Vol. 65, No. 7

JOURNAL OF VIROLOGY, JUIY 1991, p. 3496-3503

0022-538X/91/073496-08$02.00/0

Copyright C 1991, American Society for Microbiology

The Yeast GAL4 Protein Transactivates the Polyomavirus Origin of DNA Replication in Mouse Cells MOSHE BARU, MEIR SHLISSEL, AND HAIM MANOR*

Department of Biology, Technion-Israel Institute of Technology, Haifa 32,000, Israel Received 19 February 1991/Accepted

5

April 1991

We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus Ela or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55 % lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.

The polyomavirus (Py) origin of DNA replication (ori) consists of two elements: the origin core and the enhancer (7). The origin core was defined genetically as an essential and specific part of the Py ori (22, 26, 44). It extends from nucleotides 5268 to 42 in the Py A2 strain numbering system (11). The core contains three main sequence elements: (i) a central palindrome (nucleotides 5281 to 22), which includes two repeats of the large T-antigen (T-Ag) recognition pentanucleotide sequence motif [5'-(G)(A/G)GGC-3'] on each strand; (ii) an A/T-rich sequence (nucleotides 5268 to 5283) that maps on the late side of the palindrome; and (iii) a polypurine-polypyrimidine sequence (nucleotides 27 to 42) that maps on the early side of the palindrome (7). The sites of initiation of bidirectional DNA replication have been mapped on the early side of the core (15). The Py replication enhancer overlaps with the transcriptional enhancer for the early transcription unit (nucleotides

upstream promoter elements, which are also found in enhancers (28). A typical transcriptional modulator protein consists of a DNA-binding domain that specifically recognizes a short DNA sequence and a relatively nonspecific activating domain that interacts with general transcription factors and with other enhancer-binding proteins, thereby enhancing or silencing the initiation of transcription (18, 38). A number of mammalian and yeast transcriptional modulator proteins were also found to regulate DNA replication, but detailed knowledge of the molecular interactions that underlie this regulation is lacking (5, 19, 24, 33, 34, 35). We report here that the yeast GAL4 transcriptional activator protein and hybrid derivatives of this protein, which have been extensively used for studies of transcriptional activation (38), also induce initiation of replication at a modified Py ori in which the Py enhancer has been replaced with a cluster of GAL4 upstream activating sequences. We have begun a systematic study of this system and present here the data obtained so far, which indicate that transactivation of the Py ori by the GAL4 and hybrid proteins cannot be accounted for by their interactions with transcription factors that bind promoter elements.

5021 to 5265) and with the late promoter (4, 9, 14, 43, 45). Deletion of the entire enhancer inactivated the origin (9, 45). However, a functional origin could be reconstituted by reinsertion of various parts of the enhancer (3, 33, 39, 45). Moreover, functional origins were also obtained in which the Py enhancer was replaced with sequences from other viral and cellular enhancers (la, 9). Previous work on eukaryotic enhancers focused on their role in transcription. It was found that enhancer sequences bind specific protein factors which act as transcriptional modulators (18, 20, 30, 38). Similar proteins bind to some *

MATERIALS AND METHODS Plasmids. Partial maps of the various plasmids used are shown in Fig. 1 to 4. Plasmid pPyAcat26.2' (45) is a pBR322 derivative which contains a Py DNA fragment (nucleotides 4632 to 152), in which the enhancer (nucleotides 5021 to 5262) has been replaced with two repeats of a 26-bp oligonucleotide including the Py ox enhancer element (nucleotides

Corresponding author. 3496

VOL. 65, 1991

TRANSACTIVATION OF THE POLYOMAVIRUS ORIGIN

5108 to 5130). It also contains a chloramphenicol acetyltransferase (CAT) gene attached to the Py early promoter. Plasmid pPyLT1 (49) contains a Py genome from which the large T-Ag intron has been deleted, which was cloned into plasmid pAT153. Plasmid pGSE472cat (25) contains adenovirus type 5 E4 promoter sequences (-240 to +32 from the transcription initiation site) inserted upstream of the CAT gene in a pSP72 vector (Promega Biotec). In addition, this plasmid contains five repeats of a 17-mer oligonucleotide including a binding site for the yeast GAL4 protein (SxGAL4 sites) inserted upstream of the E4 promoter. Plasmids pAG4 and pAG147 (21) include genes encoding the whole GAL4 protein and the truncated N-terminal GAL4 protein [GAL4 (1-147) protein], respectively, which are expressed under the control of the adenovirus major late promoter. Plasmid pSGVP (40) includes a gene encoding a hybrid protein which consists of the N-terminal 147 amino acids of the GAL4 protein fused to the carboxyl-terminal 78 amino acids of the herpesvirus protein VP16 (GAL4-VP16 protein). Plasmid pGAL4-Ela (25) includes a gene encoding a hybrid protein which consists of the N-terminal 147 amino acids of the GAL4 protein fused to amino acids 121 to 223 of the adenovirus Ela protein (GAL4-Ela protein). Plasmid pSG147 (25) includes the gene encoding the truncated GAL4 (1-147) protein. The genes mentioned above in the last three plasmids are expressed under the control of the simian virus 40 (SV40) early promoter. Additional plasmids used were as follows. Plasmid pM96 was constructed by insertion of a fragment of Py DNA extending between the BamHI and EcoRI sites (nucleotides 4632 to 1560) into the corresponding restriction enzyme sites in plasmid pUC119 (47). In this Py DNA fragment, the enhancer (nucleotides 5021 to 5264) was replaced with a XhoI linker. Plasmid pMlOl was constructed by replacement of the Py DNA fragment extending between the BamHI and XhoI sites in plasmid pM96 with a fragment extending between the same two enzyme sites in plasmid pG5E472cat (see above). The latter fragment includes the cluster of 5xGAL4 sites. Plasmid pM107 contains the cluster of 5xGAL4 sites fused to the late side of a Py DNA segment (nucleotides 5265 to 90). To construct this plasmid, plasmid pMlOl was digested with the enzymes BamHI and BglI. A fragment extending between these two enzyme sites, which includes the above-mentioned sequences, was purified from this digest. This fragment was exposed to the enzyme T4 DNA polymerase first at 12°C and then at 37°C to digest the 3' extension formed by the BglI cleavage and to fill in the 5' extension formed by the BamHI cleavage (41) and was inserted into the SmaI site of plasmid pUC119. Plasmid pM108 contains two repeats of the above-mentioned BamHI-BglI fragment inserted in tandem into the SmaI site of plasmid pUC119. Plasmid pMlll was constructed by insertion of a Py DNA segment extending between nucleotides 5265 and 90, which was obtained by cleavage of plasmid pM108 with BamHI and XhoI, into plasmid pM107 between the BamHI and Sall sites in the multiclonal segment. To construct plasmid pM112, a fragment containing the cluster of 5xGAL4 sites was prepared by cleavage of plasmid pM108 with the enzymes EcoRI and XhoI. This fragment was purified and inserted between the recognition sites of the enzymes EcoRI and KpnI in the multiclonal segment of plasmid pM107. The insertion was performed by first ligating the matching EcoRI sites; then T4 DNA polymerase was used to digest the 3' extension produced by KpnI and to fill in the 5' extension produced by XhoI as

3497

described above; finally, the two blunt ends generated by this procedure were ligated. Transfection-replication assays. Mouse WOP cells (23) were propagated at 37°C in Dulbecco modified Eagle's medium supplemented with 5% fetal calf serum in a CO2 incubator as described previously (29). Twenty-four hours before the transfection, 3 x 105 cells were seeded in 60-mm plates in 5 ml of the same medium containing 20% fetal calf serum. Each plate was transfected with plasmids by the calcium phosphate technique (10) as follows. A 240-pAl volume of a solution containing 0.25 M CaCl2, 0.05 M N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.05), and 10 ,ug of plasmid DNA was mixed with 240 IlI of another solution containing 0.05 M HEPES, 0.28 M NaCl, 0.75 mM Na2HPO4, and 0.75 mM NaH2PO4 (pH 7.05). The mixture was incubated for 20 min at 25°C and then added to the plate. The cultures were incubated 6 h at 37°C, after which time the medium was removed and the cells were extensively washed with fresh medium. Then 5 ml of medium containing 5% fetal calf serum was added to each plate, and the cells were incubated for 48 h at either 33 or 39°C, as specified in the figures. The cells were then harvested, and small-molecular-weight DNA was selectively extracted as previously described (16, 31). The DNA was digested with the enzyme DpnI and with other enzymes as specified in the figure legends. The digests were analyzed by electrophoresis in 1% agarose gels and then subjected to Southern blotting and hybridization with a 32P-labeled plasmid probe as previously described (31). The probe used for these assays was pUC119 plasmid DNA (47) prepared by oligonucleotide labeling (17). For quantitative analysis, autoradiograms of the blots were scanned with a Cliniscan 2 densitometer (Helena Laboratories). The relative extents of replication of the test plasmids were determined by estimations of the following ratio: intensity of a band containing a test plasmid/ intensity of a band containing a reference plasmid. These ratios were normalized relative to the smallest ratio, which was given the value 1.0. CAT assays. WOP cells were transfected by the calcium phosphate technique as described above. After 48 h of incubation at 33°C, the medium was removed and each 60-mm plate was washed twice with 5 ml of a solution containing 0.13 M NaCl, 0.026 M KCl, 0.008 M Na2HPO4, and 0.001 M KH2PO4. Protein extracts were prepared at 23°C as described by Seed and Sheen (42). Briefly, the cells were scraped with a rubber policeman, suspended in 1.5 ml of the solution described above, and transferred to an Eppendorf tube. The tube was spun for 10 s at 11,000 x g in a Sorvall microfuge, and the supernatant was removed. The precipitated cells were resuspended in 1 ml of a solution containing 20 mM Tris-HCl (pH 7.5) and 2 mM MgCI2 and were incubated for 5 min at 23°C. Then the cells were precipitated again by centrifugation as described above. The precipitated cells were resuspended in 100 ,ul of a solution containing 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, and 0.1% Triton and incubated again for 5 min at 23°C. This treatment separated the nuclei from the cytoplasm. The nuclei were precipitated by spinning the tube for 5 min at 11,000 x g; the supernatant containing the cytoplasmic extract was heated for 5 min at 70°C and used for the CAT assays as follows. Fifty microliters of the extract was mixed in a plastic vial at 4°C with 200 [lA of a solution containing 1.25 mM chloramphenicol and 100 mM Tris-HCl (pH 7.8). To this mixture were added S ,ul of '4C-labeled acetyl coenzyme A (0.01 ,uCi/,A; specific activity, 4 Ci/mol) and 5 ml of the scintillation fluid Econofluor (Dupont). The mixture was incubated

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J. VIROL.

BARU ET AL.

at 37°C and counted in a Packard scintillation counter after 6 h. The acetylated chloramphenicol diffused into the organic phase; hence, the recorded radioactivity represented the extent of the reaction and gave a measure of CAT activity. It should be noted that the radioactivity increased linearly in our assays for at least 10 h.

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RESULTS Experimental strategy. Two types of shuttle plasmids were used for intracellular replication assays. Plasmids of one type, designated test plasmids, contained modified Py origins, in which the yeast GAL4 upstream activating sequences or Py enhancer elements were inserted next to the origin cores. Plasmids of the second type, designated activators, encoded the yeast GAL4 protein or various derivatives of this protein but did not contain active origins of replication. These plasmids were transfected into Py-transformed mouse cells of a line designated WOP (23), which continuously synthesize a temperature-sensitive Py large T-Ag, the viral protein required for initiation of replication at the Py ori (7). All transfection-replication assays were performed with a constant input of plasmid DNA; i.e., plasmid pUC19 or pUC119 was added to the transfection mixtures such that the cells in each 60-mm plate were transfected with 10 ,Ig of plasmid DNA. Following transfection, the cells were incubated for 48 h at either 33 or 39°C, the permissive or nonpermissive temperature, respectively, for the large T-Ag. Small-molecular-weight DNA was selectively extracted from the cells at the end of these periods and was digested with the restriction enzyme DpnI and with another enzyme that cuts the test plasmid used for each experiment once. The digests were analyzed by agarose gel electrophoresis, Southern blotting, and hybridization with a plasmid probe. The amount of DpnI-resistant plasmid DNA, as determined by blot hybridization, was a measure of the extent of replication of these plasmids (36). In some assays, reference plasmids that had been propagated in Escherichia coli dam were also cotransfected into the cells. These plasmids were DpnI resistant even though they had not replicated in the mouse cells and were used for quantitative assessments of the extents of replication of the test plasmids. The yeast GAL4 protein can induce replication of a plasmid containing a Py origin core and GAL4 upstream activating sequences. The test plasmids designated pM1Ol and pM96 were used for the first series of experiments shown in Fig. 1. As illustrated in Fig. 1C, plasmid pM1Ol contains a fragment of Py DNA including the Py origin core, the early promoter, and a part of the early region encoding the small and middle Py T-Ags and a truncated large T-Ag. The Py enhancer was replaced in this plasmid with five repeats of a 17-bp DNA oligomer containing a yeast GAL4 upstream activating sequence (5xGAL4 sites; 21). Plasmid pM96 contains a fragment of Py DNA including the part of the early region which is also included in plasmid pM101 and a part of the late region. In this plasmid, the Py enhancer has been deleted. One of the activator plasmids used for these series of assays, pAG4, encodes the GAL4 protein, which includes a DNAbinding domain and activating domains (21). A second activator plasmid, pAG147, encodes a polypeptide designated GAL4 (1-147), which consists of the N-terminal 147 amino acids of the GAL4 protein; this polypeptide includes the DNA-binding domain of the protein (21). The transfection-replication assays were performed in mouse WOP cells as described above. Autoradiograms of the blots obtained in these assays are presented Fig. 1A and

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FIG. 1. Transactivation of the Py ori by the yeast GAL4 protein. The plasmids whose structures are illustrated in panel C were transfected into WOP cells as indicated above each lane. The transfection mixtures contained 10 jig of DNA, including 1 ,ug of a test plasmid and/or 2 ,ug of each activator plasmid, the rest being pUC119 plasmid DNA, except for lane pMlOl+GAL4+GAL4(1147), in which the mixture contained 7 ,ug of plasmid pAG147, 1 ,ug of the test plasmid, and 2 ,ug of the activator plasmid. The cultures were incubated for 48 h at the indicated temperatures. Then smallmolecular-weight DNA was selectively extracted and digested with DpnI and BamHI except for lane GAL4, for which XhoI was used instead of BamHI. The digests were analyzed by agarose gel electrophoresis, Southern blotting, and hybridization with a pUC119 probe as described in Materials and Methods. Arrows indicate positions of the linear forms of the test plasmids.

B. The arrows point to the expected positions of pMlOl (Fig. 1A) and pM96 (Fig. 1B) plasmid molecules that have undergone at least one round of replication in the mouse cells, that is, the DpnI-resistant linear forms of these test plasmids. In the experiments performed at 33°C, the permissive temperature for the Py large T-Ag synthesized in WOP cells, no detectable replication of plasmid pMlOl occurred when the cells were transfected with this plasmid alone (Fig. 1A). This plasmid did replicate in cells that had also been cotransfected with the nonreplicating plasmid pAG4, encoding the whole yeast GAL4 transactivator protein. In contrast, no detectable replication of plasmid pM96 was observed when this plasmid was transfected into the cells either alone or with the plasmid encoding the GAL4 protein. We conclude

VOL. 65, 1991

TRANSACTIVATION OF THE POLYOMAVIRUS ORIGIN

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FIG. 2. Demonstration that a wild-type large T-Ag can activate a Py ori in WOP cells incubated at 39°C. Transfection-replication assays of the indicated plasmids were carried out as described in the legend to Fig. 1 except that in lane pPyLT1 the DNA was cut with DpnI and XbaI. Each transfection mixture contained 1 p.g of a test plasmid, 0.5 ,ug of plasmid pUC19 propagated in E. coli dam (ref.), and 8.5 ,ug of plasmid pUC19 propagated in E. coli dam'. The DpnI cleavage products are not shown.

that the GAL4 protein can transactivate the Py ori in a plasmid that contains GAL4 upstream activating sequences instead of the Py enhancer. In another assay shown in Fig. 1A, the cells had been cotransfected with plasmid pMlOl and plasmid pAG147, encoding the truncated protein GAL4 (1-147). It can be seen that this protein was unable to activate the Py ori. In addition, when both the plasmid encoding the whole GAL4 protein and the plasmid encoding the truncated GAL4 protein were cotransfected into the cells with plasmid pMlOl, replication induced by the whole GAL4 protein was inhibited. This result is compatible with the notion that both proteins compete for binding the cluster of 5xGAL4 sites and indicated that the inability of the truncated GAL4 protein to activate the Py ori is not due to a rapid turnover of this protein. It appears, therefore, that the GAL4 DNA-binding domain alone cannot activate the Py ori and that additional GAL4 protein sequences are required for this function. Requirement for the Py large T-Ag. The experiments described so far were carried out at 33°C, the permissive temperature for the Py large T-Ag synthesized in WOP cells. As shown in Fig. 1A, at 39°C, the nonpermissive temperature for the large T-Ag, plasmid pMlOl did not replicate even in the presence of the whole GAL4 protein transactivator. This result, however, was not necessarily due to inactivation of the T-Ag at 39°C. It was possible, for example, that replication of this plasmid was inhibited at 39°C because cell-encoded protein components of the replication apparatus were damaged at the higher temperature. To examine this question, we performed transfection-replication assays of two plasmids including functional Py origins, whose structures are illustrated in Fig. 2B. In one of these plasmids, designated pPyAcat26.2', the complete Py enhancer was a

26-bp

including a segment of the Py enhancer designated a, which provide the enhancer function needed for Py DNA replication (45). This plasmid does not encode any Py-specific proteins. The second plasmid, pPyLT1, contains a whole Py genome in which the large T-Ag intron has been deleted. Hence, this plasmid encodes the large T-Ag and the late viral proteins but not the middle and the small T-Ags. Each of these plasmids was transfected into WOP cells with a nonreplicating DpnI-resistant reference plasmid (see above). The results of the transfection-replication assays performed with these plasmids are presented in Fig. 2A, which shows just the DpnI-resistant bands found in the gels. It can be seen that plasmid pPyAcat26.2', which does not encode a Py large T-Ag, replicated at 33°C but not at 39°C. In contrast, plasmid pPyLT1, which encodes a wild-type large T-Ag, replicated at both temperatures. These results showed that the cell-encoded proteins required for DNA replication could function at 39°C. Hence, the inability of the WOP cells to support the GAL4-induced replication of plasmid pMlOl at 39°C was due to inactivation of the endogenous tempera-

ture-sensitive large T-Ag. Transactivation of the Py ori by hybrid proteins including

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3499

sequence

the yeast GAL4 DNA-binding domain and viral activating domains. Previous studies have indicated that hybrid proteins consisting of the GAL4 DNA-binding domain and activating domains derived from the viral transactivator protein adenovirus Ela (GAL4-Ela) or herpesvirus VP16 (GAL4-VP16) can enhance transcription from promoters containing GAL4 upstream activating sequences in mammalian cells (25, 40). It was therefore of interest to determine whether the hybrid proteins can also transactivate the Py ori. For this purpose, plasmids pGAL4-Ela and pSGVP encoding these proteins were cotransfected into WOP cells with test plasmid pMlOl, and the extent of replication of the test plasmid was determined. Replication of plasmid pMlOl occurred at 33 but not 39°C in the presence of either of the two hybrid transactivators (Fig. 3A). As shown previously, no replication was observed when plasmid pMlOl was transfected into the cells alone or with plasmid pSG147 encoding the truncated GAL4 (1-147) protein. Furthermore, in three plasmid cotransfection assays shown in Fig. 3A, the GAL4 (1-147) protein apparently competed with the hybrid transactivators for binding the 5xGAL4 sites and thereby reduced the extent of replication of plasmid pMlOl to an undetectable level. Relative efficiencies of transactivation of replication and transcription by the yeast GAL4 activating domains and the viral activating domains. A comparison of the intensities of the bands of test plasmid pMlOl in Fig. 1A and 3A indicated that the authentic yeast GAL4 protein transactivated the Py ori more efficiently than did the hybrid proteins including the viral activating domains. Figure 3B shows an experiment that further substantiated this conclusion and allowed a quantitative assessment of the relative potencies of these proteins as replication transactivators. In this experiment, similar assays of the GAL4 and GAL4-Ela proteins were performed in the presence of a nonreplicating DpnI-resistant reference plasmid. In parallel, the extent of replication of plasmid pPyAcat26.2' was assayed again. Scanner readings of the intensities of the bands revealed that the whole yeast GAL4 protein was 10 times more efficient in inducing the replication of plasmid pM1Ol than was the hybrid protein GAL4-Ela. The scanner readings also revealed that the extent of replication of the GAL4-activated plasmid pMlOl was 1.7 times lower than that of plasmid pPyAcat26.2', which was activated by endogenous Py enhancer-binding

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proteins. Similar estimations based on the data shown in Fig. 2 and on additional assays of the same type revealed that the extent of replication of plasmid pPyAcat26.2' was 2.5 to 3.0 times lower than that of plasmid pPyLT1, which contains a whole Py enhancer. These estimations were compatible with previous data of Veldman et al. (45), who compared the replication of plasmid pPyAcat26.2' with that of another plasmid containing a whole Py enhancer. Hence, under the conditions of our assays, the extent of GAL4-activated replication of plasmid pMl0l was 4.0 to 5.0 times lower than that of plasmids containing a complete Py ori. In view of the data described above on the relative efficiencies of the GAL4 and hybrid proteins as replication transactivators, it was of interest to determine the relative efficiencies of these proteins as transcriptional activators in WOP cells under the conditions in which the replication assays were performed. For these transcription activation assays, we used a reporter plasmid (pG5E472cat) in which the gene encoding the enzyme CAT was linked to an adenovirus E4 promoter including a cluster of 5xGAL4 sites of the type shown in Fig. 1 and 3 (25). This plasmid was cotransfected into WOP cells with the plasmids encoding the activator proteins. CAT activities were determined in extracts prepared from the transfected cells after 48 h of incubation at 33°C. In contrast to their relative activities in transactivation of the Py ori, the hybrid proteins GAL4VP16 and GAL4-Ela transactivated the modified E4 promoter much more efficiently than did the yeast GAL4 protein (Table 1). Also, the truncated GAL4 (1-147) protein did not activate the promoter. These results are compatible with the results obtained in other cell types (25, 40). Activation of the Py ori occurs in the absence of promoter elements. Since plasmid pMlOl contains the Py early promoter, it was possible that the activation of the Py ori by the GAL4 and hybrid proteins was caused by their interactions with transcriptional elements. To examine this question, we took advantage of the observation that in Py, unlike the related papovavirus SV40, the promoter elements map outside ori (7), and we deleted from plasmid pM101 the early viral promoter elements, that is, the RNA start sites and about 60 bp upstream of these sites, including the TATA box. Replication of the new plasmid, designated pM107, was then assayed as described above. As Fig. 4 shows, replication of plasmid pM107 was induced by the intact GAL4 transactivator but not by the truncated protein GAL4 (1147). Hence, the early promoter elements are not required

TABLE 1. Transactivation of the adenovirus E4 promoter by the yeast GAL4 protein and the hybrid proteins GAL4-Ela and GAL4-VP16 in mouse WOP cells Transfected DNA'

Herring DNA pG5E472cat pG5E472cat + pG5E472cat + pG5E472cat + pG5E472cat +

pAG147 pAG4 pSGVP pGAL4-Ela

Transactivator

protein6

GAL4 (1-147) GALA GAL4-VP16 GAL4-Ela

Radioactivity extracted into

organic solvent (cpm)c

1,436 ± 43 1,809 ± 103 1,557 ± 55 2,970 ± 390 4,890 ± 790 13,040 ± 580

CAT activity

(cpm)d 373 121 1,534 3,454 11,604

Relative CAT activity'

1.0 2.25 7.6

a WOp cells were transfected with herring DNA or plasmid DNAs as described in Materials and Methods. b Proteins encoded by the transfected activator plasmids. c CAT assays were performed by the phase extraction assay (42) as described in Materials and Methods. Each assay was carried out in duplicate. Numbers represent average values and standard deviations. d Values in the first row of the previous column were subtracted from values in the other rows. ' The CAT activities recorded in the two rows at the bottom were expressed relative to the CAT activity in the fourth row, which was given the value 1.0.

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assayed the replication of plasmid pM108, which contains two tandem repeats of the Py origin core and the cluster of 5xGAL4 sites but no promoter elements. As Fig. 4 shows, the extent of replication of plasmid pM108 in the presence of the GAL4 transactivator was 4.8 times higher than that of plasmid pM107. Apparently, the presence of two origin cores and two clusters of 5xGAL4 sites increased the probability of initiation of replication in a synergistic way (see Discussion). Figure 4 also shows that the extent of the GAL4-induced replication of plasmid pM108 was 2.3 times higher than the extent of replication of plasmid pMlOl and that replication of plasmid pM108 could not be induced by the truncated protein GAL4 (1-147). To assess the contribution of each of the two duplicated elements to the synergistic effect on replication of plasmid pM108, we constructed and assayed the replication of plasmids pMll1 and pM112. Figure 4 shows the structures of these plasmids and the results of these replication assays. Plasmid pM1ll includes one cluster of 5xGAL4 sites flanked by two origin cores. The extent of replication of this plasmid in the presence of the GAL4 transactivator was 1.6 times higher than that of plasmid pM107. It should also be noted that some replication of plasmid pMlll was induced by the truncated protein GAL4 (1-147). Plasmid pM112 includes one origin core flanked by two clusters of 5xGAL4 sites. No detectable replication of this plasmid was observed even when it was cotransfected with the plasmid encoding the whole GAL4 protein or the plasmid encoding the GAL4 (1-147) protein. The origin core was not mutated in this plasmid, as revealed by DNA sequencing and by the ability of the same core to cause replication of another plasmid into which it has been inserted (1). Apparently, the presence of a second cluster of 5xGAL4 sites next to the early boundary of the core inhibited the initiation of replication of plasmid pM112. It should be noted that replication of another plasmid including just one cluster of 5xGAL4 sites inserted next to the early boundary of the core was barely detectable in the presence of the GAL4 protein (not shown).

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promoter elements and of plasmids

of

a

containing

3501

plasmid

duplications

origin core and the 5xGAL4 sites. Transfection-replication the indicated plasmids were carried out 33°C legends to the Fig. 1 and 2 except that 1.4 plasmid DNA was used. The reference that had been propagated in E. coli dam. indicate the relative extents of replication for these assays, normalized relative to obtained in the assay of plasmids pM107 given the value 1.0. at

instead

pMlOl

plasmid

Numbers

of the

the

extent

plus

GAL4,

for Py origin activation by the GAL4 ever, the deletion did affect the efficiency by GAL4, since the extent of the GAL4-induced of plasmid pMl07 was apparently less plasmid pMlOl (Fig. 4). Thus, some promoter elements in the activation of

transactivator. of transactivation

than

one-half

involvement

replication

be ruled out. On the other hand, the reduction of replication of plasmid pMlO7 could strong large T-Ag binding sites which were the deleted segment (6, 37). Activation of replication of plasmids containing of the origin core and of the cluster 5xGAL4 determine whether deletion of the segment early promoter elements and the T-Ag plasmid pMlOl could be compensated elements of the origin of replication, be

due

to

also

duplications

of

containing

binding

by duplication we

constructed

DISCUSSION We have shown in this report that the yeast GAL4 transcriptional activator protein, which also enhances transcription in mammalian cells (21, 48), can induce replication of plasmids containing a Py ori in mouse cells. Replication transactivation by the GAL4 protein was found to be absolutely dependent on the presence of GAL4 upstream activating DNA sequences next to the late boundary of the Py origin core and of a functional Py large T-Ag. These data indicated that GAL4 protein molecules bound next to the origin core induced initiation of DNA replication at the core. It appears unlikely that the GAL4 protein also affected the replication indirectly by enhancement of transcription from genes whose products are required for replication, because GAL4 recognition sequences are not found in mammalian promoters. Since plasmid pMlOl, which was initially used in these studies, contained the Py early promoter, we thought that the GAL4 protein and its hybrid derivatives might activate the Py ori by virtue of their ability to stimulate transcription from the promoter, or through interactions with general transcription factors, without actually causing transcription. Such interactions could, for example, affect the chromatin structure at the adjacent Py origin core and thus facilitate assembly of the replication initiation complex. The observation that the truncated protein GAL4 (14147) could not

3502

BARU ET AL.

activate transcription and replication indicates that these two activities might be causally related. However, other results did not support this hypothesis. First, we found that the efficiencies of activation of the Py ori in plasmid pMlOl by the hybrid proteins GAL4-Ela and GAL4-VP16 were considerably lower than the efficiency of activation by the authentic yeast GAL4 protein. If activation of the Py ori were a consequence of transcriptional activation, then the relative efficiencies of these proteins as activators of transcription and replication should have been correlated. However, contrary to this expectation, the hybrid proteins were found to be more efficient transcriptional activators than the GAL4 protein in the same cells. Second, the GAL4 protein was found to efficiently induce replication of plasmid pM107, which contains the Py origin core and the cluster of 5xGAL4 sites but no Py promoter elements. Furthermore, even though the extent of the GAL4-dependent replication of the plasmid pM107 was less than half that of plasmid pMlOl, the extent of replication of plasmid pM108, which contains two repeats of the origin elements found in plasmid pM107 and also lacks promoter elements, was 2.3 times that of plasmid pMlOl (Fig. 4). Other studies on the role that transcriptional activators play in replication also supported the notion that replication transactivation by these factors may not result from the interactions that lead to transcriptional activation. In one study, it has been found that the CTF/NF-I transcriptional activators also stimulate initiation of adenovirus DNA replication (19). However, the replication enhancement activity in this system required just the N-terminal portion of the CTF/NF-I proteins, whereas transcriptional activation required, in addition, a C-terminal domain (32). Similarly, the DNA-binding domain (POU domain) of the transcription factor Oct 1 (NFIII) suffices for stimulation of adenovirus DNA replication but does not activate transcription (34, 46). Also, whereas the c-Jun/c-Fos heterodimer was found to activate both transcription and replication, the cyclic AMPresponsive element-binding protein, CREB, stimulated transcription but did not transactivate a Py ori containing a cyclic AMP-responsive element site (33). Thus, not all transcriptional activators also stimulate replication. Finally, whereas transactivator proteins that bind enhancer elements stimulate transcription even when these elements are placed at relatively long distances from the promoters on either side of the transcription units, the same proteins could activate the Py ori only when the DNA sequences binding these proteins were placed at relatively short distances from the late boundary of the core (14, 33). It is interesting to consider our data on the GAL4-induced replication of plasmids pM107, pM108, pMlll, and pM112 (Fig. 4) in relation to other models that have been proposed to account for the activation of SV40 and polyomavirus origins by transcriptional activators (8). One model assumes that binding of such proteins to a DNA-binding site inserted next to the origin core prevents chromatin assembly at the origin and thus facilitates formation of the replication initiation complex. Such a mechanism appears to account for the in vitro stimulation of SV40 DNA replication by CTF/NF-I proteins (5). This model is compatible with the positive synergistic effect of the origin duplications on the replication of plasmid pM108, for the repeat arrangement in this plasmid, 5xGAL4 sites-core-5xGAL4 sites-core, could inhibit nucleosome assembly in a synergistic way and thus cause the observed 4.8-fold increase in the extent of replication of this plasmid compared with that of plasmid pM107, which contains the single-copy 5xGAL4 sites-core. However, to

J. VIROL.

account for the observation that the truncated protein GAL4 (1-147) did not activate the Py origins in these plasmids, one would have to assume that not only the DNA-binding domain but also the activating domains of the GAL4 or hybrid proteins were required for inhibition of chromatin assembly. Furthermore, it is difficult to reconcile this model with the complete inhibition of replication of plasmid pM112 containing the repeat arrangement 5xGAL4 sites-core5xGAL4 sites, because the presence of two clusters of 5xGAL4 sites in this plasmid should presumably prevent chromatin assembly at the core at least as well as does a single cluster. A second model assumes that, in analogy to the current models of transcriptional activation, the DNA-binding transactivating proteins interact with proteins of the replication initiation complex, e.g., the Py large T-Ag, thereby stabilizing the complex and increasing the rate of initiation of replication. Such an interaction might also lead to enhancement of the large-T-Ag-dependent unwinding of the Py origin core, as suggested in studies of SV40 DNA replication (12, 13). The positive synergistic effects of the duplications of the cluster of 5xGAL4 sites and the origin core on replication of plasmids pM108 and pM1ll could result from interactions between the proteins that bind the duplicated elements. The inhibitory effect of the second cluster of 5xGAL4 sites inserted next to the early boundary of the origin core in plasmid pM112 could be accounted for if GAL4 protein molecules bound to this element were to interact with the replication initiation complex such that the initiation of replication would be inhibited rather than stimulated. Clearly, definitive experiments designed for testing these models, which are not mutually exclusive, can be performed only in an in vitro replication system. ACKNOWLEDGMENTS We thank P. Clertant, M. R. Green, R. Kamen, D. Last, J. W. Lillie, M. Ptashne, and G. M. Veldman for sending us many of the plasmids used for this study. We also thank C. Basilico and F. G. Kern for sending us the WOP mouse cells and A. Razin for providing the E. coli dam bacteria. This work was supported by a grant from the Council for Tobacco Research (USA), by grant 87-00267 from the United States-Israel Binational Science Foundation, and by a grant from the Israel Cancer Research Fund. REFERENCES 1. Baru, M. Unpublished data. la.Bennet, E. R., M. Naujokas, and J. A. Hassel. 1989. Requirements for species-specific papovavirus DNA replication. J. Virol. 63:5371-5385. 2. Brand, A. H., G. Micklem, and K. Nasmyth. 1988. A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell 51:709-719. 3. Campbell, B. A., and L. P. Villarreal. 1988. Functional analysis of the individual enhancer core sequences of polyomavirus: cell-specific uncoupling of DNA replication from transcription. Mol. Cell. Biol. 8:1993-2004. 4. Cereghini, S., P. Herbomel, J. Jouanneau, M. Saragosti, B. Katinka, B. Bourachot, B. de Crombrugghe, and M. Yaniv. 1983. Structure and function of the promoter-enhancer region of polyoma and SV40. Cold Spring Harbor Symp. Quant. Biol. 47:935-944. 5. Cheng, L., and T. J. Kelly. 1989. Transcriptional activator nuclear factor I stimulates the replication of SV40 minichromosomes in vivo and in vitro. Cell 59:541-551. 6. Cowie, A., and R. Kamen. 1984. Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA. J. Virol. 52:750-760.

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