RESUMEN
The Ccr4-Not complex is a conserved global regulator of gene expression, which serves as a regulatory platform that senses and/or transmits nutrient and stress signals to various downstream effectors. Presumed effectors of this complex in yeast are TFIID, a general transcription factor that associates with the core promoter, and Msn2, a key transcription factor that regulates expression of stress-responsive element (STRE)-controlled genes. Here we show that the constitutively high level of STRE-driven expression in ccr4-not mutants results from two independent effects. Accordingly, loss of Ccr4-Not function causes a dramatic Msn2-independent redistribution of TFIID on promoters with a particular bias for STRE-controlled over ribosomal protein gene promoters. In parallel, loss of Ccr4-Not complex function results in an alteration of the posttranslational modification status of Msn2, which depends on the type 1 protein phosphatase Glc7 and its newly identified subunit Bud14. Tests of epistasis as well as transcriptional analyses of Bud14-dependent transcription support a model in which the Ccr4-Not complex prevents activation of Msn2 via inhibition of the Bud14/Glc7 module in exponentially growing cells. Thus, increased activity of STRE genes in ccr4-not mutants may result from both altered general distribution of TFIID and unscheduled activation of Msn2.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Ribonucleasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional , Reactivos de Enlaces Cruzados/farmacología , ADN/metabolismo , Regulación de la Expresión Génica , Genotipo , Glucosa/metabolismo , Immunoblotting , Inmunoprecipitación , Modelos Biológicos , Mutación , Hibridación de Ácido Nucleico , Fosfoproteínas Fosfatasas/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteína Fosfatasa 1 , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Factor de Transcripción TFIID/química , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The Ccr4-Not complex is a multifunctional regulatory platform composed of nine subunits that controls diverse cellular events including mRNA degradation, protein ubiquitination, and transcription. In this study, we identified the yeast Saccharomyces cerevisiae osmotic and oxidative stress transcription factor Skn7 as a new target for regulation by the Ccr4-Not complex. Skn7 interacts with Not1 in a two-hybrid assay and coimmunoprecipitates with Not5 in a Not4-dependent manner. Skn7-dependent expression of OCH1 and Skn7 binding to the OCH1 promoter are increased in not4Delta or not5Delta mutants. Skn7 purified from wild-type cells but not from not4Delta cells is associated with the Srb10 kinase. This kinase plays a central role in the regulation of Skn7 by Not4, since increased OCH1 expression in not4Delta cells requires Srb10. These results reveal a critical role for the Ccr4-Not complex in the mechanism of activation of Skn7 that is dependent upon the Srb10 kinase.