The novel E2F7 and E2F8 family members are thought to function as transcriptional repressors important for the control of cell proliferation in vitro. However, as the most recently indentified and least studied E2F members, their biological functions in vivo remain unknown. Here we have analyzed the consequences of inactivating E2f7 and E2f8 in mice. While loss of either E2f7 or E2f8 did not significantly affect mouse development, their combined ablation resulted in massive apoptosis, dilated blood vessels and severe placental defects, culminating in embryonic lethality by day 11.5. E2F7 and E2F8 formed homo-dimers and hetero-dimers that could recruit various co-repressor complexes to E2F binding sites of target promoters, including E2f1. Consistent with their important role in transcriptional repression, mouse embryonic fibroblasts (MEFs) deficient for E2f7 and E2f8 expressed abnormally high levels of E2f1 and other E2F-target mRNAs. These double knockout MEFs proliferated surprisingly well, but accumulated high levels of p53 protein and were hypersensitive to DNA damage-induced cell death. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis observed in double mutant embryos but failed to rescue their embryonic lethality.
In order to identify the leading cause of fetal death, the critical tissues, related cellular processes and molecular mechanisms of E2F7 and E2F8 function, we utilized conditional knockout strategies to show that extra-embryonic function of E2F7 and E2F8 is both necessary and sufficient for embryo development, and thus define the placental abnormalities as the leading cause of embryonic lethality in E2f7-/-E2f8-/- embryos. Consistent with this genetic finding, cellular examination of double mutant placentas revealed ectopic DNA replication and inappropriate mitosis in certain cell lineages of placenta. We also provided two distinct molecular mechanisms of E2F7 and E2F8 function to explain these cellular phenotypes. On one hand, E2F7 and E2F8 could directly repress a novel transcription network that is critical for controlling DNA replication. On the other hand, E2F7 and E2F8 could indirectly regulate the expression of mitotic cyclins and therefore coordinate mitosis progression. We believe disruption of these pathways in E2f7-/-E2f8-/- placentas culminates in G1-S and G2-M specific defects and, presumably, leads to the profound placental abnormality and its associated fetal death.
In summary, this study clearly provides the first in vivo evidence for the biological functions of E2F7 and E2F8. We demonstrate, as a unique repressive arm of the E2F program, E2F7 and E2F8 are not only critical for the control of apoptotic in the fetus, but also essential for the regulation of cell cycle progression in the placenta. We conclude that the synergistic function of E2F7 and E2F8 is essential for embryo development.