RESUMEN
Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Proteínas de Homeodominio/metabolismo , Músculo Esquelético/embriología , Proteína MioD/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Animales , Secuencia de Bases/genética , Sitios de Unión/genética , Tipificación del Cuerpo/genética , Ensamble y Desensamble de Cromatina/genética , Regulación del Desarrollo de la Expresión Génica/genética , Marcadores Genéticos/genética , Proteínas de Homeodominio/genética , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Proteína MioD/genética , Células 3T3 NIH , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína/genética , Factores de Transcripción/genéticaRESUMEN
The transcriptional coactivators p300 and pCAF are necessary for the myogenic factor MyoD to initiate the expression of skeletal muscle genes. In addition to mediating histone acetylation, both of these factors can acetylate MyoD; however, the complexity of cellular systems used to study MyoD has impeded delineation of the specific roles of these two acetyltransferases. Therefore, we established a MyoD-dependent in vitro transcription system that permits us to determine the roles of p300 and pCAF during MyoD-dependent transcriptional activation. Consistent with results from cellular systems, we demonstrate that maximal levels of transactivation in vitro require both p300 and pCAF, as well as the cofactor acetyl CoA. Dissection of the steps leading to transcription initiation revealed that the activities of p300 and pCAF are not redundant. During the initial stages of transactivation, p300 acetylates histone H3 and H4 within the promoter region and then recruits pCAF to MyoD. Once tethered to the promoter, pCAF acetylates MyoD to facilitate the transactivation process. Thus, we have established that pCAF and p300 carry out sequential and functionally distinct events on a promoter leading to transcriptional activation. Further dissection of this in vitro transcription system should be highly useful toward elucidating the mechanism by which coactivators facilitate differential gene expression by MyoD.