RESUMO
BACKGROUND: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. RESULTS: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)). CONCLUSIONS: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.
Assuntos
Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismo , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/genética , Duplicação Gênica , 4-Hidroxifenilpiruvato Dioxigenase/genética , Northern Blotting , Proteínas Quinases Dependentes de AMP Cíclico/genética , Dictyostelium/metabolismo , Ácido Graxo Sintases/genética , Análise em Microsséries , NADH NADPH Oxirredutases/genética , Fenótipo , Proteômica , Esporos de Protozoários/genética , Esporos de Protozoários/metabolismoRESUMO
BACKGROUND: Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores. Cells in which the gene encoding Cdk8 has been deleted fail to enter aggregates due to a failure of early gene expression. PRINCIPAL FINDINGS: We have monitored the expression levels of cyclin C protein during development and find levels decrease after the multicellular mound is formed. This decrease is triggered by extracellular cAMP that, in turn, is working in part through an increase in intracellular cAMP. The loss of cyclin C is coincident with a reduction in the association of Cdk8 with a high molecular weight complex in the nucleus. Overexpression of cyclin C and Cdk8 lead to an increased rate of early development, consistent with the levels being rate limiting. CONCLUSIONS: Overall these results show that both cyclin C and Cdk8 are regulated during development in response to extracellular signals and the levels of these proteins are important in controlling the timing of developmental processes. These findings have important implications for the role of these proteins in controlling development, suggesting that they are targets for developmental signals to regulate gene expression.
Assuntos
Ciclina C/metabolismo , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Sequência de Aminoácidos , Animais , Fracionamento Químico , AMP Cíclico/metabolismo , Ciclina C/química , Ciclina C/genética , Quinase 8 Dependente de Ciclina/metabolismo , Dictyostelium/efeitos dos fármacos , Dictyostelium/enzimologia , Peróxido de Hidrogênio/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Dictyostelium discoideum is an excellent system in which to study developmental decisions. Synchronous development is triggered by starvation and rapidly generates a limited number of cell types. Genetic and image analyses have revealed the elegant intricacies associated with this simple development system. Key signaling pathways identified as regulating cell fate decisions are likely to be conserved with metazoa and are providing insight into differentiation decisions under circumstances where considerable cell movement takes place during development.