RESUMEN
Sonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).
Asunto(s)
Embrión de Mamíferos/metabolismo , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Polidactilia/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos/citología , Elementos de Facilitación Genéticos/genética , Factor de Transcripción de la Proteína de Unión a GA/genética , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Hibridación in Situ , Esbozos de los Miembros/citología , Ratones , Ratones Transgénicos , Mutación Puntual/genética , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos Reguladores de la Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease.