RESUMEN
Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario , ARN Mensajero/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Estructura Terciaria de Proteína , Túbulos Seminíferos/fisiopatología , Espermatocitos/fisiología , Distribución Tisular , Factores de TranscripciónRESUMEN
Proteins containing "cold shock" domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known among bacteria, plants, and animals. One of these proteins, YB-1, is widely expressed throughout development and has been implicated as a cell survival factor that regulates the transcription and/or translation of many cellular growth and death-related genes. For these reasons, YB-1 deficiency has been predicted to be incompatible with cell survival. However, the majority of YB-1(-/-) embryos develop normally up to embryonic day 13.5 (E13.5). After E13.5, YB-1(-/-) embryos exhibit severe growth retardation and progressive mortality, revealing a nonredundant role of YB-1 in late embryonic development. Fibroblasts derived from YB-1(-/-) embryos displayed a normal rate of protein synthesis and minimal alterations in the transcriptome and proteome but demonstrated reduced abilities to respond to oxidative, genotoxic, and oncogene-induced stresses. YB-1(-/-) cells under oxidative stress expressed high levels of the G(1)-specific CDK inhibitors p16Ink4a and p21Cip1 and senesced prematurely; this defect was corrected by knocking down CDK inhibitor levels with specific small interfering RNAs. These data suggest that YB-1 normally represses the transcription of CDK inhibitors, making it an important component of the cellular stress response signaling pathway.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario , Proteínas Represoras/fisiología , Animales , Proteínas de Ciclo Celular/genética , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/citología , Retardo del Crecimiento Fetal/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Marcación de Gen , Genes Letales , Ratones , Estrés Oxidativo , Biosíntesis de Proteínas/genética , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Represoras/genética , Transcripción Genética/genéticaRESUMEN
The correction of mutant beta-globin genes has long been a therapeutic goal for patients with beta-thalassemia or hemoglobinopathies. The use of homologous recombination (HR) to achieve this goal is an attractive approach because it eliminates the need to include regulatory sequences in the therapeutic construct, and it eliminates mutagenesis induced by random integration. However, HR is a very inefficient process for gene correction, and its efficiency is probably locus dependent. The length of targeting arms is thought to be a determinant of targeting efficiency, so we compared the ability of standard (8-kb) versus very long (16-, 24-, and 110-kb) regions of homology to correct a mutant murine beta-globin gene in embryonic stem cells. Increasing the length of the targeting sequences did not increase the efficiency of HR in this locus, suggesting that alternative approaches will be required to improve the efficiency of this approach for globin gene correction.