required: conserved regions 1 and 2 (CR1 and CR2), which ... Cyclin A, first expressed at the G1/S transition, is a major regulator ..... An adenovirus early region.
JOURNAL OF VIROLOGY, Apr. 1996, p. 2637–2642 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology
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Adenovirus E1A Activates Cyclin A Gene Transcription in the Absence of Growth Factors through Interaction with p107 KARIN ZERFASS,1 DIMITRY SPITKOVSKY,1 ALMUT SCHULZE,1 SILVIA JOSWIG,1 ¨ RR1* BERTHOLD HENGLEIN,2 AND PIDDER JANSEN-DU Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, D-69120 Heidelberg, Germany,1 and Institut Curie, Centre National de la Recherche Scientifique UMR 144, F-75005 Paris, France2 Received 2 January 1996/Accepted 9 January 1996
Using the infection of quiescent human fibroblasts with adenovirus type 5 and various deletion mutants, we show that E1A can stimulate transcription of the cyclin A gene in the absence of exogenous growth factors. Required for this activity is conserved region 2 (CR2), while both the N-terminal part of E1A and CR1 are dispensable. This indicates that activation of cyclin A gene expression requires the binding of E1A to p107, while binding to either pRB or p300 is not involved in transcriptional activation. We demonstrate that p107 represses the cyclin A promoter through its cell cycle-regulatory E2F binding site and that 12S E1A can activate the cyclin A promoter, essentially by counteracting its repression by p107. Since CR2 is required for cell transformation, transcriptional activation of the cyclin A gene by E1A appears to be important for its capacity to override control of cellular growth. proteins (for a recent review, see reference 36). Proteins of the pRB family act through binding to and controlling the activity of E2F transcription factors (22), and E1A activates E2Fdriven transcription, probably by dissociating E2F complexes (25). Cyclin A, first expressed at the G1/S transition, is a major regulator of cell cycle progression; it is required for S-phase entry and passage through G2 (13, 27). It was shown that expression of the cyclin A gene is turned on in response to activation of a conditional allele of E1A in a rodent fibroblast cell line; similarly, stable expression of E1A in NIH 3T3 cells prevented downregulation of cyclin A gene expression by growth factor deprivation (32). These observations suggest that the cyclin A gene is a target for transcriptional activation by E1A. To directly assay the potential of E1A to activate expression of the cyclin A gene, diploid human fibroblasts were arrested in G0/G1 by serum starvation and subsequently infected by adenovirus type 5. As expected, DNA synthesis was observed at 20 h postinfection (hpi) (data not shown). As demonstrated in immunofluorescence experiments, E1A is expressed at around 12 hpi under these conditions (data not shown) (32). Cells were harvested at 18 hpi and analyzed for expression of the E1A and cyclin A genes. The expression of both genes was monitored by Western blotting (immunoblotting; Fig. 1A). Serum starvation led to the disappearance of cyclin A in uninfected cells. At 18 hpi, the E1A protein was readily detected and the cyclin A protein levels were comparable to those observed in cells growing in the presence of serum (Fig. 1A). Similarly, the mRNA levels for cyclin A were drastically reduced in serum-starved fibroblasts but were restored upon adenovirus infection (Fig. 1B and C). Quiescent fibroblasts were also infected with the E1A-deficient virus dl312, the E1B-deficient virus dl313, and viruses carrying specific mutations in the E1A gene (Fig. 1D). E1A protein and mRNA were readily detected in cells infected by mutants dl313, dl563, dl646, and dl922 but were not detectable in cells infected with the E1A-deficient mutant dl312 (Fig. 1A and B). Infection by dl814 resulted in rather low levels of E1A mRNA (Fig. 1B) and protein (Fig. 1A). The failure of dl312 to induce cyclin A gene expression indicates that E1A is required
Adenovirus type 5 is a human DNA virus which can transform mammalian cells in culture (reviewed in reference 14). Simultaneous expression of the viral E1A and E1B genes induces cell transformation (18). The expression of E1A is sufficient to induce DNA synthesis in the presence of antiproliferative signals, e.g., growth factor depletion (4, 33). In addition to E1A, the E1B gene is required to establish continuous transformed cell lines (28). E1A cooperates with an activated ras oncogene in the transformation of rodent cells (29). In this assay, the following three domains of E1A were found to be required: conserved regions 1 and 2 (CR1 and CR2), which have high-level homology between different adenovirus serotypes, and a less conserved region at the N terminus of E1A. A third conserved region, designated CR3, is not involved in cell transformation by E1A (reviewed in reference 24) and is also dispensable for S-phase entry in quiescent human (31) and rodent (31a, 35) cells. CR3 is present on the large (289-aminoacid) E1A polypeptide encoded by the 13S mRNA but absent from the small (243-amino-acid) E1A polypeptide encoded by the 12S mRNA. E1A is a potent transactivator of viral (reviewed in reference 3) and cellular (reviewed in reference 25) genes, an activity involving mainly CR2, CR3, and the N terminus of E1A (see below). The sequence requirements for trans activation and transformation are similar but not identical. CR3 is not required for transformation but is essential for the trans activation of various adenoviral and cellular genes by E1A; trans activation may result from CR3-mediated binding to the general transcription factor TBP (23). Unlike CR3 mutants, CR2 mutants are transformation deficient. Therefore, CR2-dependent trans activation of cellular genes may have a role in transformation by E1A. CR2 mediates the interaction of E1A with proteins of the retinoblastoma gene family, pRB, p107, and p130 (reviewed in reference 11). These proteins are negative growth regulators; they are inactivated by cell cycleregulated phosphorylation or by association with viral onco* Corresponding author. Mailing address: Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Abt. 620, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: 49-6221-424628. Fax: 49-6221-424902. 2637
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FIG. 1. Activation of cyclin A gene expression in serum-starved fibroblasts upon adenovirus infection. (A) Western blot. Human diploid fibroblasts obtained from the oral cavity of a healthy human being (biopsy MS 107; provided by M.-E. deVilliers) were kept in the absence of serum for 72 h and subsequently infected by adenovirus type 5 (Ad 5) or mutant viruses, as indicated. Infections were performed by incubating starvation-synchronized MS 107 cells for 1 h with virus at 20 PFU per cell. At 18 hpi, whole-cell extracts were prepared as previously described (20), separated by polyacrylamide gel electrophoresis, and blotted on a polyvinylidene difluoride membrane. Immunoblots were performed as previously described (26) with a chemiluminescence Western blotting detection system (DuPont de Nemours). Cyclin A was detected by rabbit polyclonal antibody (a gift from M. Pagano); E1A was analyzed with monoclonal antibody M37 (a gift from Ed Harlow). MS 107 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum. Ad 5 and mutants dl312, dl313, dl563, dl646, dl814, and dl922 were grown in HeLa and 293 cells, as previously described (37). (B) Northern (RNA) blot. MS 107 cells were treated as described for panel A; at 18 hpi, RNAs were prepared and probed for the expression of E1A, cyclin A, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs, as indicated. Total cellular RNA was extracted by the guanidinium thiocyanate-acid phenol method. Ten micrograms of total RNA was electrophoresed on 1% agarose–formaldehyde gels and transferred onto nylon membranes (Hybond N1; Amersham). E1A expression was analyzed with a 0.3-kb BstXI fragment prepared from an E1A cDNA clone. Cyclin A expression was monitored with a 2.2-kb human cDNA probe (20). GAPDH expression was analyzed with a rat cDNA probe (20). (C) The cyclin A and E1A mRNA levels from panel B were quantitated by using a PhosphorImager (Molecular Dynamics) and normalized to GAPDH expression. (D) Structures of the E1A genes in mutant adenoviruses. The structures of mutant E1A genes in recombinant adenoviruses are shown. Numbers refer to amino acids, starting at the N terminus of E1A; the position of each conserved region is also shown. wt, wild type.
for growth factor-independent cyclin A gene expression. On the other hand, mutation of the E1B gene, as in mutant dl313, did not reduce cyclin A gene activation, compared with that of wild-type adenovirus, but rather led to slight increases in cyclin A levels. The reason for the latter effect was not analyzed further. Analysis of cyclin A gene expression in cells infected with E1A mutant viruses revealed that mutation of the N terminus of E1A, as in mutants dl563 and dl646, interfered only moderately with its capability to induce cyclin A gene
expression (Fig. 1B and C). The very low levels of E1A expression observed for mutant dl814, carrying a deletion of the entire N terminus and part of CR1, make it difficult to draw firm conclusions on the ability of this mutant to affect cyclin A gene expression. In cells infected by dl922, the expression of cyclin A protein and mRNA was undetectable, although E1A is strongly expressed. We conclude that mainly CR2 is required for the activation of cyclin A gene expression in serum-starved fibroblasts. These data provide a genetic link between the
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transactivating and transforming functions of E1A and identify the gene encoding cyclin A, a major regulator of the G1/S transition, as a potential target for E1A’s transforming activity. The N-terminal part of E1A, although required for transformation, is dispensable for the activation of cyclin A gene expression. It was shown that expression of the cyclin A gene is determined primarily at the level of transcriptional initiation (16). To test whether activation of the cyclin A gene promoter accounts for the observed increase of cyclin A gene expression in E1A-expressing cells, we asked whether overexpression of E1A would lead to an increase in the activity of this promoter. Since diploid human fibroblasts are difficult to transfect, NIH 3T3 cells were used for these experiments. As in human fibroblasts, cyclin A gene expression is switched off in NIH 3T3 cells upon serum starvation and can be reinduced by adenovirus infection (data not shown). NIH 3T3 cells were transiently transfected with expression vectors for adenovirus E1A, together with reporter plasmids carrying the firefly luciferase cDNA linked to fragments of the human cyclin A gene promoter. Since the ability of E1A to induce the endogenous cyclin A gene is most pronounced in the absence of external mitogens (32) (Fig. 1), transfection experiments were performed under conditions in which external growth factors were limiting (Fig. 2). Transcription from a reporter plasmid containing 7.5 kb of cyclin A 59-flanking sequence (16) was stimulated 8- to 10-fold by cotransfection of an expression vector for 12S E1A, while transfection of a construct expressing the larger (13S) E1A cDNA yielded 15-fold activation (Fig. 2A). These results suggest that CR3, which is required for the E1A-dependent activation of many genes but is dispensable for transformation (24), is not essential for activation of the cyclin A gene promoter. To identify the element(s) of the cyclin A promoter responsible for trans activation by E1A, we used a series of 59 and 39 external promoter deletion mutants. By measuring the changes in promoter activity upon E1A overexpression, we established that responsiveness to E1A is retained in a 100-bp fragment containing cyclin A promoter sequences between 289 and 111 (Fig. 2A). Further deletion of cyclin A promoter sequences, as in construct 232/111, completely abolished the inducibility of the construct by E1A (Fig. 2A and B). It was shown previously that the cyclin A minimal promoter defined by this fragment carries binding sites for the E2F (30) and ATF (9) transcription factors, both of which are potential targets for E1A-dependent trans activation (25). To analyze the role of these factors in the activation of cyclin A transcription by E1A, reporter gene constructs carrying point mutations in either site were used. Disruption of the ATF site led to a significant decrease in the response to 13S E1A, while activation of the promoter by 12S E1A was affected only slightly. Thus, the ATF binding site may contribute to E1A-dependent activation of the cyclin A promoter via CR3, which is unique to the 13S form of E1A. Disruption of the E2F binding site severely reduced the responsiveness of the cyclin A promoter to both forms of E1A (Fig. 2B). We conclude that the E2F binding site plays a critical role in the activation of cyclin A transcription by 12S E1A, which contains all the sequences required for transformation (24). To further investigate the role of the transforming domains of E1A in the activation of the cyclin A gene promoter, NIH 3T3 cells were transfected with the cyclin A reporter gene construct and expression vectors for various E1A mutants, based on the 12S form of E1A. Mutants disrupting CR2 (pm928 and pm961) were incapable of activating the cyclin A promoter, while mutants separately affecting CR1 or the Nterminal domain (RG2, D2-36, D15-35, and D38-67) retained
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the capacity to activate cyclin A transcription (Fig. 2C). Thus, the ability of E1A to promote growth factor-independent activation of the cyclin A gene promoter maps to CR2, in keeping with the results obtained in infection experiments. Similar results were obtained when E1A mutants were expressed together with cyclin A promoter-reporter gene constructs in U2-OS cells, a human osteosarcoma cell line (data not shown), further supporting the conclusion that the mechanism underlying the activation of cyclin A gene expression by E1A is conserved between human and rodent cells. The ability of CR3 mutants and N-terminal mutants of E1A to activate the cyclin A promoter demonstrates that the binding of E1A to TBP (23), Dr1 (21), and p300 (12) is not involved in the activation of cyclin A gene expression. CR2 mediates the interaction of E1A with three related proteins, pRB, p107, and p130. All three are negative regulators of S-phase entry (6, 41) and are thought to control the activity of E2F (7, 17, 38) by direct protein-protein interactions (15) (for a recent review, see reference 22). In untransformed cells, proteins of the pRB family are inactivated during G1 through cell cycle-regulated phosphorylation (reviewed in reference 36). It was shown that the interaction of E1A with members of the pRB family is differentially affected by certain E1A mutants. Binding to pRB is considerably reduced in the case of RG2 and completely abolished for mutant D38-67. Both mutants bind p107 with wild-type affinity. pm928 and pm961 are defective for pRB binding but have retained some ability to bind p107, at least in coimmunoprecipitation experiments (for recent reviews, see references 8 and 35). Therefore, the ability of RG2 and D38-67 to activate the cyclin A promoter makes it very unlikely that pRB plays any role in promoter repression, consistent with our finding that pRB is unable to bind to the E2F site of the cyclin A promoter (30). To further address the possible involvement of pRB in E1A-dependent trans activation of the cyclin A gene, we performed transient transfection experiments with Saos-2 cells, a human osteosarcoma cell line lacking functional pRB. As shown in Fig. 2D, E1A expression resulted in a fivefold increase of cyclin A promoter activity in Saos-2 cells, further supporting the conclusion that promoter activation by E1A occurs independently of interaction with pRB. As in the case of NIH 3T3 (Fig. 2C) and U2-OS (data not shown) cells, mutants affecting CR1 (D38-67) or the N terminus (RG2, D2-36, and D15-35) of E1A retained the capacity to activate cyclin A transcription, while mutants disrupting CR2 (pm928 and pm961) lost this activity (Fig. 2D). All mutants tested in the experiments reported here can bind to p107, yet only a subset of these mutants can activate the cyclin A promoter. This indicates that the ability of E1A to bind p107 may be necessary but not sufficient for promoter activation. It was shown by analysis of native E2F complexes in bandshift experiments that CR2 mediates the binding of E1A to E2F complexes containing p107 (19). In contrast to the results obtained by coimmunoprecipitation, the binding of CR2 mutants, including pm928, to p107 was not detected in the E2F interaction assay; furthermore, both pm928 and pm961 are unable to disrupt E2F-p107 complexes (19). These results indicate that CR2 mutants of E1A may bind to a subpopulation of p107 that is devoid of bound E2F, but they are unable to interact with E2F-p107 complexes. While more work is required to determine the underlying mechanism, the failure of mutants pm928 and pm961 to activate cyclin A gene expression clearly indicates that these mutants are unable to inactivate p107 function in living cells. We have previously shown that pRB is unable to bind to the E2F site of the cyclin A promoter and that this site is occupied
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FIG. 2. Activation of the human cyclin A promoter by E1A. (A) Identification of the E1A-responsive elements of the cyclin A promoter. Reporter gene constructs derived from the cyclin A promoter were tested in cotransfection experiments using expression vectors (3 mg) for 12S E1A and 13S E1A. The fold inductions by both forms of E1A are shown. NIH 3T3 cells were transfected by calcium-phosphate precipitation as previously described (5). At 16 h postincubation, cells were washed and placed in Dulbecco’s modified Eagle medium containing 0.5% calf serum. Luciferase assays were performed on cell extracts prepared 36 h after transfection. In all transfection experiments employing eukaryotic expression vectors, the amount of vector sequences was kept constant. (B) Activation of the cyclin A (cA) promoter by 12S E1A requires the E2F binding site. The transcriptional activities of constructs 289/1100, 289/111, 232/111, 289/1100DATF, and 289/111DE2F were tested in cotransfection experiments with expression vectors coding for 12S E1A and 13S E1A. Transfections were performed and analyzed as described for panel A. Cyclin A-derived reporter gene constructs were obtained from the genomic clone by restriction digests and inserted into the vector pSV0ALD59 (10), as was the DATF
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by a p107-containing E2F complex in serum-starved NIH 3T3 cells (30). To further investigate the participation of p107 in the E1A-dependent deregulation of cyclin A gene expression, we asked if p107 would repress the cyclin A promoter and if the observed trans activation by E1A could result from the relief of that repression. In a culture of asynchronously growing NIH 3T3 cells, about 70% of the cells are in G1 (data not shown). In G1 cells, the cyclin A promoter is in its repressed state, characterized by binding of a G1-specific p107-containing E2F complex and relatively low-level abundance of free E2F (30). Since free E2F is the target for repression by p107 (22), we anticipated that p107 would have a rather weak effect on the cyclin A promoter under these conditions. In keeping with this prediction, p107 expression in asynchronously growing NIH 3T3 cells resulted in twofold repression of a cotransfected cyclin A promoter-reporter construct (data not shown). To increase the amount of free E2F, asynchronously growing NIH 3T3 cells were transfected with expression vectors for DP-1 and E2F-4. Heterodimerization of DP-1 and E2F-4 is known to produce a subspecies of free E2F which is a target for p107mediated repression in vivo (34). The expression of E2F-4 and DP-1 resulted in an increase of free E2F (data not shown) and strong activation of a cotransfected cyclin A reporter gene construct (Fig. 3). Activation depends on an intact E2F binding site (data not shown). Under these conditions, promoter activity could be strongly repressed by cotransfection of an expression vector for p107. Since p107 failed to repress transcription from control reporter gene constructs, we conclude that p107 can inhibit E2F-4- and DP-1-driven cyclin A gene expression (Fig. 3). Expression vectors for both pRB and p130 are much less efficient in this assay (data not shown). These results indicate that p107 is involved in downregulation of the cyclin A promoter by serum starvation and its derepression by E1A. To investigate the ability of E1A to relieve this inhibition, cotransfection experiments were performed in the presence of an expression vector for E1A. As shown in Fig. 3, E1A expression can overcome promoter repression by p107. The ability of E1A to override p107-mediated repression was impaired by mutations disrupting CR2 but not by mutations in the N-terminal part of E1A (Fig. 3), indicating that the interaction of E1A with p107-E2F complexes appears to be required for the relief of p107-dependent promoter repression by E1A. It was shown that E1A mutant D38-67 can bind to E2F-p107 complexes but fails to disrupt such complexes in vitro (19). The ability of this mutant to activate the cyclin A promoter in transfection experiments indicates that while the binding of E1A to p107-E2F complexes is required for promoter activation, disruption of such complexes may not be essential. However, more work is required to define the interaction of E1A with the cellular proteins controlling cell cycle regulation of the cyclin A promoter. In a model derived from the findings reported here, transcription of the cyclin A gene, which is a prerequisite for S-phase entry in human diploid fibroblasts (27), is prevented in quiescent cells by p107, which inhibits E2F-driven transcription. Repression can be overcome by serum growth factors,
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FIG. 3. E1A relieves repression of the cyclin A promoter by p107. The cyclin A 289/111 reporter gene construct was cotransfected with expression vectors for E2F-4 and DP-1. Transcriptional activity was repressed by cotransfection of a p107 expression vector. As indicated, expression vectors for 12S E1A and several mutants were included (1). Transfections were performed and analyzed as described in the legend to Fig. 2. cDNAs encoding human p107, E2F-4, and DP-1 were subcloned by standard techniques into the cytomegalovirus (CMV)based expression vector pX (26).
which trigger phosphorylation of p107, presumably by cyclin D1 and cdk4 (2). Negative regulation of cyclin A gene expression is involved in many forms of G1 arrest observed in untransformed mammalian cells in response to a variety of antiproliferative signals. Transformation by DNA tumor viruses can render cells unresponsive to negative signals, such as growth factor depletion (14) or loss of cell adhesion (1). Consequently, it is conceivable that in virally transformed cells, downregulation of cyclin A gene expression by antiproliferative signals is abrogated. In support of this, we have previously shown that expression of the cyclin A gene is independent of serum growth factors in cells expressing the human papillomavirus type 16 E7 (40) and simian virus 40 large T (24a) oncogenes. Data reported here indicate that promoter repression by p107 is abolished by E1A in the absence of growth factors and p107 phosphorylation (38a), possibly involving specific targeting of E2F-p107 complexes by E1A (39). We thank Kristian Helin and Rene´ Bernards for the generous gifts of various cDNA plasmids.
construct. Construction of the DE2F construct has been described previously (30). wt, wild type. (C) Activation of cyclin A transcription by E1A mutants. Transcriptional activation of the cyclin A 289/111 promoter sequence by mutants of 12S E1A was obtained by cotransfection of vectors expressing these mutants. The fold inductions of luciferase activity are shown. Data are the means of at least three independent transfection experiments, with applications of different DNA preparations. Expression vectors for 12S E1A-derived constructs pm928, pm961, 12S D2-36, 12S D15-35, 12S D38-67, and 12S RG2 have previously been described (35). (D) Activation of cyclin A transcription in pRB-deficient Saos-2 cells. Transcriptional activation of the cyclin A 289/111wt and 289/111DE2F reporter gene constructs by 12S E1A and mutants was tested in transient cotransfections in Saos-2 cells, which lack functional pRB. The fold inductions of luciferase activity are given. (E) Structures of 12S E1A mutants. The structures of mutant 12S E1A genes in the eukaryotic expression vector are shown. Numbers refer to amino acids, starting at the N terminus of E1A; the positions of CR1 and CR2 are also shown.
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