RESUMEN
The Polycomb group (PcG) gene products form multimeric protein complexes and contribute to anterior-posterior (A-P) specification via the transcriptional regulation of Hox cluster genes. The Drosophila polyhomeotic genes and their mammalian orthologues, Phc1, Phc2, and Phc3, encode nuclear proteins that are constituents of evolutionarily conserved protein complexes designated class II PcG complexes. In this study, we describe the generation and phenotypes of Phc2-deficient mice. We show posterior transformations of the axial skeleton and premature senescence of mouse embryonic fibroblasts associated with derepression of Hox cluster genes and Cdkn2a genes, respectively. Synergistic actions of a Phc2 mutation with Phc1 and Rnf110 mutations during A-P specification, coimmunoprecipitation of their products from embryonic extracts, and chromatin immunoprecipitation by anti-Phc2 monoclonal antibodies suggest that Hox repression by Phc2 is mediated through the class II PcG complexes, probably via direct binding to the Hox locus. The genetic interactions further reveal the functional overlap between Phc2 and Phc1 and a strict dose-dependent requirement during A-P specification and embryonic survival. Functional redundancy between Phc2 and Phc1 leads us to hypothesize that the overall level of polyhomeotic orthologues in nuclei is a parameter that is critical in enabling the class II PcG complexes to exert their molecular functions.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Homeobox , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/metabolismo , Ratones , Especificidad de Órganos , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Ganglios Linfáticos/metabolismo , Proteoma , Bazo/metabolismo , Secuencia de Aminoácidos , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
Foxl1 is a winged helix transcription factor expressed in the mesenchyme of the gastrointestinal tract. In the absence of Foxl1, parietal cells fail to secrete gastric acid in response to various secretagogue stimuli including cAMP. A marked decrease in H+,K(+)-ATPase expression was observed even though a substantial number of parietal cells still existed in Foxl1-deficient mice. Ultrastructural analysis suggested that the gastric acid secretion defect in Foxl1-deficient mice is mainly due to impairment in the fusion of cytoplasmic tubulovesicular structures to the apical canalicular plasma membrane. Among the molecules involved in the membrane fusion event, only SNAP25 showed a significant decrease in mRNA expression, which likely caused the impairment in acid secretion from parietal cells in Foxl1-deficient mice, with the reduction in H+,K(+)-ATPase expression contributing to additional effect.