RESUMO
Exostosin1 (Ext1) belongs to a family of glycosyltransferases necessary for the synthesis of the heparan sulfate (HS) chains of proteoglycans, which regulate signaling of several growth factors. Loss of tout velu (ttv), the homolog of Ext1 in Drosophila, inhibits Hedgehog movement. In contrast, we show that reduced HS synthesis in mice carrying a hypomorphic mutation in Ext1 results in an elevated range of Indian hedgehog (Ihh) signaling during embryonic chondrocyte differentiation. Our data suggest a dual function for HS: First, HS is necessary to bind Hedgehog in the extracellular space. Second, HS negatively regulates the range of Hedgehog signaling in a concentration-dependent manner. Additionally, our data indicate that Ihh acts as a long-range morphogen, directly activating the expression of parathyroid hormone-like hormone. Finally, we propose that the development of exostoses in the human Hereditary Multiple Exostoses syndrome can be attributed to activation of Ihh signaling.
Assuntos
Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Heparitina Sulfato/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Osteogênese/genética , Transativadores/metabolismo , Animais , Osso e Ossos/citologia , Diferenciação Celular/genética , Condrócitos/citologia , Condrócitos/metabolismo , Exostose Múltipla Hereditária/genética , Exostose Múltipla Hereditária/metabolismo , Exostose Múltipla Hereditária/fisiopatologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog , Heparitina Sulfato/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , N-Acetilglucosaminiltransferases/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transdução de Sinais/genética , Transativadores/genética , Regulação para Cima/genéticaRESUMO
Opitz G/BBB syndrome (OS) is a congenital midline malformation syndrome characterized by hypertelorism, hypospadias, cleft lip/palate, laryngotracheoesophageal abnormalities, imperforate anus, developmental delay and cardiac defects. The X-linked form is caused by mutations in the MID1 gene, while no gene has yet been identified for the autosomal dominant form. Here, we report on a 15-year-old boy who was referred for MID1 mutation analysis with findings typical of OS, including apparent hypertelorism, hypospadias, a history of feeding difficulties, dysphagia secondary to esophageal arteria lusoria, growth retardation and developmental delay. No MID1 mutation was found, but subsequent sub-megabase resolution array CGH unexpectedly documented a 2.34 Mb terminal 4p deletion, suggesting a diagnosis of WHS, and a duplication in Xp22.31. Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion syndrome involving terminal chromosome 4p deletions, in particular 4p16.3. WHS is characterized by typical facial appearance ("Greek helmet facies"), mental retardation, congenital hypotonia, and growth retardation. While the severity of developmental delay in this patient supports the diagnosis of WHS rather than OS, this case illustrates the striking similarities of clinical findings in seemingly unrelated syndromes, suggesting common or interacting pathways at the molecular and pathogenetic level. This is the first report of arteria lusoria (esophageal vascular ring) in a patient with WHS.
Assuntos
Anormalidades Múltiplas/diagnóstico , Deleção Cromossômica , Cromossomos Humanos Par 4 , Cromossomos Humanos X , Duplicação Gênica , Anormalidades Múltiplas/genética , Adolescente , DNA/genética , Humanos , Masculino , Hibridização de Ácido Nucleico , Linhagem , SíndromeRESUMO
BACKGROUND: Mutations in the X-linked MID1 gene are responsible for Opitz G/BBB syndrome, a malformation disorder of developing midline structures. Previous Northern blot analyses revealed the existence of at least three MID1 transcripts of differing lengths. RESULTS: Here we show that alternative polyadenylation generates the size differences observed in the Northern blot analyses. Analysis of EST data together with additional Northern blot analyses proved tissue-specific usage of the alternative polyadenylation sites. Bioinformatic characterization of the different 3'UTRs of MID1 revealed numerous RNA-protein interaction motifs, several of which turned out to be conserved between different species. Furthermore, our data suggest that mRNA termination at different polyadenylation sites is predetermined by the choice of alternative 5'UTRs and promoters of the MID1 gene, a mechanism that efficiently allows synergistic function of 5' and 3'UTRs. CONCLUSION: MID1 expression is tightly regulated through concerted action of alternative promoters and alternative polyadenylation signals both during embryonic development and in the adult.
Assuntos
Regulação da Expressão Gênica , Proteínas dos Microtúbulos/genética , Proteínas Nucleares/genética , Poliadenilação/genética , Regiões Promotoras Genéticas/genética , Síndrome de Smith-Lemli-Opitz/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Northern Blotting , Sequência Conservada , Etiquetas de Sequências Expressas , Células HeLa , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Especificidade da Espécie , Ubiquitina-Proteína LigasesRESUMO
The Trps1 gene codes for an atypical member of the GATA type family of transcription factors. Mutations in human TRPS1 lead to the tricho-rhino-phalangeal syndrome types I and III, which are characterized by craniofacial and skeletal abnormalities and disturbed hair development. Correspondingly, during mouse embryonic development strong Trps1 expression is found in the cartilage condensations, the developing joints, the hair follicles and in the developing snout. In addition, Trps1 is expressed surrounding the skeletal condensations, in the trachea, the intervertebral disks, and in lung and gut mesenchyme. A complex pattern of expression is also found in the developing brain.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Animais , Condrogênese , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário , Fatores de Transcrição GATA/metabolismo , Humanos , Camundongos , Mutação , Fatores de Transcrição/metabolismoRESUMO
The Trps1 gene codes for an atypical member of the GATA type family of transcription factors. Mutations in human TRPS1 lead to the tricho-rhino-phalangeal syndrome types I and III, which are characterized by craniofacial and skeletal abnormalities and disturbed hair development. Correspondingly, during mouse embryonic development strong Trps1 expression is found in the cartilage condensations, the developing joints, the hair follicles and in the developing snout. In addition, Trps1 is expressed surrounding the skeletal condensations, in the trachea, the intervertebral disks, and in lung and gut mesenchyme. A complex pattern of expression is also found in the developing brain.
Assuntos
Proteínas de Ligação a DNA/genética , Face/fisiologia , Camundongos/embriologia , Proteínas de Neoplasias , Proteínas Nucleares/genética , Animais , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/biossíntese , Face/embriologia , Hibridização In Situ , Músculos/embriologia , Músculos/metabolismo , Proteínas Nucleares/biossíntese , Especificidade de Órgãos , Proteínas Repressoras , Pele/embriologia , Pele/metabolismo , Fatores de TranscriçãoRESUMO
BACKGROUND: Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. PRINCIPAL FINDINGS: We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-alpha4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. CONCLUSIONS: The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Análise Mutacional de DNA , Vetores Genéticos , Células HeLa , Humanos , Microtúbulos/metabolismo , Mutação , Fenótipo , Interferência de RNA , Síndrome , Transcrição Gênica , Proteína Gli3 com Dedos de ZincoRESUMO
Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), alpha4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the alpha4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the alpha4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit alpha4 and PP2A/alpha4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations.