Transcriptional repression of the E2F-1 gene by interferon-alpha is mediated through induction of E2F-4/pRB and E2F-4/p130 complexes.
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E2F is a heterodimeric transcription factor composed of one of five E2F subunits (E2F-1 to E2F-5) and a DP subunit. E2F regulates the expression of several growth-promoting genes, and thus, can be a target of antiproliferative action of interferons (IFNs). In this study, we investigated the mechanisms whereby IFN-alpha suppresses transcription of the E2F-1 gene. Transfection studies revealed that E2F-1 promoter was functionally divided into two parts: upstream activation sequences (UAS) and a downstream negative-regulatory element (E2F-binding sites). When cells were proliferating, transcription of the E2F-1 gene was primarily driven by the UAS, while E2F sites were not involved in activation. IFN-alpha markedly reduced E2F-1 promoter activity, but introduction of non-binding mutation at the E2F sites completely abrogated the inhibition. Free E2F4 was found to be the predominant species bound to the E2F sites in proliferating cells. IFN-alpha induced upregulation of E2F-4 along with dephosphorylation of pRB and p130, which resulted in the formation of E2F-4/pRB and E2F-4/p130 complexes on the E2F-1 promoter. These complexes function as transcriptional repressors to inhibit E2F-1 mRNA expression. Our findings indicate that E2F-4 is a critical regulator of E2F-1, which offer an excellent paradigm for understanding functional diversity within the E2F family. (+info)
Regulation of the G1/S transition phase in mesangial cells by E2F1.
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It has been established that E2F transcription factors are essential for the regulation of the cell cycle. The E2Fs play an important role in G1/S transition phase, as they regulate the activation of several genes whose products are required for DNA synthesis. E2Fs bind to the retinoblastoma protein family and their transcriptional activities are suppressed in the G0 and early G1 phases. The E2F family consists of a group of five closely related proteins (E2F1 through E2F5). Proliferation of the mesangial cell is a common feature of many glomerular diseases, but the regulation of mesangial cell cycle has not been clarified, nor has the participation of the E2F family in mesangial cells. To elucidate the mechanisms of G1/S transition phase in mesangial cells, we investigated the roles of the E2F family in the mesangial cell cycle. In primary cultured mesangial cells, the protein expression of E2F1 through E2F3 was induced by fetal calf serum (FCS) stimulation. E2F1 especially was strongly induced by mitogenic stimulation. The E2F4 protein was abundantly expressed in the quiescent state and was slightly increased by FCS stimulation. We considered E2F1 to be representative of the E2F family, and used adenovirus-mediated gene transfer to investigate the function of E2F1 to show that overexpression of E2F1 promoted cell cycle progression as measured by a flow cytometer. Furthermore, we investigated the effect of E2F1 overexpression to cyclin D1 and cyclin E expression. Because we previously reported that the regulation of G1 cyclins is a key factor in the G1/S transition phase in mesangial cells, we showed that overexpression of E2F1 induced protein expression of cyclin D1 and cyclin E and increased promoter activity. Thus, we conclude that E2F1 plays an important role in the G1/S transition phase and acts on the mesangial cell cycle through two distinct pathways: (1) E2F1 directly transcribes an S-phase gene, and (2) E2F1 promotes cell cycle progression via the induction of cyclin D1 and cyclin E. (+info)
Roles of E2F1 in mesangial cell proliferation in vitro.
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Roles of E2F1 in mesangial cell proliferation in vitro. BACKGROUND: The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. METHODS: To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. RESULTS: E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. CONCLUSIONS: In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins. (+info)
Subcellular compartmentalization of E2F family members is required for maintenance of the postmitotic state in terminally differentiated muscle.
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Maintenance of cells in a quiescent state after terminal differentiation occurs through a number of mechanisms that regulate the activity of the E2F family of transcription factors. We report here that changes in the subcellular compartmentalization of the E2F family proteins are required to prevent nuclei in terminally differentiated skeletal muscle from reentering S phase. In terminally differentiated L6 myotubes, E2F-1, E2F-3, and E2F-5 were primarily cytoplasmic, E2F-2 was nuclear, whereas E2F-4 became partitioned between both compartments. In these same cells, pRB family members, pRB, p107, and p130 were also nuclear. This compartmentalization of the E2F-1 and E2F-4 in differentiated muscle cells grown in vitro reflected their observed subcellular location in situ. We determined further that exogenous E2F-1 or E2F-4 expressed in myotubes at levels fourfold greater than endogenous proteins compartmentalized identically to their endogenous counterparts. Only when overexpressed at higher levels was inappropriate subcellular location for these proteins observed. At these levels, induction of the E2F-regulated genes, cyclins A and E, and suppression of factors associated with myogenesis, myogenin, and p21(Cip1) was observed. Only at these levels of E2F expression did nuclei in these terminally differentiated cells enter S phase. These data demonstrate that regulation of the subcellular compartmentalization of E2F-family members is required to maintain nuclei in a quiescent state in terminally differentiated cells. (+info)
Deregulated E2F transcriptional activity in autonomously growing melanoma cells.
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Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1-E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F-pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4-p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors. (+info)
CpG methylation as a mechanism for the regulation of E2F activity.
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Regulation of gene expression in mammals through methylation of cytosine residues at CpG dinucleotides is involved in the development and progression of tumors. Because many genes that are involved in the control of cell proliferation are regulated by members of the E2F family of transcription factors and because some E2F DNA-binding sites are methylated in vivo, we have investigated whether CpG methylation can regulate E2F functions. We show here that methylation of E2F elements derived from the dihydrofolate reductase, E2F1, and cdc2 promoters prevents the binding of all E2F family members tested (E2F1 through E2F5). In contrast, methylation of the E2F elements derived from the c-myc and c-myb promoters minimally affects the binding of E2F2, E2F3, E2F4, and E2F5 but significantly inhibits the binding of E2F1. Consistent with these studies, E2F3, but not E2F1, activates transcription through methylated E2F sites derived from the c-myb and c-myc genes whereas both E2F1 and E2F3 fail to transactivate a reporter gene that is under the control of a methylated dihydrofolate reductase E2F site. Together, these data illustrate a means through which E2F activity can be controlled. (+info)
Expression of the E2F family of transcription factors during murine development.
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The E2F family of transcription factors plays a crucial role in the control of cell cycle progression and regulation of cellular proliferation, both processes fundamental to mammalian development. In the present study, we have examined the levels of expression of the six currently identified E2F proteins in murine embryos/fetuses as a function of gestational age, compared the expression of these six proteins in selected developing and adult tissues, and examined E2F expression in the embryonic murine palate, a tissue in which perturbation of proliferation is associated with induction of cleft palate. Our results indicate that: 1) multiple forms of individual E2F family members are present in embryonic, fetal and adult cells/tissues; 2) each of the six E2Fs is expressed in a tissue specific manner in both adult and embryonic/fetal organs; 3) certain forms of individual E2F family members are preferentially detected in adult tissues, whereas others are preferentially expressed in embryonic/fetal tissues; 4) expression of the various E2Fs and their isoforms follows distinct temporal patterns during murine gestation; and 5) individual E2F family members also exhibit differential patterns of temporal expression during murine palatogenesis. (+info)
E2F proteins are posttranslationally modified concomitantly with a reduction in nuclear binding activity in cells infected with herpes simplex virus 1.
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The transition from G(1) to S phase in the cell cycle requires sequential activation of cyclin-dependent kinase 4 (cdk4) and cdk2, which phosphorylate the retinoblastoma protein, causing the release of E2F. Free E2F upregulates the transcription of genes involved in S phase and cell cycle progression. Recent studies from this and other laboratories have shown that herpes simplex virus 1 stabilizes cyclin D3 early in infection and that early events in viral replication are sensitive to inhibitors of some cdks. On the other hand cdk2 is not activated. Here we report studies on the status of members of E2F family in cycling HEp-2 and HeLa cells and quiescent serum-starved, contact-inhibited human lung fibroblasts. The results show that (i) at 8 h postinfection or thereafter, E2F-1 and E2F-5 were posttranslationally modified and/or translocated from nucleus to the cytoplasm, (ii) E2F-4 was hyperphophorylated, and (iii) overall, E2F binding to cognate DNA sites was decreased at late times after infection. These results concurrent with those cited above indicate that late in infection activation of S-phase genes is blocked both by posttranslational modification and translocation of members of E2F family to inactive compartments and by the absence of active cdk2. The observation that E2F were also posttranslationally modified in quiescent human lung fibroblasts that were not in S phase at the time of infection suggests that specific viral gene products are responsible for modification of the members of E2F family and raises the possibility that in infected cells, activation of the S phase gene is an early event in viral infection and is then shut off at late times. This is consistent with the timing of stabilization of cyclin D3 and the events blocked by inhibitors of cdks. (+info)