RESUMEN
Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle-specific and ubiquitous genes. Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl-transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program.
Asunto(s)
Acetiltransferasas/metabolismo , Músculos/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Animales , Western Blotting , Diferenciación Celular , Línea Celular , Cromatina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Proteína p300 Asociada a E1A , Genes Dominantes , Genes Reporteros , Histona Acetiltransferasas , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Mutación , Miogenina/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Activación Transcripcional , TransfecciónRESUMEN
Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína MioD/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Acetilación , Animales , Proteína de Unión a CREB , Línea Celular , Activación Enzimática , Histona Acetiltransferasas , Unión Proteica , Especificidad por Sustrato , Factores de Transcripción , Factores de Transcripción p300-CBPRESUMEN
The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells. In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo.
Asunto(s)
Proteína MioD/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteína p300 Asociada a E1A , Histona Acetiltransferasas , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Activación TranscripcionalRESUMEN
Transforming viral proteins such as E1A which force quiescent cells into S phase have two essential cellular target proteins, Rb and CBP/p300. Rb regulates the G1/S transition by controlling the transcription factor E2F. CBP/p300 is a transcriptional co-activator with intrinsic histone acetyl-transferase activity. This activity is regulated in a cell cycle dependent manner and shows a peak at the G1/S transition, suggesting a function for CBP/p300 in this crucial step of the cell cycle. Here, we have artificially modulated CBP/p300 levels in individual cells through microinjection of specific antibodies and expression vectors. We show that CBP/p300 is required for cell proliferation and has an essential function during the G1/S transition. Using the same microinjection system and GFP-reporter vectors, we demonstrate that CBP/p300 is essential for the activity of E2F, a transcription factor that controls the G1/S transition. In addition, our results suggest that CBP HAT activity is required both for the G1/S transition and for E2F activity. Thus CBP/p300 seems to be a versatile protein involved in opposing cellular processes, which raises the question of how its multiple activities are regulated.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas Portadoras , Proteínas de Ciclo Celular/metabolismo , Fase G1 , Fase S , Células 3T3 , Acetiltransferasas/genética , Animales , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Histona Acetiltransferasas , Ratones , Mutagénesis , Unión Proteica , Proteína 1 de Unión a Retinoblastoma , Eliminación de Secuencia , Proteína de Unión a TATA-Box , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Transcripción p300-CBPRESUMEN
The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure. CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways. In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP. CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases. Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity. The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain. These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule. Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways.