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...

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

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.

Deregulation of the Myc pathway and deregulation of the Rb pathway are two of the most common abnormalities in human malignancies. Recent in vitro experiments suggest a complex crossregulatory relationship between Myc and Rb that is mediated through the control of E2F. To evaluate the functional connection between Myc and E2Fs in vivo, we used a bitransgenic mouse model of Myc-induced T cell lymphomagenesis and analyzed tumor progression in mice deficient for E2f1, E2f2, or E2f3. Whereas the targeted inactivation of E2f1 or E2f3 had no significant effect on tumor progression, loss of E2f2 accelerated lymphomagenesis. Interestingly, loss of a single copy of E2f2 also accelerated tumorigenesis, albeit to a lesser extent, suggesting a haploinsufficient function for this locus. The combined ablation of E2f1 or E2f3, along with E2f2, did not further accelerate tumorigenesis. Mycoverexpressing T cells were more resistant to apoptosis in the absence of E2f2, and the reintroduction of E2F2 into these tumor cells resulted in an increase of apoptosis and inhibition of tumorigenesis. These results identify the E2f2 locus as a tumor suppressor through its ability to modulate apoptosis.YC is often amplified in human cancers, and mouse models of cancer have demonstrated a causal role for MYC overexpression in hematopoietic, mammary, and other cancer types (1-3). Deregulation of the Rb/E2F gene networks also represents common events in cancer (4). Like Myc, E2F can positively and negatively regulate the expression of hundreds of targets whose gene products are involved in a wide spectrum of biological processes, with a bias for genes that control cell cycle, apoptosis, and differentiation (5-10).Based on amino acid sequence analysis and structure-function studies in vitro, E2F family members can be artificially grouped into activator (E2F1-3) and repressor (E2F4-8) subclasses (11). Because of the intense interest in E2Fs as major regulators of the cell cycle, individual E2F family members have also been extensively studied in vivo by gene-targeting approaches in mice. E2f1 Ϫ/Ϫ mice are viable and suffer from impaired thymocyte apoptosis, defective negative selection, and testicular atrophy. E2f2 Ϫ/Ϫ mice are also viable and have a mild increase in hematopoietic and autoreactive T cells. Much later in life, a portion of E2f1 Ϫ/Ϫ and E2f2 Ϫ/Ϫ mice develop hematopoietic malignancies (12-15). These mutant phenotypes might reflect the particular bias for the expression of E2f1 and E2f2 in hematopoietic tissues. Although disruption of the E2f3 gene in a mixed genetic background yields viable mice, its disruption in pure strains results in embryonic lethality at around embryonic day 12.5 (G.L., unpublished observation). Surprisingly, embryos deficient for each of these E2Fs have no apparent defect in cellular proliferation, raising the possibility of functional redundancy among members of the activator subclass of E2Fs. There also appears to be functional redundancy among members of the repressor subclass, because disruption of E2f4,...

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