RESUMEN
Drosophila Nipped-B is an essential protein that has multiple functions. It facilitates expression of homeobox genes and is also required for sister chromatid cohesion. Nipped-B is conserved from yeast to man, and its orthologs also play roles in deoxyribonucleic acid repair and meiosis. Mutation of the human ortholog, Nipped-B-Like (NIPBL), causes Cornelia de Lange syndrome (CdLS), associated with multiple developmental defects. The Nipped-B protein family is required for the cohesin complex that mediates sister chromatid cohesion to bind to chromosomes. A key question, therefore, is whether the Nipped-B family regulates gene expression, meiosis, and development by controlling cohesin. To gain insights into Nipped-B's functions, we compared the effects of several Nipped-B mutations on gene expression, sister chromatid cohesion, and meiosis. We also examined association of Nipped-B and cohesin with somatic and meiotic chromosomes by immunostaining. Missense Nipped-B alleles affecting the same HEAT repeat motifs as CdLS-causing NIPBL mutations have intermediate effects on both gene expression and mitotic chromatid cohesion, linking these two functions and the role of NIPBL in human development. Nipped-B colocalizes extensively with cohesin on chromosomes in both somatic and meiotic cells and is present in soluble complexes with cohesin subunits in nuclear extracts. In meiosis, Nipped-B also colocalizes with the synaptonemal complex and contributes to maintenance of meiotic chromosome cores. These results support the idea that direct regulation of cohesin function underlies the diverse functions of Nipped-B and its orthologs.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Meiosis/fisiología , Mutación/genética , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Síndrome de Cornelia de Lange , Proteínas de Drosophila/inmunología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Femenino , Cobayas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunoprecipitación , Larva , Masculino , Mitosis/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Intercambio de Cromátides Hermanas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , CohesinasRESUMEN
During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. We conclude that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila.