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.

The E2F family is conserved from C. elegans to mammals with some family members having transcription activation functions and others having repressor functions 1, 2 . Whereas C. elegans 3 and Drosophila melanogaster 4, 5 have a single E2F activator and repressor proteins, mammals evolved to have at least three activator and five repressor proteins 1,2,6 . Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a 3bki ; E2f3a 1ki ) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development. KeywordsE2F3a; E2F3b; Rb; development; proliferation; transcription and apoptosis Since the identification of the founding E2F family member, E2f1 7 , two distinct genes in lower eukaryotes and eight genes in higher eukaryotes have been identified to encode the signature DNA binding domain that endow these transcription factors with E2F 1,2,6 . Among the mammalian E2F activator subset, the E2f3 gene has emerged as the critical family member involved in the control of cell proliferation and development 8,9 . The E2f3 locus was originally thought to encode a single DNA binding activity, but was later shown to drive the expression of two related isoforms, E2f3a and E2f3b, from two distinct promoters 10 . Given the critical link between the E2f3 locus and the control of cell proliferation, we used homologous recombination to individually disrupt its two isoforms in mice and rigorously evaluate how their functions are integrated with that of other E2F activators. The inactivation of E2f3a or E2f3b was achieved by targeting exon 1a or 1b sequences, respectively, using LoxP-cre technology (Fig. 1a). Mice deleted for either exon 1a or exon 1b were identified by Southern blot and genomic PCR analysis (Fig. 1b). Specific ablation of E2f3a or E2f3b was confirmed by Western blot assays using total E2F3-specific antibodies (Fig. 1c).It was previously shown that inactivation of both E2f3a and E2f3b (E2f3 −/− ) in mice with a mixed strain background yielded offspring that developed rather normally 8, 9 , but we show here that breeding these mice into a pure strain background (∼98% pure) resulted in embryonic lethality ( Fig. 1e and Supplementary Fig. 1). Intercrossing E2f3 +/− mice of different pure backgrounds restored viability of E2f3 −/− mice, albeit with some observed strain-specific biase...

The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). Here we show that the specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb resulted in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppressed Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restored placental development and extended the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb −/− embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. We conclude that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population.[Keywords: Rb; development; placenta; stem cells] Supplemental material is available at http://www.genesdev.org. The retinoblastoma (Rb) tumor suppressor gene was identified more than two decades ago as the gene responsible for retinoblastoma, but has since been implicated in most human cancers. In contrast to retinoblastoma patients, inheritance of one deleted copy of Rb in mice did not induce retinoblastoma but did increase risk of pituitary and thyroid cancers (Jacks et al. 1992;Hu et al. 1994;Maandag et al. 1994;Williams et al. 1994;Robanus-Maandag et al. 1998;Yamasaki et al. 1998). Deletion of both copies of Rb in mice resulted in a broad range of severe abnormalities that lead to lethality by embryonic day 15.5 (E15.5) (Clarke et al. 1992;Jacks et al. 1992;Lee et al. 1992;Wu et al. 2003). Because Rb is normally expressed in all tissues of the mouse embryo, it was assumed that these developmental abnormalities were due to the absence of Rb protein in the tissues affected. Subsequent analysis of chimeric embryos suggested that Rb function is likely to be much more complex than initially suspected (Maandag et al. 1994;Lipinski et al. 2001). Indeed, recent findings showed that Rb-deficient embryos supplied with a wild-type placenta could develop to term and suggested a critical function of Rb in the placenta that might underlie many of the fetal developmental abnormalities observed in Rb −/− embryos Wu et al. 2003).Because Rb is involved in so many important pro-

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 E2f3 locus encodes two Rb-binding gene products, E2F3a and E2F3b, which are differentially regulated during the cell cycle and are thought to be critical for cell cycle progression. We targeted the individual inactivation of E2f3a or E2f3b in mice and examined their contributions to cell proliferation and development. Chromatin immunoprecipitation and gene expression experiments using mouse embryo fibroblasts deficient in each isoform showed that E2F3a and E2F3b contribute to G 1 /S-specific gene expression and cell proliferation. Expression of E2f3a or E2f3b was sufficient to support E2F target gene expression and cell proliferation in the absence of other E2F activators, E2f1 and E2f2, suggesting that these isoforms have redundant functions. Consistent with this notion, E2f3a ؊/؊ and E2f3b ؊/؊ embryos developed normally, whereas embryos lacking both isoforms (E2f3) died in utero. We also find that E2f3a and E2f3b have redundant and nonredundant roles in the context of Rb mutation. Analysis of double-knockout embryos suggests that the ectopic proliferation and apoptosis in Rb ؊/؊ embryos is mainly mediated by E2f3a in the placenta and nervous system and by both E2f3a and E2f3b in lens fiber cells. Together, we conclude that the contributions of E2F3a and E2F3b in cell proliferation and development are context dependent.

fThe retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1 ⌬G ) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1 ⌬G/⌬G mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1⌬G/⌬G MEFs display a magnitude of E2F target gene derepression similar to that of Rb1 ؊/؊ cells, even though Rb1 ⌬G/⌬G cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1 ⌬G/⌬G MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G 1 to arrest proliferation. Some Rb1 ⌬G/⌬G mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.T he retinoblastoma tumor suppressor protein (pRB) has a central role in the regulation of the G 1 -to-S-phase transition. Inactivation of its control over cell cycle progression is one of the most common events in cancer (1). The RB protein is thought to regulate entry into S phase through its ability to repress E2F-dependent transcription (2). In the G 1 phase of the cell cycle, a direct interaction between the large pocket domain of pRB (RBLP) and the transactivation domain of E2Fs blocks transcription and recruits chromatin regulators that maintain the cell in G 1 (3). Activation of cyclin-dependent kinases (CDKs) results in the phosphorylation of pRB and the release of E2F transcription factors (4). Free E2Fs then activate a transcriptional program that drives the cell into S phase (3). This model of pRB regulation of E2F dominates our understanding of G 1 -to-S-phase control. Much of our knowledge of this model was derived from studies using viral oncoproteins encoded by small DNA tumor viruses (5, 6). Of particular note, the human papillomavirus E7 protein has been shown to compete for pRB-E2F interactions to deregulate proliferation (7, 8). However, E7 must also target pRB for degradation in order to induce proliferation (8). Thus, the experimental system that gave rise to the pRB-E2F regulatory axis in cell cycle control also suggests that pRB may engage other growth-suppressing activities beyond E2F regulation. By comparison with the pRB-E2F pathway, we know very little about pRB's non-E2F-dependent growth control mechanisms and their relative contribution to cell cycle regulation and tumor suppressor activities.The minimal growth-suppressive region of pRB...

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.

The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution.