Atypical E2Fs Research Articles

Atypical E2Fs either Counteract or Cooperate with RB during Tumorigenesis Depending on Tissue Context.

Simple SummaryIn virtually all human malignancies, the CDK-RB-E2F pathway is dysregulated resulting in the activation of the E2F transcriptional network. Rb and atypical E2Fs are the most important negative regulators of E2F-dependent transcription during tumorigenesis. However, it is unknown whether they cooporate or act independently in tumor development. Here we show that combined loss of RB and atypical E2Fs in mice enhances tumorigenesis in the liver, while in the pituitary gland, we observe inhibition of tumorigenesis. These findings suggest that the interaction between RB and atypical E2Fs in controlling tumorigenesis occurs in a tissue cell-type specific manner.E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and −8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo. Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb+/− mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and −8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency.

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice.

Background and AimsUp‐regulation of the E2F‐dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F‐dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking.Approach and ResultsHere, we generated transgenic mice with doxycycline‐controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine‐induced liver tumors.ConclusionCollectively, our findings demonstrate that atypical E2Fs can override cell‐cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.

Complex Cartography: Regulation of E2F Transcription Factors by Cyclin F and Ubiquitin.

The E2F family of transcriptional regulators sits at the center of cell cycle gene expression and plays vital roles in normal and cancer cell cycles. Whereas control of E2Fs by the retinoblastoma family of proteins is well established, much less is known about their regulation by ubiquitin pathways. Recent studies placed the Skp1-Cul1-F-box-protein (SCF) family of E3 ubiquitin ligases with the F-box protein Cyclin F at the center of E2F regulation, demonstrating temporal proteolysis of both activator and atypical repressor E2Fs. Importantly, these E2F members, in particular activator E2F1 and repressors E2F7 and E2F8, form a feedback circuit at the crossroads of cell cycle and cell death. Moreover, Cyclin F functions in a reciprocal circuit with the cell cycle E3 ligase anaphase-promoting complex/cyclosome (APC/C), which also controls E2F7 and E2F8. This review focuses on the complex contours of feedback within this circuit, highlighting the deep crosstalk between E2F, SCF-Cyclin F, and APC/C in regulating the oscillator underlying human cell cycles.

Cyclin F‐dependent degradation of E2F7 is critical for DNA repair and G2‐phase progression

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S‐phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/CCdh1 during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCFcyclin F during G2. Cyclin F binds via its cyclin domain to a conserved C‐terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCFcyclin F remains stable during G2. Furthermore, SCFcyclin F can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCFcyclin F nor the CY motifs in the C‐terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical‐E2F‐dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F‐dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression.

Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes providing them with the ability to induce a permanent S‐phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post‐translational mechanisms. Here, we show that atypical E2Fs can be directly phosphorylated by checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that 14‐3‐3 protein isoforms interact with both E2Fs in a Chk1‐dependent manner. Strikingly, Chk1 phosphorylation and 14‐3‐3‐binding did not relocate or degrade atypical E2Fs, but instead, 14‐3‐3 is recruited to E2F7/8 target gene promoters to possibly interfere with transcription. We observed that high levels of 14‐3‐3 strongly correlate with upregulated transcription of atypical E2F target genes in human cancer. Thus, we reveal that Chk1 and 14‐3‐3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to DNA‐damaging therapeutic reagents.

Atypical E2f functions are critical for pancreas polyploidization.

The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2f transcription factors, E2f7 and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7 or E2f8 alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age.

Abstract A105: Checkpoint kinase 1 and 14-3-3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

Abstract The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes, providing them with the ability to induce a permanent S-phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post-translational mechanisms. Here, we show that E2F7 and E2F8 can be directly phosphorylated by Checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that multiple 14-3-3 protein isoforms interact with both E2Fs in a Chk1-dependent manner. We show that 14-3-3 proteins inhibit E2F7 and E2F8 repressor function without disrupting their binding to target gene promoters or subcellular translocation. In addition, we observed that high levels of 14-3-3 strongly correlate with upregulated transcription of atypical E2Fs target genes in human cancer. Thus, we reveal that Chk1 and 14-3-3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to replication stress and DNA-damaging therapeutic reagents, which are both known inducers of Chk1 activity. Citation Format: Ruixue Yuan. Checkpoint kinase 1 and 14-3-3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A105.

Atypical E2Fs inhibit tumor angiogenesis

Atypical E2F transcription factors (E2F7 and E2F8) function as key regulators of cell cycle progression and their inactivation leads to spontaneous cancer formation in mice. However, the mechanism of the tumor suppressor functions of E2F7/8 remain obscure. In this study we discovered that atypical E2Fs control tumor angiogenesis, one of the hallmarks of cancer. We genetically inactivated atypical E2Fs in epithelial and mesenchymal neoplasm and analyzed blood vessel formation in three different animal models of cancer. Tumor formation was either induced by application of 7,12-Dimethylbenz(a)anthracene/12-O-Tetradecanoylphorbol-13-acetate or by Myc/Ras overexpression. To our surprise, atypical E2Fs suppressed tumor angiogenesis in all three cancer models, which is in a sharp contrast to previous findings showing that atypical E2Fs promote angiogenesis during fetal development in mice and zebrafish. Real-time imaging in zebrafish displayed that fluorescent-labeled blood vessels showed enhanced intratumoral branching in xenografted E2f7/8-deficient neoplasms compared with E2f7/8-proficient neoplasms. DLL4 expression, a key negative inhibitor of vascular branching, was decreased in E2f7/8-deficient neoplastic cells, indicating that E2F7/8 might inhibit intratumoral vessel branching via induction of DLL4.

Abstract B14: Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.

Abstract E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phase. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Using molecular assays, time lapse microscopy, and flow cytometry we now demonstrate that E2F7 and E2F8 are substrates of the Anaphase Promoting Cyclosome/Complex (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN-mutant E2F7 during G1 severely impairs S-phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/CCdc20. Importantly, atypical E2Fs can activate the APC/CCdh1 by repressing its inhibitors Emi1, cyclin A, and cyclin E. In conclusion, we uncovered a feedback loop between atypical E2Fs and APC/CCdh1, which ensures balanced expression of cell cycle genes and normal cell cycle progression. Citation Format: Michiel Boekhout, Ruixue Yuan, Annelotte P. Wondergem, Hendrika A. Segeren, Elsbeth A. van Liere, Nesibu Awol, Imke Jansen, Rob MF Wolthuis, Alain de Bruin, Bart Westendorp. Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B14.

Abstract B15: Synergistic functions of E2F7 and E2F8 are critical to suppress stress induced skin cancer

Abstract E2F transcription factors are important regulators of the cell cycle, and unrestrained activation of E2F-dependent transcription is considered to be an important driver of tumor formation and progression. Although highly expressed in normal skin and skin cancer, the role of the atypical E2Fs, E2F7 and E2F8, in keratinocyte homeostasis, regeneration and tumorigenesis is unknown. Surprisingly, keratinocyte-specific deletion of E2F7 and E2F8 in mice did not interfere with skin development and wound healing. However, the rate for successful isolation and establishment of E2f7/8-deficient primary keratinocyte cultures was much higher than for wild-type keratinocytes. Moreover, E2f7/8-deficient primary keratinocytes proliferate more efficiently under stress conditions, such as low/high confluence or DNA damage. Application of in vivo stress using the DMBA/TPA skin carcinogenesis protocol revealed that combined inactivation of E2f7/8 enhanced tumorigenesis and accelerated malignant progression. Loss of atypical E2Fs resulted in increased expression of E2F target genes, including E2f1. Additional loss of E2f1 did not rescue, but worsened skin tumorigenesis. We show that loss of E2F7/8 triggers apoptosis via induction of E2F1 in response to stress, indicating that the tumor-promoting effect of E2F7/8 inactivation can be partially compensated via E2F1-dependent apoptosis. Importantly, E2F7/8 repressed a large set of E2F target gene that are highly expressed in human patients with skin cancer. Together, our studies demonstrate that atypical E2Fs act as tumor suppressors, most likely via transcriptional repression of cell cycle genes in response to stress. Citation Format: Bart Westendorp, Ingrid Thurlings, Lina Martinez-Lopez, Marjolein Zijp, Raoul Kuiper, Peter Tooten, HarmJan Vos, Boudewijn Burgering, Alain de Bruin. Synergistic functions of E2F7 and E2F8 are critical to suppress stress induced skin cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B15.

Synergistic functions of E2F7 and E2F8 are critical to suppress stress-induced skin cancer

E2F transcription factors are important regulators of the cell cycle, and unrestrained activation of E2F-dependent transcription is considered to be an important driver of tumor formation and progression. Although highly expressed in normal skin and skin cancer, the role of the atypical E2Fs, E2F7 and E2F8, in keratinocyte homeostasis, regeneration and tumorigenesis is unknown. Surprisingly, keratinocyte-specific deletion of E2F7 and E2F8 in mice did not interfere with skin development and wound healing. However, the rate for successful isolation and establishment of E2f7/8-deficient primary keratinocyte cultures was much higher than for wild-type keratinocytes. Moreover, E2f7/8-deficient primary keratinocytes proliferate more efficiently under stress conditions, such as low/high confluence or DNA damage. Application of in vivo stress using the DMBA/TPA skin carcinogenesis protocol revealed that combined inactivation of E2f7/8 enhanced tumorigenesis and accelerated malignant progression. Loss of atypical E2Fs resulted in increased expression of E2F target genes, including E2f1. Additional loss of E2f1 did not rescue, but worsened skin tumorigenesis. We show that loss of E2F7/8 triggers apoptosis via induction of E2F1 in response to stress, indicating that the tumor-promoting effect of E2F7/8 inactivation can be partially compensated via E2F1-dependent apoptosis. Importantly, E2F7/8 repressed a large set of E2F target genes that are highly expressed in human patients with skin cancer. Together, our studies demonstrate that atypical E2Fs act as tumor suppressors, most likely via transcriptional repression of cell cycle genes in response to stress.

The evolutionary history of the E2F and DEL genes in Viridiplantae

The E2 promoter binding factor (E2F) proteins are present in almost all eukaryotic organisms and are essential to control several processes, such as the cell cycle progression, cell division, DNA replication, and apoptosis. The E2F family comprises two different types of proteins: the typical E2Fs and atypical E2Fs, which differ structurally and have specific functions. The E2F gene family was described for the first time in plants in 1999, and since then several studies have focused on the functional aspects, but the evolutionary history of this gene family is still unknown. Here, we investigated the evolutionary history of the E2F gene family in plants. Our findings suggest that E2F proteins arose early after the emergence of the eukaryotic species, while DEL proteins appear to have arisen before the metazoan and plants origin probably through a partial duplication of an ancient E2F protein. Our data also suggest that E2Fs activators and repressors appeared twice during evolution, once in the metazoan lineage and again in the embryophyte lineage.

Feedback regulation between atypical E2Fs and APC / C C dh1 coordinates cell cycle progression

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.

Structural insights into the DNA-binding specificity of E2F family transcription factors.

The mammalian cell cycle is controlled by the E2F family of transcription factors. Typical E2Fs bind to DNA as heterodimers with the related dimerization partner (DP) proteins, whereas the atypical E2Fs, E2F7 and E2F8 contain two DNA-binding domains (DBDs) and act as repressors. To understand the mechanism of repression, we have resolved the structure of E2F8 in complex with DNA at atomic resolution. We find that the first and second DBDs of E2F8 resemble the DBDs of typical E2F and DP proteins, respectively. Using molecular dynamics simulations, biochemical affinity measurements and chromatin immunoprecipitation, we further show that both atypical and typical E2Fs bind to similar DNA sequences in vitro and in vivo. Our results represent the first crystal structure of an E2F protein with two DBDs, and reveal the mechanism by which atypical E2Fs can repress canonical E2F target genes and exert their negative influence on cell cycle progression.

Atypical E2F Dependent Dysregulation Of Cell Cycling By Microrna-142 Regulates T-Cell Responses and Experimental Graft-Versus-Host Disease

Noncoding microRNAs (miRs) have recently been linked to immune system function. We investigated the role of miR-142, a hematopoietic specific miR, in regulating T cell responses. To understand its specific function in T cell immunity we utilized homologous recombination technology and generated mutant mice bearing a targeted deletion of the miR-142 gene on the B6 background. The homozygous miR-142 knockout (KO) animals were viable, fertile and showed no apparent developmental anomalies. Thymic analyses of the miR142-/- animals demonstrated no significant differences when compared with WT littermate controls in total thymocytes, early thymic progenitors, DP and DN cells. Bone marrow studies demonstrated similar numbers of LSK+ HSCs while analyses of secondary lymphoid organs (spleen and popliteal lymph nodes) demonstrated similar absolute numbers of naïve T cells (CD44low62L+), memory like T cells (CD44hi62L+CCR7- and CD44hi62L-CCR7- cells), CD4+25+Foxp3+ cells, CD69+VLA4+CD3+ cells and weekly peripheral blood examination demonstrated similar lymphocyte counts as the WT littermates.Functional studies, however, demonstrated that when compared with WT T-cells, the KO T-cells showed significantly slower rate of proliferation by CFSE analyses upon stimulation anti-CD3+ and 28+ antibodies (P<0.003). They showed lower IL-2, IFNγ and IL-17 but greater amount of IL-6 production (P<0.001) and demonstrated greater apoptosis (P<0.02). Cell cycling analyses with flow cytometry demonstrated that a significantly greater percent of the miR142-/- T-cells were in the S and G2 phase (P<0.01) when compared with WT T-cells suggesting altered cell cycling. Similar reduction in proliferation, cytokine secretion by the miR142-/- T-cells was also observed upon in vitro stimulation with allogeneic BALB/c DCs.To determine the in vivo relevance of miR142 deficiency in T cells, we next utilized MHC mismatched B6àBALB/c model of allogeneic BMT. BALB/c animals were lethally irradiated (9Gy) and transplanted on day 0 with 5x106 BM from WT B6 animals along with 2x106 splenic CD90+T cells from the WT or miR142-/- donors. The allogeneic animals that received KO T-cells showed significantly less severe clinical, histopathological GVHD (GI tract on days 7 and 21) and mortality (P<0.02). Analyses of donor T cells on day 7 post-BMT demonstrated reduced expansion and IFNγ secretion (P<0.04) but showed no significant differences in the ratio of Treg:conventional T-cells between the WT and KO T-cell allogeneic recipients. To further confirm the specific role of miR142 deficiency, we next treated the WT animals with miR-142 anatgomir (days 1, 3 and 7) and found that it significantly reduced GVHD mortality (P<0.003). The KO T-cells also reduced GVHD mortality in a MHC matched minor mismatched B6→C3H.sw BMT model demonstrating strain independent effects.To further determine the miR142 specific molecular mechanisms we performed extensive bioinformatic analyses. In light of a defect in T cell cycling in miR142-/- T-cells, we focused on the putative miR142 targets that are known to regulate cell cycling. Two of the three bioinformatic programs suggested the following known regulators of cell cycling, EGR2, DAG, all eight E2F transcription factor family (the typical E2F1-6 and atypical E2F7-8) members as putative targets. We next performed DNA microarray analyses to determine differential gene expression patterns in miR142-/- and WT T cells, which demonstrated an increase in the expression (>15x) of only the atypical E2Fs, namely E2F7 and E2F8, but not in any of the other above predicted cell cycle regulating molecules. The increase in the expression of the atypical E2Fs in the miR142-/- T-cells was next confirmed by PCR analyses at baseline (unstimulated) and also sequentially at 6, 12, 24 and 48 hours following in vitro stimulation. Knockdown of E2F7 and E2F8 in miR142-/- T cells with sh-RNA rescued their proliferative responses and corrected the cell cycling defects to the levels comparable to WT T-cells, thus demonstrating that the atypical E2F7 and 8 are critical for miR-142 mediated regulation of T cells.Thus our data show (a) generation of a novel miR142 knockout mouse (b) demonstrate that miR142 regulates T cell responses in vitro and in vivo by targeting atypical E2Fs and (c) suggest that targeting miR142 in vivo with its antagomir might be a novel therapeutic strategy for regulating GVHD. Disclosures:No relevant conflicts of interest to declare.

Correction: Atypical E2fs Control Lymphangiogenesis through Transcriptional Regulation of Ccbe1 and Flt4

There was information omitted from the Funding statement. The correct Funding statement should read: This work was supported by the Netherlands Organization for Scientific Research (NWO-ALW 834.11.002 and 036.002.598). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Atypical E2fs Control Lymphangiogenesis through Transcriptional Regulation of Ccbe1 and Flt4

Lymphatic vessels are derived from venous endothelial cells and their formation is governed by the Vascular endothelial growth factor C (VegfC)/Vegf receptor 3 (Vegfr3; Flt4) signaling pathway. Recent studies show that Collagen and Calcium Binding EGF domains 1 protein (Ccbe1) enhances VegfC-dependent lymphangiogenesis. Both Ccbe1 and Flt4 have been shown to be indispensable for lymphangiogenesis. However, how these essential players are transcriptionally regulated remains poorly understood. In the case of angiogenesis, atypical E2fs (E2f7 and E2f8) however have been recently shown to function as transcriptional activators for VegfA. Using a genome-wide approach we here identified both CCBE1 and FLT4 as direct targets of atypical E2Fs. E2F7/8 directly bind and stimulate the CCBE1 promoter, while recruitment of E2F7/8 inhibits the FLT4 promoter. Importantly, inactivation of e2f7/8 in zebrafish impaired venous sprouting and lymphangiogenesis with reduced ccbe1 expression and increased flt4 expression. Remarkably, over-expression of e2f7/8 rescued Ccbe1- and Flt4-dependent lymphangiogenesis phenotypes. Together these results identified E2f7/8 as novel in vivo transcriptional regulators of Ccbe1 and Flt4, both essential genes for venous sprouting and lymphangiogenesis.

Atypical E2Fs drive atypical cell cycles

It is well documented that polyploid cells exist in mammalian tissues such as the placenta and liver, but their function and the mechanisms for their formation have remained elusive. Two studies now identify a role for atypical E2F transcription factors in promoting polyploidy in mammals and challenge present theories about the function of polyploidy.

E2F8 is essential for polyploidization in mammalian cells

Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.

Canonical and atypical E2Fs regulate the mammalian endocycle

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.