RESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F6 , Proteína Potenciadora do Homólogo 2 de Zeste , Histona-Lisina N-Metiltransferase , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas/genética , Proteínas Repressoras/genética , Especificidade por Substrato , Fator de Transcrição DP1 , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of cancer. In the absence of other genetic alterations, this deregulation results in lack of differentiation, hyperproliferation and apoptosis. The pRB protein acts as a transcriptional repressor by targeting the E2F transcription factors, whose functions are required for entry into S phase. Increased E2F activity can induce S phase in quiescent cells--this is a central element of most models for the development of cancer. We show that although E2F1 alone is not sufficient to induce S phase in diploid mouse and human fibroblasts, increased E2F1 activity can result in S-phase entry in diploid fibroblasts in which the p53-mediated G1 checkpoint is suppressed. In addition, we show that E2F1 can induce S phase in primary mouse fibroblasts lacking pRB. These results indicate that, in addition to acting as an E2F-dependent transcriptional repressor, pRB is also required for the cells to retain the G1 checkpoint in response to unprogrammed proliferative signals.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fase G1/genética , Proteína do Retinoblastoma/fisiologia , Fase S/genética , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fibroblastos/citologia , Fibroblastos/fisiologia , Fase G1/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Fase S/fisiologia , Transdução de Sinais/genéticaRESUMO
Loss of function of both the p53 pathway and the retinoblastoma protein (pRB) pathway plays a significant role in the development of most human cancers. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis. In the past two years, mouse genetics and gene expression profiling have led to major advances in our understanding of how the pRB and p53 pathways regulate apoptosis and thus the development of tumours.