Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.
Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.
Wu
C L
CL
Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.
Classon
M
M
Dyson
N
N
Harlow
E
E
D000818:Animals; D001859:Bone Neoplasms; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D050684:E2F Transcription Factors; D005260:Female; D016193:G1 Phase; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008322:Mammals; D012516:Osteosarcoma; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D056465:Retinoblastoma-Binding Protein 1; C493735:TFDP1 protein, human; D050680:Transcription Factor DP1; D014157:Transcription Factors