including congenital central hypoventilation syndrome (CCHS), tumours of the sympathetic nervous system and Hirschsprung disease. In CCHS, the prevalent mutation is an expansion of a 20-alanine stretch ranging from +5 to +13 alanines, whereas frameshift and missense mutations are found occasionally. To determine the molecular basis of impaired PHOX2B function, we assayed the transactivation and DNA binding properties of wild-type and mutant PHOX2B proteins. Furthermore, we investigated aggregate formation by proteins with polyalanine tract expansions ranging from +5 to +13 alanines using immunofluorescence of transfected cells and gel filtration of in vitro translated proteins. We found that transactivation of the dopamine beta-hydroxylase promoter by PHOX2B proteins with frameshift and missense mutations was abolished or severely curtailed, as was in vitro DNA binding although the proteins localized to the nucleus. The transactivation potential of proteins with polyalanine tract expansions declined with increasing length of the polyalanine stretch, and DNA binding was affected for an expansion of +9 alanines and above. Cytoplasmic aggregation in transfected cells was only observed for the longest expansions, whereas even the short expansion mutants were prone to form multimers in vitro. Such a tendency to protein misfolding could explain loss of transactivation for alanine expansion mutations. However, additional mechanisms such as toxic gain-of-function may play a role in the pathogenic process.

Animals↗
PubMed2005

Gene expression profiling of mouse postnatal cerebellar development using oligonucleotide microarrays designed to detect differences in glycoconjugate expression.

Differences in gene expression patterns between adult and postnatal day 7 (P7) mouse cerebellum, at the peak of granule neuron migration, were analyzed by hybridization to the GLYCOv2 glycogene array. This custom designed oligonucleotide array focuses on glycosyl transferases, carbohydrate-binding proteins, proteoglycans and related genes, and 173 genes were identified as being differentially expressed with statistical confidence. Expression levels for 11 of these genes were compared by RT-PCR, and their differential expression between P7 and adult cerebellum confirmed. Within the group of genes showing differential expression, the sialyltransferases (SiaTs) and GalNAc-Ts that were elevated at P7 prefer glycoprotein substrates, whilst the SiaTs and GalNAc-Ts that were elevated in the adult preferentially modify glycolipids, consistent with a role for gangliosides in maintaining neuronal function in the adult. Also within this group, three proteoglycans--versican, bamacan and glypican-2--were elevated at P7, along with growth factor midkine, which is known to bind to multiple types of proteoglycans, and fibroblast growth factor receptor 1, whose activity is known to be influenced by heparan sulfate proteoglycans. Two sulfotransferases that can modify the extent of proteoglycan sulfation were also differentially regulated, and may modify the interaction of a subset of proteoglycans with their binding partners during cerebellar development. Bamacan, glypican-2 and midkine were shown to be expressed in different cell types, and their roles in cerebellar development during granule neuron migration and maturation are discussed.

Aging↗
PubMed2004

Molecular cloning and characterization of the promoter region of the human Phox2b gene.

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.

Animals↗
PubMed2003

Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner.

Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step of catecholamine synthesis and its expression is necessary for neurotransmitter specification of all catecholaminergic neurons, while dopamine beta-hydroxylase (DBH) is essential for the noradrenergic phenotype. In the present study, we show that Nurr1, an orphan nuclear receptor critical for dopaminergic (DA) neuron development, directly transactivates the promoter activity of the TH gene in a cell type-dependent manner, while it does not regulate the DBH promoter. Consistent with these results, only the TH promoter contains multiple sequence motifs homologous to the known Nurr1-binding motif, NBRE. TH promoter deletional analysis indicates that < 1.0 kb upstream sequences, encompassing three NBRE-like motifs (i.e. NL1, NL2 and NL3) are mostly responsible for the effects of Nurr1. Among these potential motifs, site-directed mutational analysis showed that NL1, residing from - 35 to - 28 bp, was most critical for mediating the transactivation by Nurr1. Strikingly, however, both DNase I footprinting and electrophoretic mobility shift assays showed that NL3, but not NL1 or NL2, has high binding affinity to Nurr1. To determine whether the proximity of these motifs may be important for transactivation by Nurr1 in the transient transfection assay, we generated reporter gene constructs in which NL3 is immediately proximal to the TATA box. Indeed, NL3 was more efficient in this position than NL1 or NL2 for mediating the transactivation by Nurr1. Our results suggest that Nurr1 may play a direct role for specification of DA neurotransmitter identity by activating TH gene transcription in a cell context-dependent manner.

Binding, Competitive↗
PubMed2002

A direct role of the homeodomain proteins Phox2a/2b in noradrenaline neurotransmitter identity determination.

Development of noraderenergic (NA) neurons in the vertebrate brain is dependent on the homeodomain proteins Phox2a and 2b. Here, we show that Phox2a directly controls the NA identity by activating NA-synthesizing dopamine beta-hydroxylase (DBH ) gene. Single point mutations in the homeodomain of Phox2a resulted in a failure to transactivate the DBH promoter in vitro and resulted in the loss of NA neurons in vivo. In addition, injection of Phox2a-specific antisense oligonucleotide induced the loss of NA neurons in developing zebrafish. Phox2a and 2b activate the DBH promoter and bind to three domains (PBD1-3). PBD1 is composed of two overlapping sites with which monomers of Phox2a can interact. In contrast, PBD2 and 3 interact with the dimeric form of Phox2a. Mutations in three or four, but not one or two, of the binding sites completely abolished activation of the DBH promoter by Phox2a or 2b, while the conversion of PBD3 to a consensus motif (ATTA) improved the DBH promoter activity by > 10-fold. Taken together, these findings establish that Phox2a and 2b control the development of NA neurons in part by directly transactivating DBH transcription through interactions with four binding sites clustered in the proximal promoter.

5' Untranslated Regions↗