RESUMEN
Proteins of the transforming growth factor beta(TGFbeta) superfamily regulate diverse cellular responses, including cell growth and differentiation. After TGFbeta stimulation, receptor-associated Smads are phosphorylated and form a complex with the common mediator Smad4. Here, we report the cloning of SMIF, a ubiquitously expressed, Smad4-interacting transcriptional co-activator. SMIF forms a TGFbeta/bone morphogenetic protein 4 (BMP4)-inducible complex with Smad4, but not with others Smads, and translocates to the nucleus in a TGFbeta/BMP4-inducible and Smad4-dependent manner. SMIF possesses strong intrinsic TGFbeta-inducible transcriptional activity, which is dependent on Smad4 in mammalian cells and requires p300/CBP. A point mutation in Smad4 abolished binding to SMIF and impaired its activity in transcriptional assays. Overexpression of wild-type SMIF enhanced expression of TGFbeta/BMP regulated genes, whereas a dominant-negative SMIF mutant suppressed expression. Furthermore, dominant-negative SMIF is able to block TGFbeta-induced growth inhibition. In a knockdown approach with morpholino-antisense oligonucleotides targeting zebrafish SMIF, severe but distinct phenotypic defects were observed in zebrafish embryos. Thus, we propose that SMIF is a crucial activator of TGFbeta signalling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Drosophila/genética , Endorribonucleasas , Marcación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Mutación Puntual , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína Smad4 , Técnicas del Sistema de Dos Híbridos , Pez Cebra/anomalías , Pez Cebra/genéticaRESUMEN
The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.
Asunto(s)
Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Mutación/genética , Inhibición Neural/genética , Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Proteínas de Unión al ARN/genética , Sinapsis/genética , Adolescente , Animales , Encéfalo/patología , Células Cultivadas , Proteínas de Unión al ADN , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Linaje , Sinapsis/patologíaRESUMEN
Moderate reductions in synaptic γ-aminobutyric acid(A) receptors (GABA(A)Rs) have been associated with an enhanced defensive behavioral reactivity to mild threat, sensitive to diazepam. We here tested whether a deficit in α2 subunit-containing GABAergic synapses is sufficient to cause this anxiety-related phenotype and to prevent its attenuation by the benzodiazepine. Wild type (α2+/+), heterozygous (α2+/-) and homozygous (α2-/-) knock-out littermates were tested in the free-choice exploratory (FCE) and the light/dark choice (LDC) paradigms. α2-/- mice, double mutant α1H101Rα2-/- and α3H126Rα2-/- mice, which combine a lack of α2-GABA(A)Rs with point-mutated diazepam-insensitive either α1H101R or α3H126R-GABA(A)Rs, and double point-mutated α1H101Rα2H101R and α1H101Rα3H126R mice were used to uncover the GABA(A)R subtype(s) mediating the drug effects. Data show that in the FCE, α2-/- mice exhibited more retractions (i.e. risk assessment) and longer latencies to first occurrence into the novel compartment and less transitions and time spent inside it in comparison to α2+/- and α2+/+ mice. In the LDC, α2-/- mice visited and spent less time in the lit box and stayed longer in the tunnel than the other two groups. Minor differences were found between α2+/- and α2+/+ mice in the two paradigms. Diazepam (1.5mg/kg per os) normalized retractions and latencies in the FCE in α2-/- and α3H126Rα2-/- mice, but not in α1H101Rα2-/- mice. The same drug treatment failed to attenuate behavioral aversion in both paradigms in all mutants with impaired α2-GABA(A)R function. These results reveal α2-containing GABA(A)Rs as key molecular determinants in the regulation of anxiety-related responses elicited by exposure to relative novelty and mild threat. In the absence of these receptors, diazepam through activation of α1-GABA(A)Rs remains effective in reducing risk assessment, but not behavioral aversion.