Proc. Nati. Acad. Sci. USAVol. 88, pp. 3947-3951, May 1991Biochemistry

The nuclear protooncogenes c-jun and c-fos as regulatorsof DNA replication

(polyomavirus/transcription factors API and CREB/enhancer)

YOTA MURAKAMI*, MASANOBU SATAKE*, YUKO YAMAGUCHI-IWAI*, MASAHARU SAKAIt*,MASAMI MURAMATSUt, AND YOSHIAKI ITO*§*Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606, Japan; tDepartment of Biochemistry, Facultyof Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan; and tDepartment of Biochemistry, Hokkaido University School of Medicine,Kita-ku, Sapporo 060, Japan

Communicated by Hidesaburo Hanafusa, January 2, 1991 (received for review March 14, 1990)

ABSTRACT Polyomavirus (Py) DNA replication is regu-lated by its enhancer, which contains an AP1 site. c-Jun andc-Fos, the products of nuclear protooncogenes c-jun and c-fos,form the heterodimeric transcriptional activating factor AP1.Overexpression of c-fos and c-jun genes strongly stimulated PyDNA replication from the Py origin of replication as well astranscription from the Py early promoter through the AP1binding site. The cAMP response element (CRE)-binding pro-tein CREB stimulated'only transcription, not DNA replication,through the CRE under similar conditions. The results indicatethat AP1 functions as a regulator of DNA replication and thatthe mechanism of activation of Py DNA replication by APN isdistinct from that of activation of transcription from the Pyearly promoter.

The products of the nuclear protooncogenes c-jun and c-fosare components of the transcriptional activator protein AP1,which represents one of the principal targets of signalselicited by growth factors or a tumor promoter, phorbol12-myristate 13-acetate (PMA) (see ref. 1 for review). SinceAP1 is a transcriptional regulator, deregulation of transcrip-tion in the cell has been considered as the mechanism of celltransformation by the viral oncogenes v-jun and v-fos (2). Wepresent evidence in this communication that c-jun and c-fosare also involved in the regulation of DNA replication.

Replication of polyomavirus (Py) DNA in the cell isregulated by an enhancer (3). Attempts to identify the ele-ments within the Py enhancer that are responsible for thestimulation of Py DNA replication and of transcription fromthe Py early promoter led to the conclusion that the coresequences in the Py enhancer required for the two functionswere indistinguishable (4-7). In fact, two sequence motifswithin the Py enhancer have been identified each of whichcan enhance the levels of both transcription and replicationby itself (8, 9). One of the two motifs is the AP1 binding site(10). The other binds the product of the protooncogene c-ets(11). The factor that binds to the c-Ets protein binding site isalso called PEA3 (12) or PEBP5 (13). These results suggestthat AP1 and c-Ets have dual functions. Moreover, mutationat the AP1 site within the Py enhancer significantly reducesthe efficiency of Py DNA replication (12).The present study was undertaken to demonstrate directly

the involvement of AP1 in the activation of Py DNA repli-cation and to determine whether or not the dual function ofAP1 is a general property of transcription factors.

MATERIALS AND METHODSPlasmids. pPyOICAT contains a Py DNA fragment [nucle-

otide (nt) 5268 through nt 152] and the chloramphenicol

acetyltransferase (CAT) gene coding sequence (Fig. 1 andref. 8). The Py fragment harbors the replication origin coresequence (nt 5268 to nt 56) and the early gene promoter butlacks the enhancer region. The test plasmids used for thereplication assay and CAT assay were derivatives of pPyOI-CAT. pPyAATCAT, which was used as an origin-defectivecontrol, was constructed by deleting a 33-base-pair (bp)fragment between the Bgl II and Apa I sites of pPyOICAT,which contained the A+T-rich domain of the Py origin coresequence. Synthetic oligonucleotides or Py enhancer frag-ments were inserted into the Bgl II site ofeach plasmid. Whena fragment X was inserted as a y-mer, the resulting plasmidwas designated pPy(X)yOICAT or pPy(X)yAATCAT. Forexample, when the AM oligonucleotide (see legend to Fig. 1)was inserted as a hexamer into pPyOICAT, the resultantplasmid was named pPy(AM)60ICAT. pPy(AM)6CATOI andpPyCATOI were constructed by inverting the orientation ofa Bgl 1I-BamHI fragment of pPy(AM)6OICAT or pPyOI-CAT, respectively, containing the Py origin and the CATgene (see Fig. 3a).The following plasmids were the expression vectors.

pRSV-LT expresses the Py large tumor (T) antigen by using theenhancer/promoter of the Rous sarcoma virus long terminalrepeat (LTR) (8). pHj3ARJ101 (16) is a recombinant plasmid inwhich rat c-jun derived from pRJ101 (17) is driven by the human(-actin gene enhancer and promoter. pH/BARJ101(tr) contains aframeshift mutant of rat c-jun, thereby causing the truncation ofthe translated product (16). pMMV expresses mouse c-fos byusing its genomic enhancer/promoter (18). pCG-CREB, in whichthe cDNA for the human CRE-binding protein CREB (19) hasbeen inserted between the Xba I and BamHI sites ofthe expres-sion vector pCG (20), expressed CREB by using the cytomeg-alovirus enhancer/promoter. The expression plasmid for mouseprotein kinase A, pcDSRaPKA, has been described (21).

Replication Assay. One-half microgram of test plasmid, 4pUg ofeach expression plasmid, and 0.02 ;kg ofDpn I-resistantcontrol plasmid [pHSG398 (22)] prepared from a DNA ade-nine methylation-defective (Dam-) strain of Escherichia coliwere cotransfected into F9 mouse embryonal carcinoma cellsas described (8). The control plasmid was included to monitorthe efficiency of transfection and DNA extraction. When<12.52 jig ofDNA was transfected, pBR322 DNA was addedto give 12.52 umg of total DNA. Cells were harvested 24 hrafter transfection, and replicated molecules were detected byDpn I and HindIII digestion and Southern blotting (8). DpnI cleaves only methylated input DNA and leaves both newlyreplicated DNA and the control plasmid intact. Hind1Il

Abbreviations: Py, polyomavirus; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase; CRE, cAMP re-sponse element; LTR, long terminal repeat; T antigen, tumor antigen;nt, nucleotide(s).§To whom reprint requests should be addressed.

3947

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(5268)lBgl II (152) BamHJ

Hind IH Apa I

F,PQiCAT( J|polyA PPYOICATori early rnRNA

/core

AM gatccA TGACTAACTagT ACTGATTG t c t ag (X6) PPY(AM)6OICAT

AMM 9atccAGTGACAAAGTaSTCACTGTTTCAtC tag (X6) PPY(AMM)6OICAT

CREL 9atCCGCCTCCTTGG GACGTCAG.---5CGGAGGAACCACTGCAGTCTC------AGAGAGTTTa---TCTCTCAAAtCtag (X1) PPY(CREL)iOICAT

FIG. 1. Test plasmids and inserted synthetic oligonucleotidescontaining the AP! recognition sequence (AM), its mutated version(AMM), or the cAMP response element (CRE) representing nt -60to nt -29 of the somatostatin gene (CREL; ref. 14). The binding siteof murine AP1 (PEBP1/PEAl; refs. 10 and 15) in AM and the CREconsensus sequence in CREL are boxed. ori, Origin.

linearizes both the progeny and the control plasmid DNA.The probe DNA used for hybridization was a Bgl II-Nco Ifragment of pPyOICAT containing the Py origin core se-quence and part of the CAT gene. Relative amounts ofreplicated and control DNA were quantitated by densitomet-ric scanning of autoradiograms. Transfection was repeated atleast three times for each construct, using two different DNApreparations.CAT Assay. Four micrograms of test plasmid and 4 gg of

each expression plasmid were cotransfected into F9 cells. Asan internal control, 2 gg of pRSV-fBgal (47), containing thef3-galactosidase gene, was cotransfected. When <16 gg ofDNA was transfected, pBR322DNA was added to give 16 pgof total DNA. CAT activity was measured in the cell extractobtained 24 hr after transfection, as described (9), and wasnormalized to f-galactosidase activity.

RESULTSAPi Activates Py DNA Replication Through the APi Bind-

ing Site. The murine embryonal carcinoma cell line F9 wasJA

a FF9-5000 1AT OICAT AM "'AM!11 Fl1F 1r 7r

jun+fos - + - + + ± -- +LT + + + + +- +--+t + -

replicatedpiasmid --- _

used for the replication assay in vivo. Since the expression ofc-jun and c-fos is limited in F9 (23), this cell line is useful totest the effect of exogenously introduced c-jun and c-fosgenes. pPyOICAT (Fig. 1) contains the Py origin core se-quence but lacks the Py enhancer. Various enhancer frag-ments or synthetic oligonucleotides were inserted into the BglHI site at the late side of the origin core. These test plasmidswere cotransfected with the expression plasmids, and test-plasmid replication was assayed by the Dpn I method (ref. 8;see Materials and Methods).

Wild-type Py enhancer is inert in F9 cells. Several Pystrains that are capable of growing in F9 cells, such asF9-5000 (24) and F441 (25), possess mutant enhancers capa-ble of functioning in these cells. pPy(F9-5000)OICAT, whichcontained the mutated Py enhancer of F9-5000, replicatedwell (Fig. 2a, lane 1). On the other hand, pPyOICAT, devoidof enhancer, replicated poorly (lane 3). The very low level ofpPyOICAT replication detected in lane 3 was consideredauthentic Py DNA replication, since it depended on thepresence of the origin sequence (lane 2) and Py large Tantigen (data not shown). The cotransfection of c-jun andc-fos did not affect the replication of pPyOICAT (lane 4).pPy(AM)60ICAT, which contained the tandem hexamer ofthe AP1 binding sequence as a sole enhancer element,replicated poorly as in the case of pPyOICAT (lane 7),assuring us that F9 cells had little AP1 activity. However,introduction of c-jun and c-fos into the cells enhancedpPy(AM)60ICAT replication 150-fold (lane 8). The enhancedreplication by c-jun and c-fos also represented authentic PyDNA replication, since the deletion of the origin core se-quence or the absence of large T antigen abolished replicationcompletely (lanes 5 and 6, respectively). The oligonucleotideinserted in pPy(F441)30ICAT spanned the region of themutation within the enhancer of a Py point mutant, F441 (25),and did not contain the AP1 site. This oligonucleotide wasshown previously to function as a positive element in F9 cells(26). This plasmid replicated significantly in F9 cells, but itsreplication was not affected by c-jun and c-fos (lanes 9 and10). These results suggested that the stimulation of thereplication of pPy(AM)60ICAT by c-jun and c-fos was notdue to qualitative or quantitative changes in replicationproteins, including large T antigen, but was due to the direct

b pPyF SAM IL% OCATI

__E !rjun t -- er~~fos -+ - + --- -4

replicatedplasmid

plasmid

100

control _ v ___plasmid_

0.0 3.0 3.5 0.0 0.0 0.9 1 50 21 28

I 2 3 4 56 7 8 9 1 0

RelativeActivity 1.0 5 71 -70 1. 3 5. I .1. IS i,

1 2 3 4 5 1 b 1

FIG. 2. (a) Replication of the test plasmids when cotransfected with c-jun and c-fos together with the Py large T-antigen gene. The testplasmids used were pPy(F9-5000)OICAT (lane 1), pPyAATCAT (lane 2), pPyOICAT (lanes 3 and 4), pPy(AM)6,ATCAT (lane 5),pPy(AM)6OICAT (lanes 6-8), and pPy(F441)30ICAT (lanes 9 and 10). F9-5000 represents the Py enhancer of an F9 mutant, F9-5000, with a24-bp deletion (nt 5119 to nt 5142; ref. 24). The F441 oligonucleotide represents a 24-bp region (nt 5216 to nt 5239) of the enhancer ofan F9 mutant,F441, which harbors an A -- G point mutation at nt 5233 (26). Where indicated, the expression plasmids pHf3ARJ101 (c-jun), pMMV (c-fos),

or pRSV-LT (LT) were cotransfected. Bands representing replicated test and control plasmids are shown. The relative replication activity ofeach plasmid is indicated. (b) Requirement of the AP1 binding site for the enhancement of Py DNA replication by c-jun and c-fos. The testplasmids were cotransfected with pRSV-LT with or without chfos and c-jun plasmids. Tr, pH,3ARJ101(tr). The relative replication activity ofeach plasmid is indicated.

RelativeActivity

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Proc. Natl. Acad. Sci. USA 88 (1991) 3949

effect of the products of overexpressed c-jun and c-fos geneson Py DNA replication.The activation of pPy(AM)6OICAT replication by the

simultaneous expression ofc-jun and c-fos was about 2.5- and30-fold higher, respectively, than by either alone (Fig. 2b,lanes 2-4). The synergistic activation of Py DNA replicationby c-jun and c-fos is in good agreement with the reportedproperties of the products of these genes, c-Jun and c-Fos, informing a dimer: the c-Jun/c-Fos heterodimer binds to theAP1 site more strongly than the c-Jun/c-Jun homodimer, andc-Fos does not form a stable homodimer (27-30).Plasmid pH,3ARJ101(tr), in which a mutation in the c-jun

coding region causes truncation of the product and eliminatesthe DNA-binding domain, abolished the enhancement whencotransfected with pPy(AM)6OICAT (Fig. 2b, lanes 5 and 6),suggesting the requirement of DNA binding for c-Jun toactivate Py DNA replication. The mutation in the AP1binding site drastically reduced the activation of replicationby c-jun and c-fos (lanes 7-10), firmly establishing that theeffect of c-jun and c-fos on Py DNA replication was indeedmediated through the AP1 site.

c-Jun and c-Fos Activate Transcription but Not Replicationat a Distance. The position effect of the AP1 site on Py DNAreplication as well as on transcription was examined. InpPy(AM)60ICAT, the AP1 site is next to the late boundaryof the origin core sequence and 185 bp away from the earlymRNA start site. Cotransfected c-jun and c-fos activatedboth DNA replication and transcription (Fig. 3b, lanes 3 and4). In the plasmid pPy(AM)6CATOI, the fragment containingthe origin and the CAT gene was inverted with respect to theAP1 site, so that the AP1 site was about 2.3 kbp from theorigin and the early promoter (Fig. 3a). Transcription fromthe early promoter was still activated efficiently by c-jun and

c-fos, but DNA replication ceased to be activated (Fig. 3b,lanes 9 and 10).To analyze the position effect of the AP1 site on DNA

replication more precisely, various numbers of the 20-bpoligonucleotide were inserted between the origin core se-quence and the AP1 site (Fig. 3a). This oligonucleotide, RD,has no homology to the recognition sequences of knowntranscription factors and showed no enhancer activity foreither DNA replication or transcription in F9 cells in thepresence of c-jun and c-fos (data not shown). Using theseconstructs, we found that the replication-activating functionof the test plasmids sharply decreased as the distance in-creased between the origin core sequence and the AP1binding site (Fig. 3c). In the case of pPy(AM)6(RD)60ICAT,DNA replication was no longer activated by c-jun and c-fos,whereas transcription was stimulated (Fig. 3b, lanes 5 and 6).These results suggest that the activation of Py DNA

replication depends on the position of the AP1 site relative tothe origin and that it is not a consequence of the activation oftranscription, confirming the observation by Hassell et al.(4).CREB Does Not Activate Py DNA Replication. To test

whether activation of Py DNA replication is a commonfeature of transcription factors, we examined the effect ofanother growth factor-inducible factor, CREB, on Py DNAreplication. CREB activates transcription through the CREand is stimulated by protein kinase A (31).The oligonucleotide CREL, containing the CRE sequence,

was inserted into the BgI II site of pPyOICAT as a monomer(Fig. 1). To test the effect ofCREB, pPy(CREL)1OICAT wascotransfected into F9 cells with the CREB expression vectorand the vector expressing the catalytic subunit of proteinkinase A. As reported previously (31), CREB induced CAT

aBgl If early mRNA BamnH I

Hind IIl AMx6 BaH

I Hlill|$,on CAT ( pPy(AM)6OICAT

RD ga t ccGCAAAGTGGGCTGTagCGTTTCACCCGACAtctag (Xn)

pPy(AM)6 (RD)nOICATHind I11 AM X61 l

I §}II}T ) V),aio; pPy(AM)6CATOIVN81u Apee

Hind Ill

-VOI UE iO-X pPyCATOI

C I100

X 50 -

a) A FIG. 3. Thetranscription. (I(Lower). For rewith the test plaand c-fos are inon the activa

0~~- - pPy(AM)601C)0 _API site and trespectively. Rcwith the activit

b (AM)6 (AM)6(RD)6OICAT 1OCAT OICAT

-us--- + --jun + fos - - + - +

(AM )6CATOI CATOI

-- +- - - -

Replicated--

Control -_.4 - U4am . ewe 4,m O___ L _J L ] L

Fold induction i.i 165 2.2 1.1 1.3

e 9t 9 99l

X ~ i nFold induction 1.2 48 28

1 2 3 4 5 6

lI l

1.4 587 8 9 10

e position effect of the AP1 site on the activation of DNA replication anda) Test plasmids. (b) AP1 site-dependent replication (Upper) and transcriptioneplication assay, pRSV-LT and control plasmid pHSG398 were cotransfectedasmids as indicated. Replication and transcription enhancement levels by c-junndicated. (c) Effect of the distance between the AP1 site and the origin coretion of DNA replication by c-jun and c-fos. Test plasmids wereAT, pPy(AM)6(RD),OICAT, and pPy(AM)6CATOI. The distance between thethe origin core in each plasmid was 0, 20, 40, 60, 80, 120, or 2300 bp,elative replication ofeach plasmid in the presence of c-jun and c-fos is shown,ty of pPy(AM)6OICAT set as 100.

0 20 40 60 80 100120 2300Distance from origin core (bp)

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3950 Biochemistry: Murakami et al.

activity of pPy(CREL)1OICAT in a protein kinase A-depen-dent manner (Fig. 4, lanes 4 and 6-8). CAT activity ofpPy(AM)60ICAT was also induced significantly by CREBand protein kinase A, suggesting that CREB also activatestranscription through the AP1 binding site (lane 3). Noinduction ofCAT activity was observed when the binding sitefor nuclear factor I (NFI) was used (lane 11), demonstratingthat the activation of transcription by CREB was specificallythrough the CRE and the AP1 binding site. On the other hand,replication ofpPy(CREL),OICAT and pPy(AM)60ICAT wasnot stimulated at all by overexpressing CREB and proteinkinase A (lanes 3 and 8). Overexpression of c-jun and c-fosactivated both transcription and DNA replication through theCRE at levels lower than through the AP1 site (lanes 2 and 5),confirming that the c-Jun/c-Fos heterodimer bound to theCRE to activate transcription (33).These results indicate that CREB is not a regulator of Py

DNA replication and suggest that the activation of Py DNAreplication is not a common property of transcription factors.

DISCUSSIONAdded c-jun and c-fos protooncogenes activated not onlytranscription but also By DNA replication through the AP1binding site. This activation was not due to the indirect actionofthese genes in enhancing the quantity or altering the qualityof replication proteins but was due to the direct action in cison the origin. Another enhancer-binding protein, CREB,activated only transcription. Therefore, the ability to activatePy DNA replication is not a general property of transcriptionfactors. The results indicate that the two processes aremutually independent. It remains unknown whether thedomains of c-Jun and c-Fos responsible for the activation oftranscription are also responsible for the stimulation ofreplication or whether there exist unidentified domainswithin c-Jun and c-Fos that are responsible for the activationof replication but not of transcription.

In adenoviral DNA replication, nuclear factors I and III(NFI and NFIII), which are identical to the CAAT box-

binding factor CTF and a ubiquitous form of octamer-bindingprotein, OCT1 (34-37), are required for DNA replication.These factors seem to facilitate the formation of the initiationcomplex on the replication origin by direct interaction (38). Inthe case ofNFI, only the DNA-binding domain is required forthis function (39), while the DNA-binding domain of c-Junalone is insufficient for this function (unpublished observa-tion). Cheng and Kelly (32) showed that NFI activates simianvirus 40 DNA replication in vitro by preventing chromatinassembly. It is necessary to examine whether this mechanismis also applicable to AP1.AP1 and CREB are considered as the main targets for two

major signal-transduction systems centered around proteinkinases C and A. PMA and cAMP activate protein kinases Cand A, respectively (40), resulting in the transcriptionalstimulation of many cellular genes through the binding sitesfor AP1 and CREB, respectively. CREB is closely related toc-Jun in some respects: the DNA-binding domain of bothfactors contains a basic amino acid-rich region as well as aleucine "zipper," and both proteins can form homodimers tobind to their respective binding sites, whose consensussequences differ by only one base (27, 28, 41). Recently,CRE-BP1, a CREB-related CRE-binding protein, has beenshown to form a heterodimer with c-Jun and to bind to theCRE (42, 43). CRE-BP1 therefore may have the potential toinfluence the action of c-Jun on DNA replication.PMA induces chromosomal DNA synthesis in quiescent

cells (44), while PMA stimulates Py DNA replication throughthe AP1 binding site (8). Py DNA replication mimics cellularDNA replication in several respects (45). Therefore, AP1may also be involved in the regulation of chromosomal DNAreplication. Since c-jun and c-fos are typical early responsegenes, a question arises as to whether AP1 would function inthe S phase. In this context, it is interesting that the productof a c-jun-related gene appears to be involved in the tran-scription of the Chinese hamster histone H3.2 gene, whichoccurs specifically in the S phase (46). Uncontrolled growthof cells transformed by v-jun and v-fos oncogenes could bedue, at least in part, to the deregulation of cellular DNA

pPy (AM )6 OICAT

PKA - -CREB -

jun + fos +

Replicated -

Control -

+

Reatv 1.0 1 23 1.0activity

*S.0

pPy(CREL ' COAT

_ + +-

-....-

a . 9

1.0 28 1.0 1.1 0.

DPy x iN F 0fCA-

1*J 1 .3 -'. q

Relative 1.0 51 22activity

1 2 3

1.0 32 0.9 1.2 434 5 6 7 8

1.0 0 0'.

9 10. 1.

FIG. 4. Effects of the overexpression ofCREB and protein kinase (PKA) on Py DNA replication and transcription. Replication (Upper) andtranscription (Lower) ofeach test plasmid were measured as in Fig. 3b. Test plasmids were as indicated. pPy(NFI),OICAT contains a 26-bp-longoligonucleotide, gatCATjGAATGCAGCCAAACCATGa, which has the binding site for nuclear factor I (NFI) as indicated by the underline(32). The fold induction of activity by cotransfected expression plasmids is shown.

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replication directly mediated by these genes. It remains to betested whether the gene coding forCREB becomes oncogenicwhen it is overexpressed or structurally modified.

We thank Tom Curran (Roche Institute ofMolecular Biology, NewJersey), Kozo Kaibuchi (Kobe University, Kobe), and MitsuakiYoshida (The Tokyo University, Tokyo) for the generous gifts of theexpression plasmids pMMV, pcDSRaPKA, and pCG-CREB, re-spectively. This work was supported in part by a Grant-in-Aid forSpecial Project Research on Cancer Bio-Science (subject no.02262215) from the Ministry of Education, Science, and Culture,Japan.

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