RESUMEN
Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1(-/-) mice have a nearly complete perinatal lethality. Histologic examination of Ets1(-/-) embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1(-/-) mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.
Asunto(s)
Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Corazón/embriología , Cresta Neural/citología , Proteína Proto-Oncogénica c-ets-1/metabolismo , Agrecanos/metabolismo , Animales , Western Blotting , Butadienos/farmacología , Cartílago/embriología , Cartílago/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Movimiento Celular/genética , Condrocitos/citología , Condrocitos/metabolismo , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Corazón/efectos de los fármacos , Cardiopatías Congénitas/genética , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Cresta Neural/embriología , Nitrilos/farmacología , Proteína Proto-Oncogénica c-ets-1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXD/metabolismoRESUMEN
FOG-2 is a transcriptional co-regulator that is required for cardiac morphogenesis as mice deficient in this factor die during mid-gestation of cardiac malformations. FOG-2 interacts with GATA4 to attenuate GATA4-dependent gene expression. The first 12 amino acids of FOG-2 (the FOG Repression Motif) are necessary to mediate this repression. To determine the mechanism by which the FOG Repression Motif functions, we identified 7 polypeptides from rat cardiac nuclear extracts that co-purified with a GST-FOG-2 fusion protein. All proteins identified are members of the NuRD nucleosome remodeling complex. Using in vitro binding and co-immunoprecipitation assays, we demonstrate that Metastasis-Associated proteins (MTA)-1, 2 and 3 and Retinoblastoma binding proteins RbAp46 and RbAp48 interact with FOG-2, but not with a mutant form of FOG-2 that is unable to repress transcription. Furthermore, we define a novel domain located in the C-terminal portion of MTA-1 that mediates the FOG-2/MTA-1 interaction. We also demonstrate that knockdown of MTA protein expression dramatically impairs the ability of FOG-2 to repress GATA4 activity. Finally, we show that the zinc finger domain of MTA-1 is required for FOG-2-mediated transcriptional repression and that this domain interacts with RbAp46 and RbAp48 subunits of the NuRD complex. Together, these results demonstrate the importance of FOG-2/MTA/RbAp interactions for FOG-2-mediated transcriptional repression and further define the molecular interactions between the FOG Repression Motif and the NuRD complex.