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

Logan, Thomas J.; Jordan, Kelly L.; Hall, D.

doi:10.1091/mbc.5.6.667

Cited by 27 publications

(20 citation statements)

References 35 publications

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“…Here we present evidence that the E2F1 transcription factor can directly in¯uence DNA replication, independent of any induction of apoptosis. The data are consistent with previous studies where the N terminal domain of E2F1 has been shown to a ect S phase progression in ®broblasts (Logan et al, 1994(Logan et al, , 1995. The inhibitory region of E2F1 appears to be located within its most N terminal 59 amino acids and does not require other functional domains, such as the cyclin A/cdk2 binding domain, the dimerization domain, the DNA binding domain, or the transactivation domain.…”

Section: Discussionsupporting

confidence: 93%

“…The antiFlag M2 monoclonal antibody (Kodak) or an anti-HA monoclonal antibody (Promega) for E2F1D24 was diluted to 50 ng/ml in PBS plus 0.2% gelatin. Fifty ml of diluted antibody was added to a 6 cm tissue culture plate, coverslips containing the ®xed permeabilized cells were placed cell side down on the drop of diluted antibody and incubated for 1.5 h at 378C (Logan et al, 1994). The coverslips were washed three times in PBS plus 0.2% gelatin.…”

Section: Isolation Of Plasmid Dna/southern Blotsmentioning

confidence: 99%

“…These genes encode much of the machinery involved in deoxyribonucleotide biosynthesis and DNA replication, such as DHFR, thymidine kinase, DNA Polymerase a, ORC1, and CDC6 (DeGregori et al, 1995a;Helin, 1998;Ohtani, 1999). Consistent with this ability to activate transcription, overexpression of E2F1 also has been shown to drive resting cells into S phase (Asano et al, 1996;DeGregori et al, 1995b;Johnson et al, 1993;Logan et al, 1994Logan et al, , 1995.…”

Section: Introductionmentioning

confidence: 99%

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The amino-terminus of the E2F-1 transcription factor inhibits DNA replication of autonomously replicating plasmids in mammalian cells

Stubbs

Hall

2002

Oncogene

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The E2F1 transcription factor plays a pivotal role in driving cells out of a quiescent state and into the S phase of the cell cycle, in part by transactivating genes needed for DNA replication including DHFR, thymidine kinase, and DNA Polymerase a. E2F1 has also been implicated in regulating an S phase checkpoint, however its role in this checkpoint is not well de®ned. To determine how E2F1 a ects such a checkpoint, we utilized an in vivo replication assay employing a plasmid based SV40 origin of replication, transfected into cells expressing SV40 large T antigen. Here we show that expression of full length E2F1, or only its N terminus, represses replication from plasmids containing the SV40 origin, while N terminal deletions of E2F1 do not. E2F1 appears to inhibit the elongation phase of replication and not the initiation phase since it does not a ect the replication of other cotransfected plasmids containing only the SV40 origin. Further, inhibition of replication is dependent on both the amino-terminus of the E2F1 protein and on a DNA sequence that is contained within the 3' end of the E2F1 cDNA. Additionally, both full-length E2F1, or just its N-terminus, form protein complexes with two portions of the 3' end of the E2F1 cDNA. These data provide a clue to the mechanism by which E2F1 regulates transit through the S phase checkpoint, by acting on a speci®c DNA sequence via its amino-terminal region, to inhibit elongation of DNA replication.

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Section: Discussionsupporting

confidence: 93%

Section: Isolation Of Plasmid Dna/southern Blotsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The amino-terminus of the E2F-1 transcription factor inhibits DNA replication of autonomously replicating plasmids in mammalian cells

Stubbs

Hall

2002

Oncogene

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“…Immunofluorescence, Confocal Microscopy, and Immunoprecipitation-Indirect immunofluorescence was performed as described by Logan et al (28). Briefly, COS cells were plated on 10-cm tissue culture dishes containing glass coverslips at approximately 500,000 cells/plate.…”

Section: Methodsmentioning

confidence: 99%

Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

Hann¹,

Evans²,

Fertala³

et al. 1998

Journal of Biological Chemistry

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The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabiliza- Eukaryotic DNA topoisomerase I catalyzes the relaxation of supercoiled DNA through the transient breakage and religation of a single DNA strand in a DNA duplex (reviewed in Refs. 1-3). This enzyme plays a role in a number of essential cellular processes, such as replication, recombination, and transcription (1, 3-5). Furthermore, the naturally occurring antitumor drug camptothecin specifically targets this enzyme by stabilizing the covalent enzyme-DNA intermediate (Refs. 6 and 7; reviewed in Ref. 8). During DNA replication (S phase), these stabilized enzyme-DNA adducts are converted into lethal double-stranded DNA breaks due to their interaction with the DNA replication fork (9 -12).Although this enzyme participates in numerous cellular processes, strains of the yeast Saccharomyces cerevisiae deleted for the gene encoding DNA topoisomerase I (top1⌬) are viable because other gene products, such as DNA topoisomerase II, can compensate for the loss of TOP1 (11, 13). These top1⌬ strains are completely resistant to the cytotoxic action of camptothecin (14 -16). However, expression of either S. cerevisiae or human DNA topoisomerase I restores the sensitivity of these cells to camptothecin-induced lethality (4, 14 -16). These results demonstrate the specificity of camptothecin for eukaryotic DNA topoisomerase I and the utility of using yeast as a model system for the analysis of drug-enzyme interactions. In fact, mutations in yeast and human TOP1 that render the enzyme resistant to camptothecin (17,18) or render the enzyme cytotoxic even in the absence of camptothecin (19, 20) 1,2 have been defined using this yeast system.These results indicate a significant conservation of function between the yeast and human enzymes, consistent with extensive similarities in TOP1 sequences. Nevertheless, differences between these proteins do exist. For example, expression of a camptothecin-resistant yeast or human DNA topoisomerase I mutant (top1vac) has different effects on the viability of ...

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“…These numbers were derived from the percentage of chloramphenicol acetylation in each sample 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 _ _ E2F-1 (1-437) E2F-1 (88-437) Gst E2F-1 (1-437) E2F-1 (88-437) Gst a b Figure 6 E2F-1 binds to its responsive region on the TGF-b1 promoter. Increasing concentrations of puri®ed bacterially-produced recombinant GST-E2F-1 proteins (Logan et al, 1994;Krek et al, 1995) were examined for binding to the responsive region on the TGF-b1 promoter (a, lanes 2 ± 7) and to the consensus E2F-binding site on the DHFR promoter (b, lanes 2 ± 7). E2F-1 (1 ± 437) represents the full length version of the protein and the mutant E2F-1DN contains amino acids 88 ± 437.…”

Section: E2f-1 Binds To the Tiprs On The Tgf-b1 Promotermentioning

confidence: 99%

The transcription factor E2F-1 modulates TGF-β1 RNA expression in glial cells

et al. 1997

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The cell type speci®city of the regulation of expression of the potent growth inhibitory cytokine transforming growth factor-beta (TGF-b), prompted our analyses of the regulation of TGF-b1 gene expression in glial cells by viral and cellular oncoproteins. We have shown that SV40 T-antigen diminished TGF-b1 expression in glial cells and this repression was dependent on the ability of T-antigen to interact with the tumor suppressor protein, pRb, and two structurally related proteins, p107 and p130. The cellular transcription factor E2F-1, which is a downstream e ector of T-antigen, was unable to in¯uence expression from the TGF-b1 promoter by itself. Interestingly, E2F-1 could overcome viral Tantigen-mediated repression of the TGF-b1 promoter, suggesting potential feedback loop between TGF-b and E2F in virally transformed glial cells. Using deletion analyses, we have mapped two E2F-1-responsive regions on the TGF-b1 promoter: a T-antigen-dependent negative regulatory sequence (TdNRS) between 7323 and 7175, and a T-antigen-independent positive regulatory sequence (TiPRS) between 734 and +10 on the TGF-b1 promoter. Further examination of TiPRS revealed the presence of a functional E2F binding site. Interestingly, the amino terminus of E2F-1 was required for its activation of TGF-b1 expression, as mutations in that domain abolished the ability of E2F-1 to increase TGF-b1 expression. These data suggest that yetuncharacterized interaction between the amino terminus of E2F-1 and cellular proteins regulates TGF-b1 expression. The mechanism for E2F-1-mediated Tantigen-dependent regulation of TGF-b1 expression from TdNRS awaits further characterization.

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

Cited by 27 publications

References 35 publications

The amino-terminus of the E2F-1 transcription factor inhibits DNA replication of autonomously replicating plasmids in mammalian cells

The amino-terminus of the E2F-1 transcription factor inhibits DNA replication of autonomously replicating plasmids in mammalian cells

Increased Camptothecin Toxicity Induced in Mammalian Cells Expressing Saccharomyces cerevisiae DNA Topoisomerase I

The transcription factor E2F-1 modulates TGF-β1 RNA expression in glial cells

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