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Molecular and Cellular Biology, July 2002, p. 4952-4964, Vol. 22, No. 13
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.13.4952-4964.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Koichi Matsuo,2,
Marta Wisniewska,1,3 Erwin F. Wagner,2 and Moshe Yaniv1*
Unité Expression Génétique et Maladies, CNRS URA 1644, Institut Pasteur, 75724 Paris Cedex 15, France,1 Research Institute of Molecular Pathology (IMP), 1030 Vienna, Austria,2 Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland3
Received 7 March 2002/ Accepted 3 April 2002
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ABSTRACT |
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Fra2, but not c-JunFra1 and c-JuncFos (the tilde indicates a tethered dimer), inhibited G0 arrest at confluency and under low-serum conditions and specifically activated cyclin A expression. These data suggest that the choice of dimerization partner defines the role of c-Jun in gene activation and cell cycle regulation and that single-chain AP-1 molecules provide a powerful tool for assessing the role of specific AP-1 dimers. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AP-1 complexes in exponentially growing mouse NIH 3T3 fibroblasts are predominantly dimers containing c-Jun, JunD, and Fra2 (19, 20). In serum-starved fibroblasts, JunD accumulates while the level of the other members decreases. Mitogenic stimulation induces the synthesis of c-Jun, JunB, c-Fos, Fra1, Fra2, and FosB in a defined order (19, 20). In addition, we have recently shown that the abundance and phosphorylation of Jun proteins vary during the cell cycle (2).
The combinatorial character of the AP-1 transcriptional complex makes the interpretation of overexpression experiments difficult. Introduction of a new bZIP species into the cell can produce primary effects from homodimerization of itself or from heterodimerization with usually unidentified endogenous bZIP proteins, which cause potentially widespread effects by titrating out certain monomers and shifting the equilibrium within the AP-1 pool. Furthermore, the expression level and phosphorylation status of individual bZIP proteins in the recipient cell can be dramatically different depending on cell type and cell context. To overcome these difficulties, we developed a tethering strategy. Inspired by successful examples using other dimeric molecules, we joined two AP-1-forming monomers by a flexible polypeptide so that they formed an intramolecular dimer. In this study, we characterized the biochemical properties and biological activities of tethered JunFos proteins (the tilde indicates a tethered dimer). These single-chain AP-1 molecules revealed differences in the transcriptional regulation of specific promoters and showed that c-Jun in combination with Fra2 inhibits cell cycle arrest under both confluency and low-serum conditions for cultured NIH 3T3 mouse fibroblasts.
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MATERIALS AND METHODS |
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Fos molecules, several restriction sites and a FLAG epitope were introduced by high-fidelity PCR using specific oligonucleotide primers and cloned into pBluescript. For c-Jun, JunB, and JunD, a HindIII site was generated at the 3' end of each coding sequence, removing the stop codon. For c-Fos, Fra1, and Fra2, an XhoI site was created in front of the initiation codon, while the FLAG sequence with a stop codon (5'-GAC TAC AAG GAC GAC GAC GAC AAG TGA-3') was introduced at the end of the coding sequence followed by an EcoRI site. An XbaI-HindIII fragment from the pBluescript-cJun plasmid was ligated with a HindIII-XhoI double-stranded oligonucleotide linker encoding the polypeptide tether (5'-AAG CTT GGG GGA TCA GGC GGA GGT GGA GGA TCC GGT GGC GGT GGC TCG AGC-3') and then inserted into XbaI-XhoI-linearized pBluescript-cFosFLAG vector. Other tethered molecules were constructed in the same way. Constructed coding regions were verified by sequencing. The c-Fos template used throughout our studies contained five amino acid substitutions present in v-Fos. Miller and colleagues have shown that these substitutions do not modify the properties of mouse c-Fos; in particular, they did not confer a transformed phenotype (23).
The mutated version of c-JunFra1 (c-Jun*Fra1) was constructed by the replacement of an AccI-Psp 1406 fragment from pBluescript-cJun-Fra1 by the corresponding mutated fragment from RSVcJunDB-4 (a kind gift from D. Bohmann), where the critical Lys-Cys amino acid pair, located in the c-Jun DNA binding domain, was mutated to Ile-Asp (3).
To construct the JunFos expression vectors, the corresponding cDNA coding sequences were excised from pBluescript constructs by using the appropriate restriction sites and ligated into either a cytomegalovirus (CMV)-driven expression vector (pCG; a kind gift from F. Thierry) or the pBabe-puro vector (24). Retroviral supernatants were prepared as previously described (22) using transient transfection into the pNXeco packaging cell line. The 
Jun expression vector was described previously (13).
Culture conditions and generation of cell lines.
NIH 3T3 and 293 cells were cultured in Dulbecco minimal essential medium (DMEM) containing 7% (or 0.5%) fetal calf serum (FCS). To generate NIH 3T3/c-JunFos and NIH 3T3/puro control cells, NIH 3T3 cells were seeded at a density of 105 cells in a 6-cm-diameter dish and infected with approximately 106 CFU of each pBabe virus in 2 ml of DMEM containing 8 µg of Polybrene/ml for 3 h. The infection medium was subsequently diluted by addition of an equal volume of fresh medium, and cells were cultured for an additional 12 h. Cells were then rinsed and split in three 10-cm-diameter dishes, and 5 µg of puromycin/ml was added to the culture medium after 24 h. Puromycin-resistant colonies were then pooled.
Fuorescence microscopy. Fluorescence microscopy was described previously (20). Cells were costained with DAPI (4',6'-diamidono-2-phenylindole) (Sigma) for DNA and with a mouse monoclonal anti-FLAG antibody (Kodak) followed by fluorescein isothiocyanate (FITC)-coupled anti-mouse antibody (Amersham) for the expressed protein. Cells were observed under a Zeiss Axiophot epifluorescence microscope, and photographs were taken with Kodak films.
Flow cytometry and measurement of DNA synthesis. For flow cytometry cell cycle analysis, cells were tripsinized and collected by centrifugation at 2,000 rpm for 5 min and resuspended in 0.1% Na citrate, 0.1% Triton X-100, 50 µg of propidium iodide/ml, and 50 µg of DNase-free RNase (HSS buffer)/ml. To discriminate sub-G1 cells, trypsinized cells were eventually fixed overnight in ice-cold 70% ethanol and rinsed with phosphate-buffered saline prior to resuspension in HSS buffer. Analysis was carried out on an Epics XL Flow Cytometer (Coulter), and cell cycle phases were estimated using the Multiplus Software (Phoenix Systems).
In vitro protein synthesis and EMSA. Proteins were prepared for in vitro studies using the TNT rabbit reticulocyte coupled transcription-translation system (Promega). The mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose filters, and probed using the indicated antibodies or used for electromobility shift assays (EMSA).
EMSA for AP-1 were performed as described previously (30). Briefly, equivalent amounts of in vitro-synthesized proteins or cell extracts were preincubated for 10 min in 10 mM HEPES (pH 7.0), 100 µg of bovine serum albumin (BSA)/ml, 20% glycerol, 2 mM dithiothreitol, 0.5 mM spermidine, 2 mM MgCl2, and 1 µg of poly(dI-dC). Then, 50 ng of double-stranded 32P-labeled oligonucleotide was added and the mixture was incubated on ice for 15 min. DNA-protein complexes were resolved on 8% native polyacrylamide-Tris-borate-EDTA (TBE) gels, dried, and visualized by autoradiography and phosphorimager (Molecular Dynamics).
The following oligonucleotides were used: MMP1, corresponding to the TRE element present in the collagenase promoter (17) (5'-GGC TAG CTG ACT CAG ATG TCC-3'); Jun1 and Jun2, corresponding to the two CRE-like element present in the human c-jun promoter (38) (5'-AGC TGG GGT GAC ATC ATG GG-3' and 5'-AGC TAG CAT TAC CTC ATC CC-3', respectively); CCND1-1 and CCND1-2, corresponding to the TRE and CRE elements presents in the human cyclin D1 promoter (15) (5'-TTA AAA TGA GTC AGA ATG GAG ATC ACT GT-3' and 5'-CTT AAC AAC AGT AAC GTC ACA CGG ACT AC-3', respectively); and CCNA2, corresponding to the CRE element present in the human cyclin A promoter (14) (5'-CGC CTT GAA TGA CGT CAA GGC CGC-3').
Preparation of protein extracts and immunoblotting. Whole-cell extracts were prepared in a mixture of 50 mM Tris (pH 7.6), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 0.5 µg of of leupeptin/ml, 0.5 µg of aprotinin/ml, 0.5 µg of pepstatin A/ml, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma). Western blotting procedures and c-Jun antibodies were described previously (20). Cyclin A-, cyclin D1-, cyclin E-, p21-, p27-, actin-, and tubulin-specific antibodies were purchased from Santa Cruz.
Reporter assays.
NIH 3T3 and 293 cells were transfected by calcium phosphate coprecipitation with Renilla luciferase expression vector and one of the following reporter plasmids: MMP1-luc (Coll517luc [17]) provided by P. Herrlich, CCND1-luc (973CD1luc [15]) provided by R. Müller, Jun-Luc (Junluc [38]) provided by H. van Dam, and CCNA2-luc (pWTluc [14]) provided by J. Sobczac-Thepot. In the case of 293 cells, Jun, Fos, or JunFos, expression vectors were included. Luciferase assays were performed using the Promega dual luciferase kit by following the manufacturer's instructions.
Proliferation curves.
Triplicate cultures of NIH 3T3/puro (control) or NIH 3T3/c-JunFos cells were seeded in medium containing 7 or 0.5% serum. Adherent cells were trypsinized and counted daily using a Coulter ZI cell counter.
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RESULTS |
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Fra1 polyprotein were the predominant translation products, migrated at the expected size, and were recognized by the appropriate antibodies (Fig. 1B). Moreover, the tethered c-JunFra1 protein showed similar mobility on gel filtration columns as mixed, nondenaturated monomers (data not shown). EMSA showed that c-JunFra1 protein bound the consensus TRE element with a mobility shift similar to that of a mixture of c-Jun and Fra1 (Fig. 1C). These results strongly suggest that the single-chain molecules fold correctly to form genuine pseudodimers. Mutation of the critical Lys-Cys amino acid pair in the basic region of the c-Jun part (Fig. 1C) or excess of unlabeled AP-1 probe abolished formation of the complex between c-JunFra1 and the TRE (data not shown), further demonstrating the maintenance of specific DNA recognition after intramolecular dimerization. Moreover, the c-JunFra1 protein showed nuclear localization in transiently transfected HeLa cells, as visualized by indirect immunofluorescence using the anti-FLAG antibody (Fig. 1D) as well as anti-c-Jun and anti-Fra1 antibodies (data not shown). Similar constructs were generated for all combinations between Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, Fra1, and Fra2) proteins and showed nuclear localization and DNA binding properties comparable to those of mixtures of the corresponding monomers (not shown).
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Fos polyproteins are more resistant to interfering monomers.
We next investigated whether tethered JunFos molecules bind DNA predominantly as intramolecular pseudodimers or promiscuously as intermolecular dimers with other single chains or with free monomers in the cell. To distinguish between these two possibilities, sensitivity to intervening monomers was compared between c-JunFra1 and a mixture of c-Jun and Fra1 monomers. We used Jun, a dominant-negative form of c-Jun lacking amino acids 1 to 168, as the intervening monomer. Since it retains the bZIP domain of c-Jun, Jun dimerizes with Jun and Fos proteins and binds to the TRE but shows reduced transactivation capacity (16). If tethered c-JunFra1 folds into a stable intramolecular dimer, it should be less sensitive to excess Jun than intermolecular dimers. A c-JunFra1 vector or a mixture of c-Jun- and FLAG-tagged Fra1-expressing vectors (c-Jun plus Fra1) were cotransfected with an expression vector encoding FLAG-tagged Jun in 293 cells, and proteins of the expected sizes were visualized by anti-FLAG and anti-c-Jun antibodies (Fig. 2A). In the absence of Jun, nuclear extracts from cells transfected with either c-JunFra1 or c-Jun plus Fra1 shifted the TRE oligonucleotide similarly to in vitro-translated c-JunFra1 (Fig. 2B, lanes 4, 8, and 14). When Jun was cotransfected, the c-Jun-plus-Fra1 monomer mixture was efficiently disrupted as judged by the quasi disappearance of the major retarded band and the concomitant appearance of a faster migrating species corresponding to Jun-containing dimers (Fig. 2B, lanes 9 to 11). In contrast, DNA binding of the tethered c-JunFra1 molecule was far more resistant to the addition of Jun (Fig. 2B, lanes 4 to 7), and a decrease of DNA binding was observed only at the highest Jun level tested (Fig. 2B, lane 7). This suggests that some interaction between the tethered protein and Jun can occur when the latter is present in vast excess. Transfected Jun seems to preferentially associate with free Fra1 monomer since homodimerization with itself (lanes 2 and 3) or with transfected c-Jun (lanes 9 to 11) was not detected under the experimental conditions used.
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Jun on c-JunFra1 was further examined using a luciferase reporter construct driven by TREs within the 517-bp promoter fragment of the human collagenase gene (MMP1-luc [17]). As shown in Fig. 2C, both c-JunFra1 and c-Jun plus Fra1 activated the collagenase promoter to a similar extent in the absence of Jun. When the Jun expression vector was cotransfected, the forced dimer was substantially resistant to added Jun, whereas low doses of Jun efficiently inhibited transcriptional activation by c-Jun plus Fra1 (Fig. 2C). Only at higher levels of Jun was forced dimer activation progressively reduced, but to a lesser extent. This could have been due to competitive occupation of the AP-1 target sites by Jun dimers or to interactions between Jun and c-JunFra1. Similar results were obtained using the tethered c-JuncFos molecule (not shown). These data support the view that tethering Jun to Fos molecules strongly reduces their direct interaction with endogenous bZIP monomers and can be used as a tool to study molecular and phenotypic effects of individual AP-1 dimers.
Transcriptional specificity of single-chain AP-1 molecules.
A major unsolved issue concerns the differences in transcriptional activation and target gene specificity of different AP-1 heterodimers. We therefore examined the binding and transactivation selectivities of five c-Jun-containing tethered AP-1 proteins by using gel retardation and reporter assays. c-JunATF2, c-Junc-Jun, c-JunFra1, c-JunFra2, and c-JuncFos were produced in reticulocyte lysate and assayed for binding to five oligonucleotides derived from three different promoters: a consensus TRE element present in the human collagenase promoter (MMP1) actively bound by Jun-Fos dimers, two CRE-like elements (Jun-1 and Jun-2) present in the human c-jun promoter that have been shown to bind efficiently Jun-ATF2 dimers and not Jun-Fos dimers (38), and two elements (CCND1-1 and CCND-2) present in the human cyclin D1 promoter that have been proposed to be targets for Jun-containing dimers (15). The relative amounts of synthesized proteins were controlled by anti-Flag Western blotting (Fig. 3A), and the results of a representative gel retardation experiment is shown in Fig. 3B: noticeably, c-JuncFos dimers showed an exclusive binding to TRE elements (MMP1 and CCND1-1), compared with CRE elements. The two other JunFos tethered dimers showed a milder site selectivity since c-JunFra1 and c-JunFra2 could bind the CRE elements present in Jun-1, Jun-2, and CCND1-2. On the other hand, and in agreement with the previously published studies (37, 38), the c-JunATF2 dimer showed a clear preference for CRE-type elements, especially Jun-1. Finally, we could not detect any binding of the c-Junc-Jun tethered homodimer in the binding conditions we used.
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ATF2 and c-Junc-Fos being less abundant (Fig. 3C). Similar results were obtained using either HeLa (not shown) or 293 (Fig. 3D) cells. For the MMP1 reporter, and in agreement with the gel retardation results, c-Junc-Fos was the strongest activator, followed by c-JunFra2, c-JunFra1, and the two non-Fos dimers. Since c-Junc-Fos is present at lower levels than c-JunFra1 or c-JunFra2 (Fig. 3C), the difference among these three constructs may be even greater. In contrast, c-JunATF2, which was a poor activator of the MMP1 promoter, was the strongest activator for the Jun promoter, and c-Junc-Fos was the least potent activator of this promoter. Finally, three constructs containing Fos proteins, c-JunFra1, c-JunFra2, and c-JuncFos, activated the CCND1 promoter, while transactivation by c-JunATF2 and c-Junc-Jun was only marginal. The molecular basis for the observed decreased selectivity of the CCND1 promoter towards the three different Fos proteins might be due to the lower binding selectivity of the CCND1-1 TRE element present in this promoter toward the three Fos dimers, as well as to the contribution of the CCND1-2 CRE element (Fig. 3B).
Taken together, these data indicate that c-JunFos dimers bind and efficiently activate TRE-type motifs while JunATF2 prefers the atypical CRE-like motifs that are found in the Jun promoter. Moreover, the different JunFos dimers show subtle differences in their relative binding and activation of AP-1-controlled promoters. c-JunFra1 and c-JunFra2 dimers bind and activate the Jun promoter more efficiently that c-JunFos-forced dimers.
Phenotypes of cultured cells stably expressing tethered Jun-Fos proteins.
To analyze cellular phenotypes associated with specific AP-1 composition, we established NIH 3T3-derived cell lines stably expressing tethered JunFos proteins. Coding sequences for c-JunFra1, c-JunFra2, and c-Junc-Fos were cloned into the pBabe-puro retroviral vector (24). After retroviral gene transfer and puromycin selection, pools of infected cells were analyzed by immunofluorescence using an anti-FLAG antibody, and the transduced proteins showed the expected nuclear localization (Fig. 4A). The expression level was also analyzed by Western blotting using either anti-c-Jun antibody (Fig. 4B) or anti-FLAG antibody (not shown). Compared to the amount of endogenous c-Jun expressed in these cells, exogenous proteins were estimated to be expressed at levels of one- to threefold relative to the endogenous c-Jun (Fig. 4B). In spite of its low expression level, c-Junc-Fos activated a transiently transfected MMP1-luc reporter up to sixfold, while c-JunFra1 and c-JunFra2 activated the same reporter only two- and fourfold, respectively (Fig. 4C). The strong activation by c-Junc-Fos is consistent with the data obtained using transient cotransfection in 293 cells (Fig. 3D), even though these pools of infected cells are likely to express less tethered proteins per cell than transiently transfected cells.
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Fra2-expressing cells reached higher saturation density than the others. To investigate this further, cell cultures expressing vector, c-JunFra1, c-Junc-Fos, and c-JunFra2 were seeded at low density in medium containing 7% serum and adherent cells were counted daily. During the first 4 days of culture, all growth curves were virtually identical (Fig. 4E). However, at later time points, when cell numbers for control, c-JunFra1, and c-Junc-Fos became constant, the c-JunFra2 cells continued growing for another 2 days and reached a substantially higher saturation density (Fig. 4E). Moreover, estimated doubling times were shorter for c-JunFra2 cells (approximately 19 h) than for the three other cell lines (22 to 24 h). To explore the molecular basis for the different sensitivity to confluency, cell cycle parameters were determined by flow cytometry on days 2, 5, and 9. Control and c-JunFra1 cells were already growth arrested after 5 days of culture, as judged by the low percentage of cells in S phase at days 5 and 9. In contrast, c-JunFra2 and c-Junc-Fos cells were still actively synthesizing DNA at day 5, based on higher S-phase cells than at day 9 (Fig. 4F). The higher S-phase cell population of cells expressing c-JunFra2 (on days 2 and 5) was associated with higher percentage of G2/M cells and a lower percentage of G0/G1 cells (data not shown). These data suggest that c-JunFra2 promotes cell proliferation at otherwise saturation density. The shortened doubling time was correlated with an increase in S, G2, and M compartments of the cell cycle. In other words, cells expressing c-JunFra2, but not other tethered dimers, are less sensitive to contact inhibition.
An alternative to confluency, deprivation of serum or growth factors is widely used to render NIH 3T3 cells quiescent. We therefore cultured the control and the three c-JunFos cells in medium containing 0.5% serum. After 48 h, c-JunFra2 cultures showed fewer floating, shrunken cells than control, c-Jun-Fra1 or c-Junc-Fos cultures (Fig. 5A). While the population of adherent control, c-JunFra1 or c-Junc-Fos cells did not increase in serum with a low concentration, c-Jun-Fra2 cells substantially increased in number (Fig. 5B). In addition, flow cytometry analyses indicated that this increase correlated with an increase in S and G2/M cells (Fig. 5C and D) and with a substantial decrease in cells with less than diploid DNA content, representative of dead cells (Fig. 5C and D). To visualize replicating cells, cultures were pulse labeled with BrdU for 2 h after 48 h of serum deprivation. While nuclear labeling was undetectable in the control cells and low in c-JunFra1 and c-Junc-Fos cultures, numerous BrdU-positive cells were detected in c-JunFra2 cultures, confirming that these cells actively synthesize DNA in low-serum culture conditions (Fig. 5E). These results indicate that c-JunFra2 expressing cells are able to maintain cell cycle progression in both high-density and low-serum culture conditions and to better resist cell death induced by growth factor deprivation.
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Fra2 cells.
The cell cycle is regulated by cyclins, CDKs, and CDK inhibitors. Cyclins D and E regulate G1 progression, and cyclin A is required for both G1/S and G2/M transitions. Expression of cyclins declines when mitogens are withdrawn, leading to a relative excess of CDK inhibitors, which blocks S phase progression in cells deprived of growth factors (for review, see reference 34). To obtain further insights into the molecular basis for the resistance to growth arrest of the c-JunFra2 cells, we performed immunoblot analyses for cyclins and CDK inhibitors involved in G1 and G1/S progression. We prepared extracts from exponentially growing cells and serum-deprived cells. Protein levels of cyclin D1, cyclin A, cyclin E, p21, and p27 were rather similar between the different cells when they were exponentially growing in normal medium (Fig. 6A). In a low level of serum, however, cyclin E, cyclin A, and p21 protein levels were higher in the c-JunFra2 cells (Fig. 6B). These results suggest that the reduced sensitivity to growth factor deprivation of c-JunFra2 expressing cells is correlated with a failure to modulate cyclin expression.
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Fra2 is directly involved in transcriptional regulation of cyclins, we analyzed the activation of transfected reporter promoters constructs for cyclin D1 (CCND1-luc) and cyclin A (CCNA2-luc) in exponentially growing and serum-deprived cells. Both constructs contain human promoter fragments (14, 15), which show similar regulation as their murine counterparts (8, 29). The CCND1 promoter is activated most efficiently by c-Junc-Fos in normal serum conditions while activation of the CCNA2 promoter is only marginal (Fig. 6C). In a low serum concentration, c-JunFra2 activated the CCND1 promoter almost as efficiently as c-Junc-Fos. Furthermore, c-JunFra2 substantially and reproducibly activated the CCNA2 reporter significantly better than the other dimers (Fig. 6D). Consistently, transient introduction of c-JunFra2 in the vector-infected control cells also activated the CCNA2 reporter (Fig. 6D, vector + c-JunFra2). These data suggest that c-JunFra2 is directly responsible for high cyclin A protein levels in serum-deprived c-JunFra2 cells. We also performed parallel experiments using a cyclin A reporter construct mutated in the CRE site located at -76 bp in the promoter. This site has been shown to be important for cyclin A regulation at the G1/S transition (7) and required for the downregulation of cyclin A transcription upon contact inhibition (41). The mutated reporter construct did not respond to c-JunFra2, in both low- and high-serum-level conditions (data not shown), suggesting that c-JunFra2 activates the cyclin A promoter through binding to the CRE element. Moreover, we showed, using in vitro-synthesized proteins and gel retardation assays, that c-JunFra2 can indeed bind an oligonucleotide derived from the cyclin A promoter and containing an intact CRE element (data not shown). Taken together, these expression studies indicate that c-JunFra2 can specifically affect, probably through the regulation of cyclin A expression, the cell cycle progression of mouse fibroblasts. |
DISCUSSION |
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Design and characterization of tethered JunFos molecules.
To reduce the complexity of the AP-1 network, we attempted to construct forced JunJun, JunFos, and JunATF dimers that cannot exchange with free monomers. Following the successful examples in the literature, using the MyoD and E47 bHLH transcription factors (25, 35), we designed synthetic molecules in which two complete AP-1 dimerization partners are placed in close proximity so that they preferentially form an intramolecular forced pseudodimer. One could suspect that linking one monomer to another might impair proper protein folding. However, since entire monomer units remain intact, tethering two monomers may be no more disruptive than routine domain shuffles that combine DNA-binding domains from one factor with a transactivation domain from another. Nor is it more disruptive than dimerization domain swapping or mutagenesis in order to restrict dimerization to one or more partners (1, 4, 31, 37).
It is possible that two linked molecules might associate with each other through intermolecular bZIP-bZIP interactions to generate tetrameric (or even higher-order) forms. Alternatively, linker proteolysis could produce free constituent monomers from the tethered construct. However, the DNA-binding properties of our single-chain proteins suggest that tethered JunFos constructs fold into active pseudodimers rather than forming tetramers or dimers of proteolytic products. In support of this, we observed a similar mobility on gel filtration columns for tethered AP-1 dimers as their unlinked dimeric counterparts (result not shown) and demonstrated that the major TRE-binding activity in c-JunFra1 expressing cells were resistant to Jun, except when in large molar excess. Resistance to the effect of dominant-negative c-Jun was also observed at the transcriptional level, since activation of the collagenase promoter by the c-JunFra1 protein was far less sensitive to Jun inhibition than activation by the corresponding monomer mixture. Resistance to Jun challenge implies that once introduced into cells, such tethered molecules are insulated from endogenous bZIP proteins. Conversely, resistance of the tethered form to Jun can be used to discriminate between direct activation by forced AP-1 dimers or secondary activation through induced expression of endogenous Jun and Fos proteins.
Our studies are in line with previous work using tethered MyoDE47 proteins that showed that intramolecular folding of this dimeric transcription factor could be achieved as soon as the tether was of sufficient length and flexibility and that such linked molecules behave like natural dimers (25). Moreover, structural studies performed with a tethered MASH-1 homodimer further supported the hypothesis that such constructs form intramolecular dimers (35).
Sequence and promoter selectivity.
In the course of this study, we have generated single-chain dimers between c-Jun and c-Fos, Fra1, Fra2, and ATF2 as well as c-Jun homodimers and confirmed that the different tethered proteins retain selectivity toward DNA target sites in a series of gel retardation and reporter assays. Using in vitro-synthesized proteins, we showed that tethered JunFos dimers prefer TRE elements while c-JunATF binds better to CRE sequences. We confirmed these observations by using transient-expression and reporter assays. In transient transfection, JunFos dimers activated the previously characterized Jun-Fos-controlled collagenase promoter more efficiently than c-Junc-Jun or c-JunATF2 dimers, while the latter dimers were more potent activators of the Jun-ATF-controlled c-Jun promoter. Furthermore, mutation of the previously characterized AP-1 responsive elements on these reporter constructs abolished activation by the tethered dimers (not shown). These experiments showed that the tethering manipulations did not produce regulators with decreased selectivity when compared to free dimers, at least for these previously defined DNA target sites.
It is noteworthy that the activation of the cyclin D1 reporter by c-Jun-Fos dimers shows less sensitivity to the nature of the Fos partner than the collagenase reporter (Fig. 3). Moreover, the binding selectivity of Jun-Fos dimers toward TRE elements depends on the identity of the Fos partner since c-JunFra1 and c-JunFra2 dimers bind CRE elements better than c-Junc-Fos. This could constitute the first indication of a reduced selectivity for Fos proteins of the AP-1 responsive sites on the cyclin D1 promoter together with the first indication of a different binding and transactivation activity among Jun-Fos dimers. Additional careful analyses, including comparison with corresponding mixtures of monomers, need to be performed, but this type of study could be extended to other promoters and will help in understanding the site specificity and transactivation properties of individual AP-1 dimers.
Phenotypic effects of stable JunFos expression in mouse fibroblasts.
We further characterized the biological properties of the different tethered c-JunFos dimers by establishing pools of fibroblast cells expressing tethered c-JunFra1, c-JunFra2, and c-Junc-Fos proteins. The levels of overexpressed tethered proteins were modest compared to that of the endogenous c-Jun but sufficient to activate the collagenase reporter construct. Noticeably, the expression of the tethered c-Jun-c-Fos dimer was rather low, consistent with transient-transfection experiments. Repeated observation that c-Junc-Fos is expressed much less than other c-Jun-forced dimers seem to reflect inherent instability of c-Fos messages and proteins (12, 36).
NIH 3T3/c-JunFra2 cultures reached higher saturation densities than control, c-JunFra1, and c-JuncFos cells and were more resistant to serum withdrawal. Analysis of cell cycle distribution by flow cytometry indicated that under conditions where control cells stopped proliferating and accumulated in the G0/G1 compartment of the cell cycle, c-JunFra2-expressing cells were still able to progress through S and G2/M phases and further divide. This appeared to be the case both when cells were cultured to confluency and when they were deprived from growth factors by serum withdrawal. Nevertheless, sensitivity to cell-to-cell contacts was delayed rather than completely lost, since the c-JunFra2 cells cultures arrested at later time points (Fig. 4), remained in monolayers, and did not grow in soft agar (not shown).
Upon serum withdrawal, c-JunFra2 cultures showed a reproducible increase in cell numbers compared to the other cultures, together with a decrease in the G0/G1 phase, indicating that the c-JunFra2-expressing cells are indeed able to proliferate in a low level of serum. Moreover, we observed a substantial decrease in the sub-G1 compartment, representative of dead cells, in c-JunFra2 cultures relative to the control cell cultures. In conclusion, the increase in c-JunFra2 cell numbers in low-serum-level culture conditions can be due to a combination of increased proliferation and decreased cell death.
The two other cell populations showed milder and different phenotypes when results were analyzed more carefully. For example, even though the proliferation curve of the c-Junc-Fos cells was identical to the control in both culture conditions, the percentage of S phase cells in the c-Junc-Fos cultures was always slightly higher than in the control cultures. This could point to a possible role of this particular dimer in cell cycle progression. On the other hand, both c-Junc-Fos and c-JunFra1 cells seemed to be slightly more sensitive to serum withdrawal than control cell lines, as judged by both morphology and the sub-G1 proportion (Fig. 5). It is possible that certain JunFos dimers alter cell cycle distribution, which was not apparent from their proliferation curves due to increased cell death. This has already been observed in other situations; for example, upon overexpression of the c-myc transcription factor (9). An interesting issue for future experiments would be to compare the resistance of these cell lines to death-inducing stimuli, as it has been proposed that both c-Jun and c-Fos are important mediators of the cellular response to DNA damage-induced apoptosis (18, 32, 40).
It is noteworthy that while Fra2 has been described as a less potent transactivator than c-Fos (for a review, see reference 10), overexpression of c-JunFra2 and not tethered c-Junc-Fos dimers is sufficient to override, at least partially, the growth arrest signals induced by both increased cell density and serum withdrawal. We have previously shown that in NIH 3T3 cells, Fra2 is the predominant Fos monomer expressed when cells are growing exponentially and the other Fos proteins can be detected only when cells are subjected to extracellular stimulation, such as addition of serum (20). Since Fra2 is the major Fos species in nonstimulated exponentially growing NIH 3T3 fibroblasts, a restricted set of AP-1 dimers therefore seems sufficient to allow cell cycle progression. In other 3T3 cell lines, however, the set of possible AP-1 dimers can be larger since Fra1 has been shown to be expressed together with Fra2 in exponentially growing cells (19). The three Jun proteins are expressed in fibroblasts (19, 20), and numerous reports have suggested essential and opposite roles for Jun proteins in cell cycle progression; JunD and JunB monomers having a negative, antiproliferative role while c-Jun is the positive counterpart (2, 6, 27, 28, 39). We have previously shown that, when NIH 3T3 fibroblasts are deprived of serum or are grown to saturating densities, the relative expression levels of Jun and Fos proteins are modified since only JunD remains detectable, while c-Jun and Fra2 levels drop significantly (20). In NIH 3T3 mouse fibroblasts, down-regulation of c-Jun and Fra2 upon serum deprivation or cell-to-cell contact could therefore be a prerequisite for growth arrest. The phenotype of our c-JunFra2-expressing cells support this model. Consistent with the idea that full transformation requires several interacting Jun and Fos monomers, overexpression of the particular c-JunFra2 dimer in NIH 3T3 cells seems sufficient to initiate some aspects of transformation, like growth factor and contact-inhibition independence, but is not sufficient to completely bypass negative-growth regulators nor it is enough for full transformation. A combination of several Jun-Fos dimers might be necessary to achieve transformation. Alternatively, this partial effect may reflect the need for additional positive regulators other than AP-1.
We have analyzed the molecular basis of c-JunFra2-mediated growth factor independence. The results suggest that c-JunFra2-expressing cells continue to express cyclin A protein in a low-serum-level conditions. Moreover, a cyclin A promoter-reporter construct displayed a substantially higher activity when transfected in c-JunFra2 serum-deprived cells than in other cell lines and ectopic expression of c-JunFra2 increased cyclin A reporter activity. Therefore, the ability of this specific tethered dimer to override growth arrest signals and activate division-promoting pathways might be mediated, at least in part, through maintained expression of cyclin A in these cells. The molecular mechanisms leading to the modulation of cyclin D1, cyclin E, and p21 protein levels in the different c-JunFos-expressing cells remain to be further clarified but will certainly provide more insights into the role of individual AP-1 dimers in the control of cell cycle progression.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the EEC Biomed and Training and Mobility Programs. L.B. was the recipient of awards from the Pasteur Weitzmann Council, the Fondation des Treilles, and the EMBO Short Term Fellowship. M.W. was the recipient of an initial award from the French Foreign Ministry and was supported by the Association for International Cancer Research (AICR).
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FOOTNOTES |
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Present address: Research Institute of Molecular Pathology (IMP), 1030 Vienna, Austria.
Present address: Department of Geriatric Research, National Institute for Longevity Sciences, Morioka, Obu, Aichi 474-8522, Japan.
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REFERENCES |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bakiri, L., D. Lallemand, E. Bossy-Wetzel, and M. Yaniv. 2000. Cell cycle-dependent variations in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression. EMBO J. 19:2056-2068.[CrossRef][Medline]
3. Bohmann, D., and R. Tjian. 1989. Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell 59:709-717.[CrossRef][Medline]
4. Castellazzi, M., L. Loiseau, F. Piu, and A. Sergeant. 1993. Chimeric c-Jun containing an heterologous homodimerization domain transforms primary chick embryo fibroblasts. Oncogene 8:1149-1160.[Medline]
5. Chinenov, Y., and T. K. Kerppola. 2001. Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 20:2438-2452.[CrossRef][Medline]
6. Chiu, R., P. Angel, and M. Karin. 1989. Jun-B differs in its biological properties from, and is a negative regulator of, c-Jun. Cell 59:979-986.[CrossRef][Medline]
7. Desdouets, C., C. Ory, G. Matesic, T. Soussi, C. Brechot, and J. Sobczak-Thepot. 1996. ATF/CREB site mediated transcriptional activation and p53 dependent repression of the cyclin A promoter. FEBS Lett. 385:34-38.[CrossRef][Medline]
8. Eto, I. 2000. Molecular cloning and sequence analysis of the promoter region of mouse cyclin D1 gene: implication in phorbol ester-induced tumour promotion. Cell Prolif. 33:167-187.[CrossRef][Medline]
9. Evan, G. I., A. H. Wyllie, C. S. Gilbert, T. D. Littlewood, H. Land, M. Brooks, C. M. Waters, L. Z. Penn, and D. C. Hancock. 1992. Induction of apoptosis in fibroblasts by c-myc protein. Cell 69:119-128.[CrossRef][Medline]
10. Foletta, V. C. 1996. Transcription factor AP-1, and the role of Fra-2. Immunol. Cell Biol. 74:121-133.[Medline]
11. Glover, J. N., and S. C. Harrison. 1995. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature 373:257-261.[CrossRef][Medline]
12. Grosset, C., C. Y. Chen, N. Xu, N. Sonenberg, H. Jacquemin-Sablon, and A. B. Shyu. 2000. A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex. Cell 103:29-40.[CrossRef][Medline]
13. Ham, J., C. Babij, J. Whitfield, C. M. Pfarr, D. Lallemand, M. Yaniv, and L. L. Rubin. 1995. A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 14:927-939.[CrossRef][Medline]
14.
Henglein, B., X. Chenivesse, J. Wang, D. Eick, and C. Brechot. 1994. Structure and cell cycle-regulated transcription of the human cyclin A gene. Proc. Natl. Acad. Sci. USA 91:5490-5494.
15. Herber, B., M. Truss, M. Beato, and R. Müller. 1994. Inducible regulatory elements in the human cyclin D1 promoter. Oncogene 9:1295-1304.[Medline]
16. Hirai, S., B. Bourachot, and M. Yaniv. 1990. Both Jun and Fos contribute to transcription activation by the heterodimer. Oncogene 5:39-46.[Medline]
17. Jonat, C., B. Stein, H. Ponta, P. Herrlich, and H. J. Rahmsdorf. 1992. Positive and negative regulation of collagenase gene expression. Matrix Suppl. 1:145-155.[Medline]
18.
Kolbus, A., I. Herr, M. Schreiber, K. M. Debatin, E. F. Wagner, and P. Angel. 2000. c-Jun-dependent CD95-L expression is a rate-limiting step in the induction of apoptosis by alkylating agents. Mol. Cell. Biol. 20:575-582.
19.
Kovary, K., and R. Bravo. 1992. Existence of different Fos/Jun complexes during the G0-to-G1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins. Mol. Cell. Biol. 12:5015-5023.
20. Lallemand, D., G. Spyrou, M. Yaniv, and C. M. Pfarr. 1997. Variations in Jun and Fos protein expression and AP-1 activity in cycling, resting and stimulated fibroblasts. Oncogene 14:819-830.[CrossRef][Medline]
21.
Landschulz, W. H., P. F. Johnson, and S. L. McKnight. 1988. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759-1764.
22. Matsuo, K., J. M. Owens, M. Tonko, C. Elliott, T. J. Chambers, and E. F. Wagner. 2000. Fosl1 is a transcriptional target of c-Fos during osteoclast differentiation. Nat. Genet. 24:184-187.[CrossRef][Medline]
23. Miller, A. D., T. Curran, and I. M. Verma. 1984. c-fos protein can induce cellular transformation: a novel mechanism of activation of a cellular oncogene. Cell 36:51-60.[CrossRef][Medline]
24.
Morgenstern, J. P., and H. Land. 1990. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:3587-3596.
25. Neuhold, L. A., and B. Wold. 1993. HLH forced dimers: tethering MyoD to E47 generates a dominant positive myogenic factor insulated from negative regulation by Id. Cell 74:1033-1042.[CrossRef][Medline]
26. O'Shea, E. K., R. Rutkowski, and P. S. Kim. 1992. Mechanism of specificity in the Fos-Jun oncoprotein heterodimer. Cell 68:699-708.[CrossRef][Medline]
27. Passegué, E., and E. F. Wagner. 2000. JunB suppresses cell proliferation by transcriptional activation of p16(INK4a) expression. EMBO J. 19:2969-2979.[CrossRef][Medline]
28. Pfarr, C. M., F. Mechta, G. Spyrou, D. Lallemand, S. Carillo, and M. Yaniv. 1994. Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras. Cell 76:747-760.[CrossRef][Medline]
29. Philips, A., S. Chambeyron, N. Lamb, A. Vie, and J. M. Blanchard. 1999. CHF: a novel factor binding to cyclin A CHR corepressor element. Oncogene 18:6222-6232.[CrossRef][Medline]
30.
Piette, J., S. Hirai, and M. Yaniv. 1988. Constitutive synthesis of activator protein 1 transcription factor after viral transformation of mouse fibroblasts. Proc. Natl. Acad. Sci. USA 85:3401-3405.
31.
Pomerantz, J. L., P. A. Sharp, and C. O. Pabo. 1995. Structure-based design of transcription factors. Science 267:93-96.
32. Schreiber, M., B. Baumann, M. Cotten, P. Angel, and E. F. Wagner. 1995. Fos is an essential component of the mammalian UV response. EMBO J. 14:5338-5349.[Medline]
33. Shaulian, E., and M. Karin. 2001. AP-1 in cell proliferation and survival. Oncogene 20:2390-2400.[CrossRef][Medline]
34.
Sherr, C. J. 1996. Cancer cell cycles. Science 274:1672-1677.
35.
Sieber, M., and R. K. Allemann. 2000. Thermodynamics of DNA binding of MM17, a "single chain dimer" of transcription factor MASH-1. Nucleic Acids Res. 28:2122-2127.
36. Tsurumi, C., N. Ishida, T. Tamura, A. Kakizuka, E. Nishida, E. Okumura, T. Kishimoto, M. Inagaki, K. Okazaki, and N. Sagata. 1995. Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases. Mol. Cell. Biol. 15:5682-5687.[Abstract]
37.
van Dam, H., S. Huguier, K. Kooistra, J. Baguet, E. Vial, A. J. van der Eb, P. Herrlich, P. Angel, and M. Castellazzi. 1998. Autocrine growth and anchorage independence: two complementing Jun-controlled genetic programs of cellular transformation. Genes Dev. 12:1227-1239.
38. van Dam, H., D. Wilhelm, I. Herr, A. Steffen, P. Herrlich, and P. Angel. 1995. ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J. 14:1798-1811.[Medline]
39. Weitzman, J. B., L. Fiette, K. Matsuo, and M. Yaniv. 2000. JunD protects cells from p53-dependent senescence and apoptosis. Mol. Cell 6:1109-1119.[CrossRef][Medline]
40. Wisdom, R., R. S. Johnson, and C. Moore. 1999. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 18:188-197.[CrossRef][Medline]
41. Yoshizumi, M., C. M. Hsieh, F. Zhou, J. C. Tsai, C. Patterson, M. A. Perrella, and M. E. Lee. 1995. The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells. Mol. Cell. Biol. 15:3266-3272.[Abstract]
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