The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here, we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis.

In the classic paradigm of mammalian cell cycle control, Rb functions to restrict cells from entering S phase by sequestering E2F activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase1, 2. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examine the effects of E2f1, E2f2 and E2f3 triple deficiency in murine ES cells, embryos and small intestines. We show that in normal dividing progenitor cells E2F1-3 function as transcriptional activators, but contrary to current dogma, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells they function in complex with Rb as repressors to silence E2F targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2F1-3 from repressors to activators, leading to the superactivation of E2F responsive targets and ectopic cell divisions, and loss of E2f1-3 completely suppressed these phenotypes. This work contextualizes the activator versus repressor functions of E2F1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles in vivo.

SUMMARYThe endocycle is a variant cell cycle consisting of successive DNA synthesis and Gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using novel lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals.

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SUMMARY The evolutionarily ancient arm of the E2f family of transcription factors consisting of the two atypical members E2f7 and E2f8 is essential for murine embryonic development. However, the critical tissues, cellular processes and molecular pathways regulated by these two factors remain unknown. Using a series of fetal and placental lineage-specific cre mice we show that E2F7/E2F8 functions in extra-embryonic trophoblast lineages are both necessary and sufficient to carry fetuses to term. Expression profiling and biochemical approaches exposed the canonical E2F3a activator as a key family member that antagonizes E2F7/E2F8 functions. Remarkably, the concomitant loss of E2f3a normalized placental gene expression programs, corrected placental defects and fostered the survival of E2f7/E2f8 deficient embryos to birth. In summary, we identified a placental transcriptional network tightly coordinated by activation and repression through two distinct arms of the E2F family that is essential for extra-embryonic cell proliferation, placental development and fetal viability.

The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.The orderly progression through the cell cycle is governed by the coordinated activity of the E2F transcription factors (16). The E2Fs are a family of DNA binding proteins whose activity is regulated through their interaction with the retinoblastoma family (pRb, p107, and p130) (6). The E2Fs regulate a cohort of cell cycle regulatory genes, and they thereby coordinate the transitions through the different phases of the cell division cycle. The 8 E2F proteins (E2F1 to E2F8) are subdivided into two classes based on their transcriptional regulatory activities: the transactivating members (E2F1 to E2F3) and the transrepressing members (E2F4 to E2F8) (6). In general, the transactivating members drive cell cycle progression by inducing the expression of proliferation-associated genes, whereas the transrepressing members impede cell growth by repressing these genes.In addition to regulating cell growth, the E2F transcription factors can promote apoptosis through the activation of deathinducing genes, such as p73, caspases, Apaf1 and Bcl-2 homology region 3 (BH3)-only proteins (9,14,24,25,27,36). Ectopic E2f1 expression can induce both p53-dependent and p53-independent apoptosis (16). Although initial studies had suggested that only E2F1 could induce apoptosis, other studies have demonstrated that E2F2 and E2F3a also possess proapoptotic functions (7,17,31,38). Indeed, the ectopic expression of E2F3a has been shown to promote apoptosis both in vitro and in vivo (5,17,31,38). Interestingly, apoptosis induced by ectopic E2F3 expression can occur in a p53-independent manner (17, 31). However, the absence of E2f1 eliminates the proapoptotic function of E2F3a (17, 31). Thus, one interpretation of these studies is that among the activating E2Fs, only E2F1 has a specific proapoptotic function and that the induction of apoptosis by other E2Fs may be a consequence of deregulated E2F1 activity.Analysis of Rb null embryos has revealed that E2Fs can exhibit proapoptotic activities in a physiological setting (13). Loss of Rb results in extensive apoptosis in developing embryos (15, 21). Genetic deletion of either E2f1 or E2f3 suppresses cell death in the lens of Rb null embryos (39)...

E2F transcription factors regulate the progression of the cell cycle by repression or transactivation of genes that encode cyclins, cyclin dependent kinases, checkpoint regulators, and replication proteins. Although some E2F functions are independent of the Retinoblastoma tumor suppressor (Rb) and related family members, p107 and p130, much of E2F-mediated repression of S phase entry is dependent upon Rb. We previously showed in cultured mouse embryonic fibroblasts that concomitant loss of three E2F activators with overlapping functions (E2F1, E2F2, and E2F3) triggered the p53-p21Cip1 response and caused cell cycle arrest. Here we report on a dramatic difference in the requirement for E2F during development and in cultured cells by showing that cell cycle entry occurs normally in E2f1-3 triply-deficient epithelial stem cells and progenitors of the developing lens. Sixteen days after birth, however, massive apoptosis in differentiating epithelium leads to a collapse of the entire eye. Prior to this collapse, we find that expression of cell cycle-regulated genes in E2F-deficient lenses is aberrantly high. In a second set of experiments, we demonstrate that E2F3 ablation alone does not cause abnormalities in lens development but rescues phenotypic defects caused by loss of Rb, a binding partner of E2F known to recruit histone deacetylases, SWI/SNF and CtBP-polycomb complexes, methyltransferases, and other co-repressors to gene promoters. Together, these data implicate E2F1-3 in mediating transcriptional repression by Rb during cell cycle exit and point to a critical role for their repressive functions in cell survival.