The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

The E2F transcription factor family plays a crucial and well established role in cell cycle progression. Deregulation of E2F activities in vivo leads to developmental defects and cancer. Based on current evidence in the field, mammalian E2Fs can be functionally categorized into either transcriptional activators (E2F1, E2F2, and E2F3a) or repressors (E2F3b, E2F4, E2F5, E2F6, and E2F7). We have identified a novel E2F family member, E2F8, which is conserved in mice and humans and has its counterpart in Arabidopsis thaliana (E2Ls). Interestingly, E2F7 and E2F8 share unique structural features that distinguish them from other mammalian E2F repressor members, including the presence of two distinct DNA-binding domains and the absence of DP-dimerization, retinoblastoma-binding, and transcriptional activation domains. Similar to E2F7, overexpression of E2F8 significantly slows down the proliferation of primary mouse embryonic fibroblasts. These observations, together with the fact that E2F7 and E2F8 can homodimerize and are expressed in the same adult tissues, suggest that they may have overlapping and perhaps synergistic roles in the control of cellular proliferation.

The mammalian E2F family of transcription factors plays a crucial role in the regulation of cellular proliferation, apoptosis, and differentiation. Consistent with its biological role in a number of important cellular processes, E2F regulates the expression of genes involved in cell cycle, DNA replication, DNA repair, and mitosis. It has proven difficult, however, to determine the specific roles played by the various known family members in these cellular processes. The work presented here now extends the complexity of this family even further by the identification of a novel E2F family member, which we now term E2F7. Like the expression of the known E2F activators, E2F1, E2F2, and E2F3, the expression of E2F7 is growth-regulated, at least in part, through E2F binding elements on its promoter, and its protein product is localized to the nucleus and associates with DNA E2F recognition sites with high affinity. A number of salient features, however, make this member unique among the E2F family. First, the E2F7 gene encodes a protein that possesses two distinct DNA-binding domains and that lacks a dimerization domain as well as a transcriptional activation and a retinoblastoma-binding domain. In contrast to the E2F activators, E2F7 can block the E2F-dependent activation of a subset of E2F target genes as well as mitigate cellular proliferation of mouse embryo fibroblasts. These findings identify E2F7 as a novel member of the mammalian E2F transcription factor family that has properties of a transcriptional repressor capable of negatively influencing cellular proliferation.

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The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.

The E2F family of transcription factors is believed to have an essential role in the control of cellular proliferation by regulating the transcription of genes involved in cell cycle progression. Previous work has demonstrated that the targeted inactivation of E2f1, E2f2, and E2f3 results in elevated p21 CIP1 protein levels, loss of E2F target gene expression, and cell cycle arrest at G 1 /S and G 2 /M, suggesting a strict requirement for these E2Fs in the control of normal cellular proliferation. We now demonstrate that E2f1, E2f2, and E2f3 are also required for oncogene-mediated transformation of mouse embryonic fibroblasts. Analysis of synchronized populations of mouse embryonic fibroblasts revealed that the inactivation of p21 CIP1 restores the ability of E2f1-3-deficient cells to enter and transit through G 1 /S (but not G 2 /M). In contrast, loss of p53 restored the ability of these cells to progress through both G 1 /S and mitosis, leading to their continued proliferation. The inactivation of p53 (but not p21 CIP1 ) rendered E2f1-3-deficient cells sensitive to transformation and tumorigenesis. These results suggest that the negative regulation of the p53-p21 CIP1 axis by the E2F1-3 factors is critical for cell cycle progression and cellular transformation.The timely entry and proper progression through the cell division cycle requires an ordered series of cyclin-dependent phosphorylation events and the execution of a well orchestrated transcriptional program. Growth factor-dependent activation of cyclin-dependent kinases (cdks) 3 cdk4/6 and cdk2 results in the sequential phosphorylation of the Retinoblastoma (Rb) family of pocket proteins. This phosphorylation results in the release of E2F transcription factors from Rb-E2F complexes and activation of E2F target gene expression, leading to cell cycle progression (1, 2). In addition to the temporal association with the Rb family of pocket proteins, E2F activity is also regulated through cell cycle-dependent expression, post-translational modifications, interactions with cofactors, and protein degradation (1-3).Several decades of work support the view that E2F represents an important activity that controls cellular proliferation (4 -6). More recent studies suggest a particularly important role for the E2F3 family member in the control of gene expression during the G 1 /S transition. Inhibitory antibodies against E2F3 (but not E2F1) blocked the entry of cells into S phase (7). Consistent with these results, gene-targeting strategies in mice demonstrated a significant role for E2F3 in the proliferation of MEFs (8, 9). Some functional redundancy among family members is apparent, however, as the combined inactivation of E2f1, E2f2, and E2f3 led to a much more profound proliferation arrest that was manifested at multiple stages of the cell cycle (9). Interestingly, this block was accompanied by the induction of p53 transcriptional activity and the elevation of its target genes including p21 CIP1 (9, 10). These observations raise the possibility that E2F1-3 activa...

Graphical Abstract Highlights d E2F expression during cell division, differentiation, and quiescence is measured in vivo d E2F3A, E2F8, and E2F4 accumulate sequentially in the nucleus of cycling cells d E2F3A-4 nuclear accumulation controls gene expression during cell-cycle exit d Deep learning tools are applied to nuclear segmentation of complex mammalian tissues In Brief The study of E2Fs in vivo has been challenging. Cuitiñ o et al. reconstruct the spatiotemporal expression of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the mammalian cell cycle and propose that orchestrated accumulation of different E2F combinations control gene expression in proliferating (E2F3A-8-4) and differentiating (E2F3A-4) cells. SUMMARYOrchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cellcycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G 0 , G 1 -S, and S-G 2 -M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the singlecell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.

E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.