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Results for “E2F2 Transcription Factor”

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At least 19 recordsLinked to original sources
PubMed2005

Induction of replication in human corneal endothelial cells by E2F2 transcription factor cDNA transfer.

PURPOSE: Corneal endothelial cells in humans do not replicate to any meaningful extent. Diminishing density of the cell monolayer with age and in the disease states is a major cause of loss of corneal transparency. This study was conducted to test the hypothesis that overexpression of the transcription factor E2F2 results in replication in nonproliferating human corneal endothelial cells. METHODS: Whole human corneas were incubated for 2 hours in a solution of recombinant E1(-)/E3(-) adenovirus incorporating cDNA encoding E2F2 and green fluorescent protein (GFP) under control of a bidirectional promoter and subsequently maintained in ex vivo culture. Control specimens were incubated with an identical virus bearing the GFP sequence only, or virus-free medium. Efficiency of gene transfer and localization was examined by fluorescence microscopy. En face confocal microscopy of the corneal endothelial surface was used to image recombinant E2F2 expression. 5-bromodeoxyuridine (BrdU) incorporation was used to examine progression to the S phase. Changes in density of the corneal endothelium were quantified by specular microscopy and counting of trypan-blue-stained cells. Apoptosis was tested with a TUNEL assay. RESULTS: Recombinant proteins were expressed predominantly in the endothelium and in a high proportion of endothelial cells in the first week after exposure to virus, diminishing thereafter. Compared with the control, transduction with E2F2 resulted in progression from the G(1) to the S phase in a significant number of cells and in increased cell density. Apoptosis was not found to any significant extent. CONCLUSIONS: Overexpression of the transcription factor E2F2 in nonmitotic human corneal endothelial cells results in short-term expression, cell-cycle progression, and increased monolayer cell density.

Adenoviruses, Human↗
PubMed2004

Effect of overexpressing the transcription factor E2F2 on cell cycle progression in rabbit corneal endothelial cells.

PURPOSE: To test the hypothesis that overexpression of the transcription factor E2F2 promotes cell cycle progression in nonproliferating corneal endothelial cells. METHODS: Ex vivo rabbit corneas were transfected with a lipid transfection reagent and either a control plasmid containing full-length cDNA for enhanced green fluorescent protein (pIRES2-EGFP) or a plasmid containing full-length cDNA for both E2F2 and EGFP (pIRES2-E2F2/EGFP). Transfection control experiments consisted of corneas incubated in buffer without transfection reagent or plasmid or incubated in reagent without plasmid. After transfection, corneas were incubated for various periods in 0.1% FBS (a concentration that maintains cell health, but does not promote proliferation). Immunocytochemical (ICC) localization tested for overexpression of E2F2 in transfected corneal endothelial cells and permitted calculation of transfection efficiency. Endothelial cell viability was tested in transfected ex vivo corneas and confluent cultures by using a cell-viability assay. Apoptosis was detected in confluent cultures by TUNEL assay. RT-PCR tested for mRNA expression of Ki67 (a marker of actively cycling cells) and cyclin B1 (a marker for the G2-phase of the cell cycle). Semiquantitative densitometric analysis compared the relative amounts of PCR reaction products. RESULTS: ICC demonstrated the colocalization of E2F2 and EGFP in corneal endothelium with a transfection efficiency of 10% to 12%, using the pIRES2-based plasmid and transfection reagent. The cell-viability assay revealed very few dead cells in ex vivo corneal endothelium that overexpressed E2F2. Cell viability and TUNEL assays of confluent cultures revealed that approximately 27% of cells died in all cultures incubated with transfection reagent, but death appeared not to be due to apoptosis. No additional cell death was noted by either assay in cells that overexpressed E2F2. RT-PCR of endothelial samples obtained 48 hours after transfection showed the presence of higher levels of reaction product for Ki67 (a 5.1-fold increase) and cyclin B1 (a 2.3-fold increase) in cells that overexpressed E2F2 than in control samples. CONCLUSIONS: Overexpression of the transcription factor E2F2 in nonproliferating rabbit corneal endothelial cells induces cell cycle progression without inducing significant apoptosis.

Animals↗
PubMed2002

Transcriptional regulation of the human tumor suppressor p14(ARF) by E2F1, E2F2, E2F3, and Sp1-like factors.

The human ARF/INK4a locus encodes two cell cycle inhibitors, p16(INK4a) and p14(ARF), by using separate promoters. A variety of mitogenic stimuli upregulate ARF but a direct modulation at the transcriptional level has been reported only for E2F-1. We show here that the ARF promoter is strongly responsive also to E2F2 and E2F3, thus providing a strong support to their suggested role in the induction of apoptosis. Through the usage of both deletion mutants and/or site-directed mutants, we surprisingly found that none of the four putative E2F consensus sites is strictly necessary for the upregulation of ARF expression, as a minimal deletion mutant, lacking all the putative E2F binding sites, is still transactivated by E2F. Moreover, our data suggest that the ARF promoter is regulated by E2F through both direct binding to the promoter sequences and indirectly, probably by being tethered to the ARF promoter by Sp1-like factors.

3T3 Cells↗
PubMed2000

Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2.

PURPOSE: Previous studies have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause lens fiber cell proliferation and apoptosis. Because pRb is thought to block cell cycle progression by inhibition of E2F transcription factors, experiments were conducted to test whether overexpression of different E2F family members would be sufficient to induce fiber cell proliferation and subsequent apoptosis. The in vivo functions of the transcription factor E2F2 have not previously been analyzed or described in transgenic mice. METHODS: Human E2F1 and E2F2 cDNAs were linked to the alphaA-crystallin promoter. Transgenic mice were generated by microinjection. Changes in cell cycle regulation were assayed by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation and by in situ hybridization. Cell death was assayed using the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS: At embryonic day (E)15.5, strong expression of the E2F1 and E2F2 transgenes was detected in lens fiber cells with little or no expression in epithelial cells. BrdU incorporation and TUNEL assays showed that overexpression of either E2F1 or E2F2 in lens fiber cells was sufficient to cause cell cycle entry and subsequent apoptosis. Expression of either E2F1 or E2F2 was sufficient to induce the transcription of cyclins (A2, B1, and E), as well as p53 and Bax in the lens fibercells. CONCLUSIONS: Expression of either E2F1 or E2F2 can induce postmitotic lens fiber cells to re-enter the cell cycle. Inappropriate cell cycle entry is recognized by p53 in each case, and programmed cell death ensues.

Animals↗
PubMed2005

Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation.

The E2F transcription factor family plays a crucial and well established role in cell cycle progression. Deregulation of E2F activities in vivo leads to developmental defects and cancer. Based on current evidence in the field, mammalian E2Fs can be functionally categorized into either transcriptional activators (E2F1, E2F2, and E2F3a) or repressors (E2F3b, E2F4, E2F5, E2F6, and E2F7). We have identified a novel E2F family member, E2F8, which is conserved in mice and humans and has its counterpart in Arabidopsis thaliana (E2Ls). Interestingly, E2F7 and E2F8 share unique structural features that distinguish them from other mammalian E2F repressor members, including the presence of two distinct DNA-binding domains and the absence of DP-dimerization, retinoblastoma-binding, and transcriptional activation domains. Similar to E2F7, overexpression of E2F8 significantly slows down the proliferation of primary mouse embryonic fibroblasts. These observations, together with the fact that E2F7 and E2F8 can homodimerize and are expressed in the same adult tissues, suggest that they may have overlapping and perhaps synergistic roles in the control of cellular proliferation.

Amino Acid Sequence↗
PubMed1998

[Interaction of E2F/Rb family members with factor binding to co-repressor element on B-myb and E2F1 promoters].

The E2F transcription factor plays an important role in controlling the expression of genes required for cell cycle progression. The transcription of a number of these genes, including E2F1 and B-myb, is repressed in G0/early G1 at E2F DNA binding sites mediated by interaction of E2F with the Rb family member proteins. It was shown that a corepressor element CHR, which was originally identified in the B-myb promoter, is also responsible for the repression of the E2F1 promoter. The mutation of the CHR element adjacent to E2F sites leads to a derepression of the E2F1 promoter in quiescent cells. The CHR-mutated promoter is activated by the E2F family of proteins (E2F1, E2F2, E2F3, and E2F4) but unable to be repressed by any of the Rb family members (Rb, p107, and p130) to the level of the wild-type promoter activity in G0, indicating that the repression by the Rb family members is required for the corepressor element. Moreover, it was shown that a factor specifically bound to the CHR element is co-purified with E2F by DNA affinity purification and co-immunoprecipitated with E2F4 and the Rb family members. These results seggested that E2F and the Rb family member proteins regulate the transcription of the E2F1 and B-myb genes by associating with an additional corepressor protein.

Animals↗
PubMed2006

JPO1/CDCA7, a novel transcription factor E2F1-induced protein, possesses intrinsic transcriptional regulator activity.

JPO1/CDCA7 was originally identified as a c-Myc-responsive gene that participates in neoplastic transformation. Here, we report the identification of JPO1/CDCA7 as a direct transcriptional target of transcription factor E2F1. We demonstrated that overexpression of E2F1 by adenoviral-mediated gene transfer upregulated JPO1/CDCA7 mRNA expression in human cells. Analysis of human and mouse JPO1/CDCA7 promoter constructs showed that an E2F-responsive sequence was necessary for E2F1-induced activation of the JPO1/CDCA7 gene transcription. Among the members of the E2F family, E2F1 to E2F4, but not E2F5 or E2F6, activated the JPO1/CDCA7 reporter construct. Chromatin immunoprecipitation analysis demonstrated that E2F1, E2F2, and E2F4 specifically bound to an E2F-responsive sequence of the human JPO1/CDCA7 gene. Like JPO2/R1, which has a homologous transcriptional regulator domain, the C-terminal cysteine-rich region of JPO1/CDCA7 protein induced transcriptional activity in a mammalian one-hybrid assay. Taken together, our results suggest that JPO1/CDCA7 is a unique transcription regulator whose expression is activated by E2F1 as well as c-Myc.

Animals↗
PubMed2002

Activation of ARF by oncogenic stress in mouse fibroblasts is independent of E2F1 and E2F2.

The ARF tumour suppressor protein (p14(ARF) in human and p19(ARF) in mouse) is a major mediator of the activation of p53 in response to oncogenic stress. Little is known about the signalling pathways connecting oncogenic stimuli to the activation of ARF. Regulation of ARF occurs primarily at the transcriptional level and several modulators of ARF transcription have been identified. Notably, ectopic expression of E2F1 upregulates ARF transcriptionally, and both E2F1 and ARF have been implicated in apoptosis and cell-cycle arrest. We have used primary mouse fibroblasts deficient for E2F1, E2F2, or both to determine the possible role of these E2F proteins as upstream regulators of ARF in response to oncogenic stimuli and other stresses. In particular, we have studied the effects of oncogenic Ras and the viral oncoprotein E1A on ARF levels, neoplastic transformation, and sensitization to apoptosis. We have also examined the behaviour of the E2F-deficient MEFs with respect to immortalization and sensitivity to DNA damage. None of the ARF-mediated responses that we have analysed is significantly affected in E2F1(-/-), E2F2(-/-) or E2F1/2(-/-) MEFs, and ARF is upregulated normally in all cases. Taken together, our results indicate that the activation of ARF in response to oncogenic stress can occur by E2F1- and E2F2-independent mechanisms. This challenges previous suggestions implicating E2F factors as key mediators in the activation of ARF by oncogenic stress.

Adenovirus E1A Proteins↗
PubMed2007

E2f1, E2f2, and E2f3 control E2F target expression and cellular proliferation via a p53-dependent negative feedback loop.

E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21(CIP1). Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21(CIP1) and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Animals↗
PubMed2001

Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity.

E2Fs are important regulators of proliferation, differentiation, and apoptosis. Here we characterize the phenotype of mice deficient in E2F2. We show that E2F2 is required for immunologic self-tolerance. E2F2(-/-) mice develop late-onset autoimmune features, characterized by widespread inflammatory infiltrates, glomerular immunocomplex deposition, and anti-nuclear antibodies. E2F2-deficient T lymphocytes exhibit enhanced TCR-stimulated proliferation and a lower activation threshold, leading to the accumulation of a population of autoreactive effector/memory T lymphocytes, which appear to be responsible for causing autoimmunity in E2F2-deficient mice. Finally, we provide support for a model to explain E2F2's unexpected role as a suppressor of T lymphocyte proliferation. Rather than functioning as a transcriptional activator, E2F2 appears to function as a transcriptional repressor of genes required for normal S phase entry, particularly E2F1.

Animals↗
PubMed2001

E2F1 and E2F2 determine thresholds for antigen-induced T-cell proliferation and suppress tumorigenesis.

E2F activity is critical for the control of the G(1) to S phase transition. We show that the combined loss of E2F1 and E2F2 results in profound effects on hematopoietic cell proliferation and differentiation, as well as increased tumorigenesis and decreased lymphocyte tolerance. The loss of E2F1 and E2F2 impedes B-cell differentiation, and hematopoietic progenitor cells in the bone marrow of mice lacking E2F1 and E2F2 exhibit increased cell cycling. Importantly, we show that E2F1 and E2F2 double-knockout T cells exhibit more rapid entry into S phase following antigenic stimulation. Furthermore, T cells lacking E2F1 and E2F2 proliferate much more extensively in response to subthreshold antigenic stimulation. Consistent with these observations, E2F1/E2F2 mutant mice are highly predisposed to the development of tumors, and some mice exhibit signs of autoimmunity.

Age Factors↗
PubMed2001

Reconstitution of dna synthetic capacity in senescent normal human fibroblasts by expressing cellular factors E2F and Mdm2.

Unraveling the mechanisms underlying cellular senescence will contribute to the understanding of processes involved in aging and cancer. We sought to determine whether expression of cellular factors in senescent WI-38 human fibroblasts was sufficient to induce nuclear DNA synthesis. Expression by recombinant adenovirus of E2F1, E2F2, E2F3, cyclin E/cdk2, and Mdm2 individually resulted in DNA synthesis in 10-30% of cells. However, combination of Mdm2 with E2F or cyclin E/cdk2 resulted in 50 to 75% of cells synthesizing DNA. DNA synthesis occurred approximately 30 h following infection. We conclude that expression of normal cellular factors is sufficient to induce DNA synthesis in senescent normal human fibroblasts.

Adenoviridae↗
PubMed2004

Aberrant regulation of survivin by the RB/E2F family of proteins.

Survivin is a putative oncogene that is aberrantly expressed in cancer cells. It has been hypothesized to play a central role in cancer progression and resistance to therapy in diverse tumor types. Although some of the transcriptional processes regulating its expression have been established, the diversity of genes that may be controlling the levels of its expression in both normal cells as well as in cancer cells has not been fully explored. The most common genetically mutated pathways in human malignancies are the p53 tumor suppressor pathway and the RB/E2F pathway. Both of these pathways, when intact, provide essential checkpoints in the maintenance of normal cell growth and protect the cell from DNA damage. Using non-transformed embryonic fibroblasts, we provide evidence of a molecular link between the regulation of survivin transcription and the RB/E2F family of proteins. We demonstrate that both pRB and p130 can interact with the survivin promoter and can repress survivin transcription. We also show that the E2F activators (E2F1, E2F2, and E2F3) can bind to the survivin promoter and induce survivin transcription. Genetically modified cells that harbor deletions in various members of the RB/E2F family confirm our data from the wild-type cells. Our findings implicate several members of the RB/E2F pathway in an intricate mechanism of survivin gene regulation that, when genetically altered during the process of tumorigenesis, may function within cancer cells to aberrantly alter survivin levels and enhance tumor progression.

Adenoviridae↗
PubMed2005

E2F1 and E2F2 are differentially required for homeostasis-driven and antigen-induced T cell proliferation in vivo.

Homeostasis-driven T cell proliferation occurs in response to a lymphopenic environment and is mediated by TCR and IL-7 signaling. In this report, we demonstrate a defect in the proliferation of murine naive and memory T cells lacking both E2F1 and E2F2 in response to lymphopenic conditions, suggesting that E2F1 and E2F2 function redundantly downstream of TCR and/or IL-7 signaling during homeostasis-driven proliferation. In contrast, T cell proliferation in response to antigenic stimulation is either unaffected (in vivo) or potentiated (ex vivo) by loss of E2F1 and E2F2, indicating divergent requirements for these E2F factors in T cell proliferation mediated by distinct stimuli. E2F1/E2F2 double knockout (DKO) T cells enter S phase in response to homeostatic signaling, but fail to divide, suggesting that S phase progression is either incomplete or defective. In addition, E2F1/E2F2 DKO mice do not recover normal T cell numbers following exposure to a sublethal dose of radiation, indicating that this defect in homeostasis-driven proliferation is physiologically relevant. Consistent with their failure in cell cycle progression, the differentiation of DKO T cells into memory T cells in response to homeostatic signals is significantly reduced. These observations support the idea that proliferation is required for memory T cell formation and also have implications for the development of clinical strategies to minimize the occurrence of lymphopenia-induced autoimmunity.

Animals↗
PubMed1996

Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence.

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (-381 to -17) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that 1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetectable in both early passage and senescent cells; and 3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human fibroblasts. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells.

Base Sequence↗
PubMed1998

dE2F2, a novel E2F-family transcription factor in Drosophila melanogaster.

Mammalian E2F transcription factors comprise a family of proteins encoded by distinct genes which function in the form of heterodimers with DP proteins. In Drosophila melanogaster, only a single E2F-related transcription factor, dE2F, has been reported. We have now identified and characterized a cDNA encoding another E2F family member in Drosophila, termed dE2F2. The predicted amino acid sequence shares 38.8% identity with dE2F, including the QKRRIYDITNVLEGI motif which is highly conserved in mammalian E2F family members and dE2F. The 18 amino acids, located in the carboxy-terminal region of the mammalian E2F family, sufficient for binding to pRb are also conserved in dE2F2. Band mobility shift analyses with glutathione S-transferase fusion proteins revealed dE2F2 binding to E2F-recognition sites to be dependent on the presence of dDP protein, in apparent contrast to dE2F. Furthermore, cotransfection experiments in Kc cells demonstrated dE2F2 repression of the PCNA gene promoter activity, while dE2F caused activation, the target site for the repression being identical to the dE2F-recognition site.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of CpG islands of the human chromosome 1p36 region.

CpG islands were identified and localized to chromosome 1p36 by means of pulsed-field gel blot hybridization with 1p36-specific microclone probes. Five CpG islands, designated CpG17, CpG28, CpG60, CpG112a, and CpG112b, were molecularly cloned from corresponding cosmids. All five islands are associated with transcribed sequences, as shown by RNA blot hybridizations. Screening of cDNA libraries with the island-specific genomic probes led to the isolation of two cDNA clones to date. These encode the human transcription factor E2F-2 and the dominant-negative helix-loop-helix gene ID3, respectively. Pulsed-field gel electrophoresis analysis also revealed that these two genes are located next to each other at a distance of about 25 kb.

Carrier Proteins↗