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Results for “E2F1 Transcription Factor”

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At least 19 recordsLinked to original sources
PubMed1999

An early S phase checkpoint is regulated by the E2F1 transcription factor.

The E2F1 transcription factor regulates transit of cells through the S phase checkpoint, dependent on its association with cyclin A/cdk2. Expression in cells of a mutant E2F1 lacking the cyclin A/cdk2 binding domain leads to partial arrest of cells at the S phase checkpoint. When subconfluent growing cells expressing this mutant E2F1 are analyzed in detail, it is shown here that they display a significantly reduced incorporation of 3H-thymidine into the DNA of each S phase cell, compared to control cells or to cells overexpressing full-length E2F1. Further, when cells are blocked at the G1/S phase border and released, there is a clear reduction in the amount of 3H-thymidine incorporated into the DNA of S phase cells by 1.5 hours post release. Considering a normal 6 hour S phase duration, the results show that the S phase checkpoint mediated by E2F1 is not a late S phase event but an early one.

3T3 Cells↗
PubMed2003

Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor.

Necdin is a growth suppressor expressed predominantly in postmitotic neurons and implicated in their terminal differentiation. Necdin shows a moderate homology to the MAGE family proteins, the functional roles of which are largely unknown. Human genes encoding necdin, MAGEL2 (necdin-like 1), and MAGE-G1 (necdin-like 2) are located in proximal chromosome 15q, a region associated with neurodevelopmental disorders such as Prader-Willi syndrome, Angelman syndrome, and autistic disorder. The necdin and MAGEL2 genes are subjected to genomic imprinting and suggested to be involved in the etiology of Prader-Willi syndrome. In this study, we compared biochemical and functional characteristics of murine orthologs of these necdin-related MAGE proteins. The colony formation and bromodeoxyuridine incorporation analyses revealed that necdin and MAGE-G1, but not MAGEL2, induced growth arrest. Necdin and MAGE-G1 interacted with the transcription factor E2F1 via its transactivation domain, repressed E2F1-dependent transcription, and antagonized E2F1-induced apoptosis of N1E-115 neuroblastoma cells. In addition, necdin and MAGE-G1 interacted with the p75 neurotrophin receptor via its distinct intracellular domains. In contrast, MAGEL2 failed to bind to these necdin interactors, suggesting that MAGEL2 has no necdin-like function in developing brain. Overexpression of p75 translocated necdin and MAGE-G1 in the proximity of the plasma membrane and reduced their association with E2F1 to facilitate E2F1-induced death of neuroblastoma cells. These results suggest that necdin and MAGE-G1 target both E2F1 and p75 to regulate cell viability during brain development.

Animals↗
PubMed2001

The transcription factor E2F1 promotes dopamine-evoked neuronal apoptosis by a mechanism independent of transcriptional activation.

The E2F1 transcription factor plays an important role in promoting neuronal apoptosis; however, it is not clear how E2F1 does this. Here we show that E2F1 is involved in dopamine (DA)-evoked apoptosis in cerebellar granule neurons (CGNs). E2F1 -/- CGNs and CGNs expressing an antisense E2F1 cDNA were significantly protected from DA-toxicity relative to controls. The neuronal protection was accompanied by significantly reduced caspase 3 activity. E2F1-mediated neuronal apoptosis did not require activation of gene transcription because: (1) ectopic expression of E2F1 or its mutants lacking the transactivation domain induced neuronal apoptosis, whereas an E2F1 mutant lacking the DNA-binding domain did not; (2) under all of these conditions, known E2F1 target genes including cyclin A, cdc2 and p19(ARF) were not induced; and (3) DA-evoked neuronal apoptosis was associated with up-regulated E2F1, but not transcription of its target genes. Finally, E2F1-mediated neuronal apoptosis was associated with reduced nuclear factor (NF)-kappaB DNA-binding activity. Taken together, these data suggest that E2F1 promotes DA-evoked caspase 3-dependent neuronal apoptosis by a mechanism independent of gene transactivation, and this may possibly occur through inhibition of anti-apoptotic genes including NF-kappaB.

Animals↗
PubMed1997

Reduction in fibronectin expression and alteration in cell morphology are coincident in NIH3T3 cells expressing a mutant E2F1 transcription factor.

Fibronectin within the extracellular matrix plays a role in cell attachment, spreading, and shape, while it also affects aspects of cell proliferation. Transcription factors such as E2F1 are also known to regulate cell shape and cell proliferation. Yet, to date no linkage has been established between fibronectin expression and E2F1. We show here that cells constitutively expressing a mutant E2F1 protein (E2F1d87) produce reduced amounts of fibronectin mRNA and protein. The altered expression of fibronectin seen in the E2F1d87 expressing cells is due, in part, to a reduction in transcription from the fibronectin promoter. Providing exogenous fibronectin, but not Type I collagen or laminin, as a substrate for cell adhesion is sufficient to revert the altered morphology and reestablish actin-containing microfilaments lost in the mutant cell line. An additional characteristic of the cells expressing the mutant E2F1 is that they demonstrate slow growth and a doubling in S phase duration. While providing exogenous fibronectin as an adhesion substrate did not shorten the S phase duration in the mutant line, it did significantly shorten the S phase duration in the parental NIH3T3 cell line, implicating a role for the extracellular matrix in regulating S phase transit in normal cells.

3T3 Cells↗
PubMed1995

Physical and functional interactions between p53 and cell cycle co-operating transcription factors, E2F1 and DP1.

One way in which wild-type p53 is able to regulate cell cycle progression is thought to be via the induction of its downstream target gene Waf1/CIP1, thus indirectly regulating the transcriptional activity of E2F. The E2F transcription factors are known to be key effectors of the cell cycle. We report here that there is a physical and functional interaction between p53 and two of the components of the E2F transcription factors, E2F1 and DP1. The expression of wild-type p53 can inhibit the transcriptional activity of E2F, and the expression of both E2F1 and DP1 can also downregulate p53-dependent transcription. The transcriptional activity of p53 is known to be inhibited by the direct binding of mdm2, but we demonstrate here that both E2F1 and DP1 can inhibit p53 transcriptional activity independently of mdm2. Detailed studies of protein-protein interactions have provided evidence that E2F1 and its co-operating factor DP1 can complex with p53 both in vitro and in vivo.

Animals↗
PubMed2000

The transcription factor E2F1 modulates apoptosis of neurons.

The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 -/- mice) were resistant to staurosporine-induced apoptosis as evidenced by the significantly lower caspase 3-like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild-type mice. Furthermore, overexpressing E2F1 alone using replication-deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3-like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis.

Adenoviridae↗
PubMed2002

Impaired adult neurogenesis in mice lacking the transcription factor E2F1.

During nervous system development the fate of neural stem cells-whether to undergo proliferation, differentiation, or apoptosis-is controlled by various signals, such as growth factors. Here, we demonstrate that the transcription factor E2F1, which is targeted by several signaling cascades that are activated by growth factors, is involved in neurogenesis in the adult brain. When analyzing the brains of E2F1-deficient mice, we found significantly decreased stem cell and progenitor division in the proliferative zones of the lateral ventricle wall and the hippocampus. As a consequence, the production of newborn neurons in the adult olfactory bulb and dentate gyrus was decreased. Neuronal cell counts of the adult cerebellum revealed a mild but significant cerebellar atrophy, whereas neocortical neurons were unaffected, suggesting that E2F1 deficiency produces a predominantly postnatal phenotype. The results indicate an involvement of E2F1 in controlling proliferation and neuronal cell numbers in the postnatal and adult brain.

Animals↗
PubMed1996

Isolation of two novel cDNAs whose products associate with the amino terminus of the E2F1 transcription factor.

The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Absence of the transcription factor E2F1 attenuates brain injury and improves behavior after focal ischemia in mice.

Because of observations that cultured neurons from mice deficient in the transcription factor E2F1 exhibit resistance after treatment with a wide variety of cell-death inducers, the authors investigated whether resistance extended to a cerebral ischemic insult. No differences in cerebral blood flow or physiologic parameters were observed in the mutant E2F1 littermates after the focal ligation. After 2 hours of left middle cerebral artery occlusion and 1 day of reperfusion, a 33% smaller infarct (P < 0.05) was observed by 2,3,5-triphenyltetrazolium staining in the brains of E2F1-null mice compared with their E2F1+/+ and +/- littermates. A milder ischemic insult produced by 20 minutes of middle cerebral artery occlusion and 7 days of reperfusion produced a greater difference in the E2F1-null animals with a 71% smaller infarct (P < 0.001) compared to littermate controls. A decrease in neuronal damage after mild ischemia in E2F1-null mice was observed by immunohistochemical monitoring of the loss in neuronal-specific microtubule-associated protein 2 cytoskeletal protein and the appearance of nuclear DNA fragmentation by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling. This decreased brain damage was evidenced by improved behavior in motor function of E2F1 -/- mice compared with their E2F1 +/+ littermates by 7 days of reperfusion. In an effort to address the underlying molecular mechanism of the resistance of E2F1-null mice, the expression of several downstream proapoptotic target genes (p73, Apaf1, Arf) of the E2F1 transcription factor was measured by quantitative polymerase chain reaction. Although an attenuated increase in Hsp68 mRNA was found in E2F1 -/- mice, no changes in the proapoptotic transcripts were found after ischemia, and a mechanistic inference was not possible. The authors conclude that the transcription factor E2F1 does modulate neuronal viability in brain after cerebral ischemia and corroborates the findings with cultured neurons.

Animals↗
PubMed1994

Altered shape and cell cycle characteristics of fibroblasts expressing the E2F1 transcription factor.

To gain an understanding of the role the E2F1 transcription factor plays in cell physiology, the full length protein (E2F1) and an amino terminal deletion of 87 amino acids (E2F1d87) were constitutively expressed in NIH3T3 fibroblasts. Multiple cell lines were generated for each construct. These cells do not proliferate in media containing low serum and do not proliferate in soft agar, indicating that they are likely not transformed. However, both sets of cell lines show increased DNA synthesis and increased numbers of cells in S phase when cultured in media containing low serum, compared to the control cell lines. Cells expressing E2F1d87 (but not E2F1) have an extremely rounded morphology when cultured in 10% serum-containing media. These rounded cells lack detectable microfilaments, microtubules, and focal contacts. However, when these cells are cultured in low serum-containing media (0.5%), they attain the flattened morphology and cytoskeletal structure of normal NIH3T3 cells.

3T3 Cells↗
PubMed2001

Expression and purification of the DNA-binding domain of the human transcription factor E2F1.

The DNA-binding domain of the human transcription factor E2F1 was expressed in Escherichia coli. Through a single purification step using a Ni2+ column, 40-50 mg of the highly purified recombinant protein was obtained from 1 liter of bacterial culture. In addition, it was shown that the recombinant protein had higher stability and solubility under acidic conditions than at a neutral or alkaline pH. The gel shift assay showed that the recombinant E2F1 DNA-binding domain was active in binding a fragment containing E2F sites. Circular dichroism measurements revealed that the recombinant protein approximately contains 33% alpha-helix, 11% beta-sheet, 5% turn, and 51% random coil at pH 7.0, and there was no obvious change for the secondary structure of the recombinant protein between pH 4.0 and pH 9.0. A 3D model was obtained by comparative protein modeling with a homologous protein whose structure was known by program Modeller 4. With the program DSSP, the predicted secondary structure content of the model was consistent with the result of circular dichroism spectrum.

Amino Acid Sequence↗
PubMed1999

Decreased brain infarct following focal ischemia in mice lacking the transcription factor E2F1.

E2F1+/- mice subjected to 2 h middle cerebral artery occlusion developed an infarct of 77.0 +/- 3.2 mm3 (mean +/- s.e.m., n = 15) in the ischemic hemisphere after 24 h reperfusion. A significantly smaller infarct of 58.8 +/- 4.8 mm3 (n = 15; p < 0.01) was found in E2F1-/- animals. Both deficient and normal mice had similar cerebral angioarchitecture and intra-ischemic decreases in regional blood flow. Similar areas of hypoxia in both groups of ischemic animals were demonstrated directly by immunohistochemical detection of nitroimidazole adducts. It was concluded that all animals received the same ischemic insult, yet the subsequent damage was different in the mutant mice. This is the first indication that the E2F1 gene plays a role in ischemic death of post-mitotic neurons.

Animals↗
PubMed1994

Detailed analysis of the basic domain of the E2F1 transcription factor indicates that it is unique among bHLH proteins.

The E2F1 transcription factor binds to sites within the promoters of a number of cell cycle regulated genes through a basic-helix-loop-helix motif (bHLH). It is shown here that the basic region of E2F1 is distinct from that of all other bHLH proteins. The center of the basic region contains a helix breaking proline-glycine pair, (P122, G123), implying a turn within this region. This is in contrast to the known bHLH containing proteins where the basic region is alpha-helical. Substitution of P122 and G123 with alanines results in a significant reduction in DNA binding levels, with the predicted formation of an alpha-helix. Also in contrast to other bHLH proteins, mutations generated in conserved basic residues of E2F1 do not effect DNA binding. In addition, a single leucine (191) between helix no. 2 and the leucine zipper is required for DNA binding while the leucine zipper itself is not necessary. Finally, E2F1 interacts with all of the G-residues in the sequence GGCGGGAAA while the A-residues are not required for DNA binding. The uniqueness of the E2F1 DNA binding domain is likely to play a role in its binding a DNA site that is distinct from that of all other bHLH proteins (CACGTG).

Base Sequence↗
PubMed2001

Increased expression of the transcription factor E2F1 during dopamine-evoked, caspase-3-mediated apoptosis in rat cortical neurons.

The transcription factor E2F1 mRNA and protein levels increased in rat cortical neurons in response to dopamine (DA)- or 6-hydroxydopamine (OHDA)-evoked apoptosis. Increased E2F1 protein was detected in the nucleus of neurons by double fluorescent immunocytochemistry using antibodies to E2F1 and NeuN. DA and 6-OHDA induced caspase-3-mediated apoptosis of cortical neurons which was attenuated by the addition of antioxidants or caspase-3 inhibitors to the cultures. Antioxidants prevented DA-evoked neuronal apoptosis, and also attenuated the increase in E2F1 expression. These findings suggest that increased expression of the transcription factor E2F1 may serve as a death signal during DA-evoked neuronal apoptosis.

Animals↗
PubMed1996

Constitutive expression of the E2F1 transcription factor in fibroblasts alters G0 and S phase transit following serum stimulation.

The E2F1 transcription factor was constitutively expressed in NIH3T3 fibroblasts to determine its effect on the cell cycle. These E2F1 cell lines were not tightly synchronized in G0 phase of the cell cycle following serum starvation, as are normal fibroblasts. Instead, the cells are spread throughout G0 and G1 phase with a portion of the population initiating DNA synthesis. Upon serum stimulation, the remaining cells in G0/G1 begin to enter S phase immediately but with a reduced rate. Constitutive expression of E2F1 appears to primarily affect the G0 phase, since transit of proliferating E2F1 cell lines through G1 phase is the same as control cells. Consistent with a shortened G0 phase, the E2F1 cell lines have a significantly reduced cellular volume. Additionally, the first S phase after serum stimulation, but not subsequent S phases, is nearly doubled in the E2F1 cell lines compared with control cells. Cell lines expressing a deletion mutant of E2F1 (termed E2F1d87), known to significantly affect cell shape, have cell cycle and volume characteristics similar to the E2F1 expressing cells. However, all S phase durations are considerably lengthened and the cells demonstrate delayed growth after plating.

3T3 Cells↗
PubMed1995

Expression of the E2F1 transcription factor overcomes type beta transforming growth factor-mediated growth suppression.

Inhibition of cell growth by type beta transforming growth factor (TGF-beta) occurs in mid-G1 and is associated with decreased G1 cyclin-dependent kinase activity and maintenance of the retinoblastoma tumor suppressor protein Rb in an underphosphorylated, growth-suppressive state. A variety of recent experiments suggest that a functional target of Rb is the E2F transcription factor. In addition, the growth-suppressive effects of TGF-beta can be overcome by expression of viral oncogene products that dissociate E2F from Rb and Rb-related polypeptides. These results suggest the possibility that control of E2F may be a downstream event of TGF-beta action. Consistent with that possibility is the observation that E2F1 RNA levels are drastically reduced in TGF-beta-treated cells. We have also used a recombinant adenovirus containing the human E2F1 gene to overexpress the E2F1 product in mink lung epithelial cells that were growth arrested with TGF-beta. We find that overexpression of E2F1 can overcome the TGF-beta-mediated effect as measured by the activation of cellular DNA synthesis. These results suggest that a likely downstream target for the cyclin-dependent kinases, which are controlled by TGF-beta, is the activation of E2F.

Adenoviridae↗
PubMed1995

Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes.

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.

Adenoviridae↗
PubMed1995

Regulation of the cyclin E gene by transcription factor E2F1.

A variety of results point to the transcription factor E2F as a critical determinant of the G1/S-phase transition during the cell cycle in mammalian cells, serving to activate the transcription of a group of genes that encode proteins necessary for DNA replication. In addition, E2F activity appears to be directly regulated by the action of retinoblastoma protein (RB) and RB-related proteins and indirectly regulated through the action of G1 cyclins and associated kinases. We now show that the accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin D1 promoters, both promoters are activated by E2F gene products, and at least for cyclin E, the E2F sites contribute to cell cycle-dependent control. Most important, the endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in an autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through G1.

Animals↗