Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

ABSTRACT

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. TheCDC6 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 G₁controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.

ACKNOWLEDGMENTS

We thank Karin Holm, Alexandra Charlesworth, Stefania Lupo, Heiko Müller, and Emanuela Frittoli for technical assistance in sequencing, FACS, elutriation, and microinjection. Pier Paolo di Fiore is thanked for providing reagents, and we thank the members of the Lattanzio family who donated the microinjection setup. We thank Peter Cartwright and Simonetta Piatti for critical reading of the manuscript.

This work was supported in part by grants from the Human Frontiers Science Program and the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by fellowships from the European Community (G.H.), the Fondazione Italiana per la Ricerca sul Cancro (E.V.), the Fondazione per la Formazione Oncologica (A.W.), and the Danish Research Academy (B.O.P.).