Coordinated activation and repression of E2F-responsive genes is believed to be pivotal for progression of normal cell cycle. Studies using lower organisms and mammalian cell culture systems support the idea that this balanced cumulative E2F activity in different phases of cell cycle is executed by canonical E2F activators (E2F1-E2F3) and repressors (E2F4-E2F6). But, recent studies in mouse models with ablation of these factors have failed to provide substantial molecular and phenotypic evidence to support this notion. The evolutionarily ancient arm of E2F family consisting of newly identified atypical E2F repressors, E2F7 and E2F8, is known to be critical for mammalian embryonic development. Germline ablation of E2f7 and E2f8 leads to severe placental defects culminating in embryonic lethality by embryonic age 11.5. Remarkably, the concomitant loss of E2f3a normalized placental and fetal gene expression programs, corrected placental defects and fostered the survival of E2f7/E2f8 deficient embryos to birth. Using expression profiling and biochemical approaches, we show that atypical E2F repressors and canonical E2F activator E2F3a form key antagonistic regulators of G1-S transcriptional program during placental development. We thus provide the first in vivo evidence to show that balanced repression by E2F7 and E2F8 and activation by canonical E2F activators coordinate expression of E2F-resonsive genes in mitotic cell cycle during mammalian development.
Utilizing a combination of novel and established lineage-specific cre mice we also demonstrate that these 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), converge on the regulation physiological polyploidy in vivo. Ablation of atypical repressors diminished ploidy in the trophoblast giant cells in the placenta, hepatocytes in the liver and megakaryocytes in bone marrow, whereas ablation of canonical activators in the trophoblast giant cells and hepatocytes augmented genome ploidy. In addition, the severe reduction of ploidy caused by E2f7/E2f8 deficiency could be rescued significantly by added loss of E2f1 and E2f3a. Taken together, the results presented within provide the first in vivo evidence for a direct role of E2Fs in regulating non-traditional cell cycles in mammals. Though polyploidy has been demonstrated to be essential for metazoan development and is widely believed to have conserved physiological functions in mammals, to our surprise, reduction of ploidy caused by loss of E2f7 and E2f8 had no apparent adverse impact on placental, hepatic or megakaryocyte physiology. In summary, our studies reveal novel functions of mammalian atypical E2Fs and provide significant insight into mechanism of cell cycle phase dependent regulation of E2F-responsive genes in normal mitotic cell cycle and variant cell cycles in mammals.