RESUMEN
O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors. A single CR point mutation, Thr-88 --> Ala, results in a form of the protein that is not fucosylated and has substantially weaker activity in cell-based CR/Nodal signaling assays, indicating that fucosylation is functionally important for CR to facilitate Nodal signaling.
Asunto(s)
Factor de Crecimiento Epidérmico , Fucosa/metabolismo , Proteínas de Homeodominio , Glicoproteínas de Membrana , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Factores de Transcripción , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN , Proteínas Ligadas a GPI , Glicosilación , Humanos , Péptidos y Proteínas de Señalización Intercelular , Espectrometría de Masas , Proteínas de la Membrana , Datos de Secuencia Molecular , Mutagénesis , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Mapeo Peptídico , Mutación Puntual , Homología de Secuencia de Aminoácido , Solubilidad , XenopusRESUMEN
Vertebrate Nodal-related factors play central roles in mesendoderm induction and left-right axis specification, but the mechanisms regulating their expression are largely unknown. We identify an element in Xnr1 intron 1 that is activated by activin and Vg1, autoactivated by Xnrs, and suppressed by ventral inducers like BMP4. Intron 1 contains three FAST binding sites on which FAST/Smad transcriptional complexes can assemble; these sites are differentially involved in intron 1-mediated reporter gene expression. Interference with FAST function abolishes intron 1 activity, and transcriptional activation of Xnrs by activin in embryonic tissue explant assays, identifying FAST as an essential mediator of Xnr autoregulation and/or 'signal relay' from activin-like molecules. Furthermore, the mapping of endogenous activators of the Xnr1 intronic enhancer within Xenopus embryos agrees well with the pattern of Xnr1 transcription during embryogenesis. In transgenic mice, Xnr1 intron 1 mimics a similarly located enhancer in the mouse nodal gene, and directs FAST site-dependent expression in the primitive streak during gastrulation, and unilateral expression during early somitogenesis. The FAST cassette is similar in an ascidian nodal-related gene, suggesting an ancient origin for this regulatory module. Thus, an evolutionarily conserved intronic enhancer in Xnr1 is involved in both mesendoderm induction and asymmetric expression during left-right axis formation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inhibinas/metabolismo , Intrones , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/genética , Proteínas de Xenopus , Activinas , Animales , Sitios de Unión , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Nodal , Elementos de Respuesta , Factor de Crecimiento Transformador beta/metabolismo , Transgenes , Xenopus laevisRESUMEN
The left-right (L-R) asymmetric expression of lefty2 and nodal is controlled by a left side-specific enhancer (ASE). The transcription factor FAST2, which can mediate signaling by TGF beta and activin, has now been identified as a protein that binds to a conserved sequence in ASE. These FAST2 binding sites were both essential and sufficient for L-R asymmetric gene expression. The Fast2 gene is bilaterally expressed when nodal and lefty2 are expressed on the left side. TGF beta and activin can activate the ASE activity in a FAST2-dependent manner, while Nodal can do so in the presence of an EGF-CFC protein. These results suggest that the asymmetric expression of lefty2 and nodal is induced by a left side-specific TGF beta-related factor, which is most likely Nodal itself.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Embrión no Mamífero , Elementos de Facilitación Genéticos , Retroalimentación , Factores de Transcripción Forkhead , Factores de Determinación Derecha-Izquierda , Ratones , Ratones Transgénicos , Proteína Nodal , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Xenopus laevisRESUMEN
Smads are signal transducers for the transforming growth factor-beta superfamily of factors. In early Xenopus embryos, the transforming growth factor-beta member activin induces the gene Mix.2 by stimulating the formation of a multiprotein complex, activin-responsive factor (ARF). This complex contains Smad2 or Smad3, Smad4, and a novel forkhead transcription factor, FAST-1, and binds to an enhancer (activin-responsive element; ARE) that confers activin regulation of Mix.2 transcription. Both FAST-1 and Smads can bind directly to the ARE; we have investigated 1) the role of FAST-1 and Smad DNA binding sites in ARF recognition of the ARE, 2) the contributions of FAST-1 and Smad binding to ARF binding in vitro and to ARE regulation in early Xenopus embryos, 3) the extent to which different Smads can replace Smad4 in regulation of the ARE. We find that ARF binds to ARE through both FAST-1 and Smad binding sites. FAST-1 recognition of the ARE is essential both for ARF binding in vitro and activin regulation in vivo. In contrast, Smad binding of ARE is unnecessary for ARF binding or activin regulation but does enhance the binding and regulatory activity of ARF. Also, Smad3 can partially substitute for Smad4 in the regulation of the ARE. These observations elucidate how broadly expressed signal transducers (Smads) regulate a developmentally specific transcriptional response in conjunction with a temporally restricted transcription factor, FAST-1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Inhibinas/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Activinas , Animales , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , Metilación de ADN , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Factores de Transcripción Forkhead , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso , Unión Proteica , Proteínas Smad , Proteína Smad2 , Proteína smad3 , Proteína Smad4 , Transcripción Genética/fisiología , Xenopus/embriologíaRESUMEN
The apical ectodermal ridge of the vertebrate limb bud lies at the junction of the dorsal and ventral ectoderm and directs patterning of the growing limb. Its formation is directed by the boundary between cells that do and cells that do not express the gene Radical fringe. This is similar to the establishment of the margin cells at the Drosophila wing dorsoventral border by fringe. Radical fringe expression in chick-limb dorsal ectoderm is established in part through repression by Engrailed-1 in the ventral ectoderm.