RESUMEN
Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-beta-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of beta-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.
Asunto(s)
Transducción de Señal/fisiología , Factor 1 de Transcripción de Linfocitos T/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Inmunoprecipitación de Cromatina , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Polyamines play an important role in cell growth and differentiation. We studied changes in morphogenesis and the expression of the developmental control genes in the embryonic mouse kidney in response to polyamine depletion, using a kidney organ culture approach and reducing the polyamine pools with alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of ornithine decarboxylase (ODC). We found that inhibition of ODC results in a systematic kidney organogenesis phenotype, in that the DFMO-treated kidney specimens were of smaller size, had less epithelial ureteric bud branches, and their mesenchymal-derived tubule formation was retarded. These dysmorphologies were shown to be associated with changes in cell proliferation. Whole-mount in situ experiments revealed that inhibition of ODC causes increases in epithelial c-ret and E-cadherin and a decrease in mesenchymal Pax-8 expression, whereas levels of epithelial Wnt-11, mesenchymal GDNF, FoxD1, and Pax-2 transcripts remain unchanged. We studied regulation of the Pax-2 gene by analyzing a mouse line in which lacZ was driven by an 8.5 kb Pax-2 enhancer in the epithelial ureteric bud, and found that Pax-2 expression, as indicated by lacZ expression, increased after DFMO treatment. Transient transfection experiments in HEK 293 cells with the minimal Pax-2 promoter showed enhanced transcription upon reduction of the polyamine pools. We propose that ODC and polyamines have an important role in kidney organogenesis, being involved in the regulation of the expression of genes implicated in epithelial-mesenchymal tissue interactions.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Inhibidores de la Ornitina Descarboxilasa , Poliaminas/metabolismo , Animales , Cadherinas/genética , Línea Celular , Proteínas de Unión al ADN/genética , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Riñón/citología , Riñón/enzimología , Ratones , Proteínas Nucleares/genética , Organogénesis/efectos de los fármacos , Organogénesis/genética , Ornitina Descarboxilasa/genética , Factor de Transcripción PAX2 , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Acetyl-CoA synthetase (AceCS) provides acetyl-CoA for different physiological processes, such as fatty acid and cholesterol synthesis, as well as the citric acid cycle. We show here that the cytosolic isoform of this enzyme, AceCS1, is expressed during mouse development. In the embryonic stage E9.5 AceCS1 transcripts localize in the cephalic region. At E10.5 the cephalic expression intensifies and transcripts appear also in the spinal cord and in the dorsal root ganglions. During organogenesis AceCS1 is expressed in the liver from E11.5. The AceCS1 gene is expressed also in the testes from E12.5 onwards and expression localizes in the interstitial Leydig cells. In the ovaries, expression is transient and AceCS1 transcripts are detected from E13.5 to E15.5 in the ovarian interstitial component. In the kidneys AceCS1 transcripts appear in a subset of the renal tubules at E16.5 and remains in these structures in newborns. Hence, expression of AceCS1 is developmentally regulated suggesting a role for AceCS1 during embryogenesis.
