RESUMO
The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore-spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.
Assuntos
Cromossomos/genética , Genes cdc/fisiologia , Mitose/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Fuso Acromático/genética , Anáfase/genética , Proteínas de Ciclo Celular/genética , Centrômero/genética , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , DNA/biossíntese , Proteínas Fúngicas , Genes cdc/efeitos dos fármacos , Hidroxiureia/farmacologia , Cinetocoros/fisiologia , Mitose/efeitos dos fármacos , Mutação/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fase S/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , CoesinasRESUMO
Identification of Pygopus in Drosophila as a dedicated component of the Wg (fly homolog of mammalian Wnt) signaling cascade initiated many inquiries into the mechanism of its function. Surprisingly, the nearly exclusive role for Pygopus in Wg signal transduction in flies is not seen in mice, where Pygopus appears to have both Wnt-related and Wnt-independent functions. This review addresses the initial findings of Pygopus as a Wg/Wnt co-activator in light of recent data from both fly and mammalian studies. We compare and contrast the developmental phenotypes of pygopus mutants to those characterized for known Wg/Wnt transducers and explore the data regarding a role for mammalian Pygopus 2 in tumorigenesis. We further analyze the roles of the two conserved domains of Pygopus proteins in transcription, and propose a model for the molecular mechanism of Pygopus function in both Wg/Wnt signaling and Wnt-independent transcriptional regulation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Montagem e Desmontagem da Cromatina , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Evolução Molecular , Feminino , Genes de Insetos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade da Espécie , Transativadores/genética , Transativadores/metabolismo , Proteínas Wnt/genética , Proteína Wnt1 , beta Catenina/metabolismoRESUMO
Canonical Wnt signaling involves complex intracellular events culminating in the stabilization of beta-catenin, which enters the nucleus and binds to LEF/TCF transcription factors to stimulate gene expression. Pygopus was identified as a genetic modifier of Wg (Wnt homolog) signaling in Drosophila, and encodes a PHD domain protein that associates with the beta-catenin/LEF/TCF complex. Two murine pygopus paralogs, mpygo1 and mpygo2, have been identified, but their roles in development and Wnt signaling remain elusive. In this study, we report that ablation of mpygo2 expression in mice causes defects in morphogenesis of both ectodermally and endodermally derived tissues, including brain, eyes, hair follicles, and lung. However, no gross abnormality was observed in embryonic intestine. Using a BAT-gal reporter, we found Wnt signaling at most body sites to be reduced in the absence of mpygo2. Taken together, our studies show for the first time that mpygo2 deletion affects embryonic development of some but not all Wnt-requiring tissues.