RESUMEN
The transcriptional repressor E2F6 has been identified as a component of two distinct polycomb group protein (PcG)-containing complexes, suggesting a mechanism for the recruitment of repressive complexes to target sequences in DNA. Whereas one complex is involved in the repression of classic E2F target genes in G0, a role for E2F6 within the cell cycle has yet to be defined. We searched for novel E2F6-binding proteins using a yeast two-hybrid screen and identified the PcG protein, EPC1. We showed that, both in vitro and in vivo, E2F6, DP1, and EPC1 form a stable core complex with repressive activity. Furthermore, we identified the proliferation-specific PcG, EZH2, as an EPC1-interacting protein. Using affinity purification, we showed that E2F6, DP1, EPC1, EZH2, and Sin3B co-elute, suggesting the identification of a novel E2F6 complex that exists in vivo in both normal and transformed human cell lines. EZH2 is required for cellular proliferation and consistent with this, EZH2 elutes with the E2F6-EPC1 complex only in proliferating cells. Thus we have identified a novel E2F6-PcG complex (E2F6-EPC1) that interacts with EZH2 and may regulate genes required for cell cycle progression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Factor de Transcripción E2F6 , Proteína Potenciadora del Homólogo Zeste 2 , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas/genética , Proteínas Represoras/genética , Especificidad por Sustrato , Factor de Transcripción DP1 , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.