The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway.

Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G 0 -G 1 phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G 1 arrests mediated by the p16 INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G 0 and early G 1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G 0 until mid-to late G1 phase whereas E2F-1 is active in late G 1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.E2F was originally defined as a cellular activity required for the transactivation of the adenovirus E2 promoter by the E1A oncoproteins (34). E1A binds directly to pRB, the product of the retinoblastoma susceptibility gene, and to two pRB relatives, p107 and p130 (40). These proteins, often referred to as pocket proteins, are regulators of the E2F family of transcription factors. Five E2F family members have so far been isolated by virtue of their ability to bind directly to pocket proteins and by homology cloning (38). The affinity of the E2Fs toward pocket proteins and DNA is greatly enhanced by their binding to one of two heterodimeric partners, DP-1 and DP-2/3 (38). The DNA tumor virus oncoproteins E1A, human papillomavirus E7, and simian virus 40 (SV40) large T antigen all regulate E2F-dependent transcription by binding and dissociating the pocket proteins from the E2F heterodimers (4).The E2Fs are believed to regulate the correct timing of transcription of several genes whose products are required for DNA replication (dyhydrofolate reductase, DNA polymerase ␣, and thymidine kinase) and progression through the cell cycle (cyclin A, cyclin E, CDC2, E2F-1, B-Myb...

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 G 1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.Although E2F was originally defined as a factor that binds specifically to an element in the adenovirus E2 promoter (42), it is now evident that E2F is essential for coordinating transcription during the mammalian cell cycle (for reviews, see references 12 and 72). A number of genes are found to be regulated by E2F, particularly during the transition from G 1 to S phase. To date, six members of the E2F family are known: E2F-1 through E2F-5 and the recently identified E2F-6 (11). Furthermore, two heterodimerization partners of the E2Fs, DP-1 and DP-2, have been isolated. The E2F transcription factors appear to be key downstream targets for the retinoblastoma protein pRB and two pRB-related proteins, p107 and p130 (reviewed in references 12 and 70). Binding of pRB family members (also called pocket proteins) to the E2F transcription factors results in transcriptional repression of E2F-regulated genes. Phosphorylation of the pocket proteins by cyclin-dependent kinases releases the pocket proteins from E2F, leading to derepression and/or activation of E2F-dependent genes and subsequent entry into S phase. The demonstration that deregulated E2F activity is sufficient to induce S phase in quiescent cells has provided a m...

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105Rb , and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105Rb complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105 Rb complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.Cells have evolved complex regulatory mechanisms to ensure orderly progression through the cell cycle. One of the major regulatory systems entails the interactions between members of the retinoblastoma susceptibility (Rb) protein family and E2F transcription factors. p105Rb and other members of the Rb family, p107 and p130, form complexes with various members of the E2F family and regulate their activity (15,43). E2F transcription factors exist as stable heterodimers with DP subunits. During the G 1 and G 0 phases of the cell cycle, complexes consisting of E2F-DP heterodimers and hypophosphorylated Rb proteins actively repress promoters that contain E2F binding sites (21,25,27,33,35,40,42,58,61). Many of the genes repressed in this fashion encode proteins that are required for entry into and transit through S phase, and E2F4-p105Rb and E2F4-p130 complexes are particularly active in transcriptional repression (9,39,53,54,57). In addition, complex formation with Rb family members protects E2F proteins from degradation by the ubiquitin-proteosome pathway and promotes the localization of E2F4 to the nucleus (22,26,37,38). In contrast, phosphorylation of Rb family members by cyclin-dependent kinases during cell cycle progression disrupts Rb-containing E2F complexes and releases free E2F-DP heterodimers that may t...