Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F.
The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.
Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.
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G
Hateboer
A
A
Wobst
B O
BO
Petersen
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L
Le Cam
E
E
Vigo
C
C
Sardet
K
K
Helin
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D003001:Cloning, Molecular; D019927:Cyclin E / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D018983:DNA Footprinting; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000502:physiology; D056465:Retinoblastoma-Binding Protein 1; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000502:physiology*
C533431:Arid4a protein, mouse; C066503:CDC6 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D050680:Transcription Factor DP1; D014157:Transcription Factors; C082462:cdc18 protein, S pombe; D012313:RNA; D004247:DNA