The transcription factor E2F-1 is a downstream target of RB action.
Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular transcription factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated transcriptional repression.
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Dana Farber Cancer Institute, Boston, Massachusetts 02115.
Livingston
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Ewen
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Sellers
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Arany
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Kaelin
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D001483:Base Sequence; D001665:Binding Sites; D001859:Bone Neoplasms; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016193:G1 Phase; D016161:Genes, Retinoblastoma; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D012516:Osteosarcoma; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D015345:Oligonucleotide Probes; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase