Transrepression of RNA polymerase II promoters by the simian virus 40 small t antigen.

Microbiology

Transrepression

of

RNA

Polymerase

II

Promoters by the

Simian Virus 40 Small

t

Antigen

WON-BO WANG,t ILAN BIKEL, ERIKA MARSILIO, DAVID NEWSOME,ANDDAVID M. LIVINGSTON*

Dana-FarberCancer Institute and Harvard Medical School, Boston, Massachusetts 02115

Received23 August1993/Accepted23 June 1994

Simian virus 40 (SV40)small tantigen (t)canactivatetranscription from certain RNApolymeraseII and

IIIpromoters (M. Loeken,I.Bikel,D. M.Livingston,andJ. Brady, Cell55:1171-1177, 1988). Herewereport

a new function oft,itsabilitytorepresshumanc-fos promoterandAP-1transcriptionalactivityinCV-1Pcells.

This function is theproduct ofa discreteN-terminaldomainoft,because thelargeTantigen (T)/t-common polypeptide, whichcontainsonlythe first 82amino acids common toboth TandtofSV40,was,like theintact

protein, an active repressor.The datafurthersuggestthat the t-andT/t-common-mediated repressionofc-fos

expressionwas mostlikely manifestatthe level oftranscription. Inkeepingwith thepossibilitythat taffects

theexpression ofthegenomic c-fos promoter,it also led torepressionof AP-1formation.Thus,SV40tis both anactivatorand arepressor oftranscription.Itsabilitytoinhibitc-fos expressionshould beconsidered inlight ofthe natural historyof SV40initsnatural host.

The early region of simian virus 40 (SV40) encodes two

proteins, large T and small t antigens (T and t) (54). t is a

-20-kDapolypeptide found both in the nucleus andcytoplasm

ofinfected cells(15,54).Tis theprimary transforming element ofthevirus,whiletserves as atransformationhelper, enhanc-ing the action of T (4, 5, 7, 12, 17, 18, 21, 30, 41, 51). The N-terminal 82 amino acids oftand T areidentical,while the

remaining 92 amino acids of t are unique. Although t alone appearstolackoverttransforming activity,itssynthesismaybe required for the fullexpression ofan SV40-transformed

phe-notype (4,5, 7, 12, 17, 18, 21, 30,41, 51).

The molecularmechanism which underlies the

transforma-tion-enhancing effect oft is notknown. Inaddition to the T

transformation helper effect,tis knowntohavecertain effects invivo. These include theabilitytopromotethedissolution of the actincytoskeletoninsomecellsand theabilityto overcome

growth-arrestingeffectsoftheophyllineinCV-1cells(6, 19, 21,

41, 42). Recently, thasbeen found to associatewithprotein phosphatase2A(37) and inhibit theenzymatic activity of the latter(46,59).Moreover,twasshowntoactivatetranscription from certainpolymerase II (pol II) andpol III promoters (3,

27, 50). Whether any of these properties is related to the transformation-enhancing activity of t isnotclear.

The c-fos proto-oncogene is the cellular homolog of the transforming gene of Finkel-Biskis-Jinkins osteosarcoma virus

(10, 11)and encodesanuclearprotein active in normal cellular

growth and differentiation (11, 44). The c-Fos protein is a member of a family of transcription factors that contain highly conserved, positively charged DNA-binding domains and leucinezipper motifs. It forms complexes with a member of the Jun family of transcription factorsto form AP-1, a complex which can activate genes containing the

12-O-tetradeca-noylphorbol-13-acetate-responsive element (TRE) (9,39). The control oftranscription by c-fos is believed to play a key role in the cellular response togrowth factors (9, 31). Supporting this view is the observation that introduction of anti-Fos antibodies

(25,

40)

orc-fosantisense RNA(22, 35) into fibroblasts inhibits

*Corresponding author.

tPresentaddress: Graduate Institute ofMicrobiology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.

their proliferation and their ability to reenter the cell

cycle

fromquiescence. Further evidence for the role ofc-fos in the

control of cell growth is suggested by the finding that alter-ations in the normal pattern of c-fos expression can lead to

oncogenesis (57).

SV40infectioncanstimulatequiescentcellstoreenterthe S phase of the cell cycle (20, 21, 49). An analysis of the

transcriptional activation of certain loci in cells infected

by

SV40 revealed enhanced transcription of a common set of

proto-oncogenes, including c-myc and c-fos (33). Since t

pos-sesses a transcription activation function, we examined

whether itcanstimulatec-fos expression and,if so, whetherit

contributes to enhanced c-fos transcription after infection.

Surprisingly,wefound that transfection oft ledto repressed c-fos expressionand AP-1 transcription activation activityand that this repression was most likely manifest at the level of

transcription.

MATERUILSAND METHODS

Plasmids. Plasmid pSV-t/cDNA, which contains an intact SV40tcDNAlinkedtotheSV40early promoter/enhancer,has been described elsewhere (27). The vector plasmid

pRSV.3-BglII (27) (Fig. 1),which contains a Rous sarcoma virus(RSV) long terminal repeat (LTR) and SV40 polyadenylation site,

was used as a recipient for the construction of the three

effector plasmids, pRSV-t/cDNA,

pRSV-TItCommonw

and

pRSV-TEX. pRSV-t/cDNA(Fig.1)wasgenerated by inserting theHindIIIfragment ofpSV-t/cDNA, which contains an intact SV40 t cDNA coding unit, into the unique HindIII site of

pRSV.3-BglII.

pRSV-T/tcommon

(Fig. 1) was obtained by

in-serting the BamHI fragment of

pSV-T/tCommon

(27), which containsT/t-commoncodingunit, into the unique BglII site of

pRSV.3-BglII.

The derivation of pRSV-TEX has been

de-scribed elsewhere(27) (Fig.1).

pRSV_T/tCommonApromoter

was constructed by deleting the

HindIII-NruI

fragment, which contains RSV LTR, from

pRSVJTItCommon.

pSV-tA34-39,

pSV-tA40-45, pSV-tA46-51, pSV-tA65-70,

and

pSV-tsb59-64 were constructed by replacing the

BstXI-SfiI

fragment of pSV-t/cDNA, which contains the N-terminal re-gion of t, with the corresponding fragments from

EMdl34-39,

EMdl40-45, EMdl46-51, EMdl65-70,

and

EMsbS9-64,

respec-6180

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TRANSREPRESSION BY SV40 SMALL t ANTIGEN 6181

MULTIPLE

CLONING

SITE

PRODUCTS

RSV LTR

1 82 174

Hindm

1 82

i I

poly

Asit

pRSV.3-BgIH

pRSV-t/cDNA

pRSV-T/t Common

BamHI

1 82 _-ILi 70 B

samHI

pRSV-TEX

FIG. 1. Structureof thebackbonevectorand thet-,T/t-common-, and T-encoding plasmids.The construction of these plasmids is described inMaterialsandMethods. Thenamesand protein products of these plasmidsarelistedontheright. Codingsequences: E,tunique; _,T/t

common; OII, T unique.

tively(28). The deletionmutantshave each sustaineddeletions of six amino acidsattheindicatedpositionsinthetsequence.

In pSV-tsb59-64, residues 59 to 64 oft were substituted in

framebythesequence NAAIRS (28).

PSVpromoter

was

gener-atedby insertingthe EcoRI-HindIIIfragmentofpSV-t/cDNA,

which containstheSV40 promoter/enhancer unit into pUC18. pSV-t/cDNAAPromoterwasgenerated by deleting the

KpnI-Sfil fragmentfrompSV-t/cDNA. Plasmid

pSV-T/tcommon

has been describedelsewhere (27).

PlasmidspRSV-HGH, which containsanRSVLTR-driven

human growth hormone (HGH) gene, was constructed by replacing the NdeI-HindIII fragment ofp0GH (48) with the NdeI-HindIII fragmentofpRSV.3-BglII.

PlasmidspFC700,pL16-CAT, pEC113-CAT, pcoll-517/+63, pcoll-73/+63, plxcollTRE tata, p5xcoll TREtata, and ptata havebeendescribed elsewhere (16, 22a, 24, 24a, 36).

Cell cultureandtransfection.Low-passage-number monkey kidney CV-1P or CV-1 cells were routinely grown in

mono-layercultures in Dulbecco'smodifiedEagle medium (DMEM) supplementedwith 10%fetal calfserum (FCS)in 10% CO2.

Alltransfectionswereperformedwithyoungcellswhichwere

lessthan 20daysoldafterthawing. Twenty-fourhoursbefore transfection, cells were reseeded in 35-mm-diameter plates.

Cells were transfected at 70% confluency by the calcium

phosphate coprecipitation technique (45); 0.3 jig of pRSV-HGH, a plasmidwhich contains an RSV LTR-driven HGH

gene,was also cotransfected as an internal control for

trans-fectionefficiency. The cellswere exposed to the transfection

precipitatefor8h,washed twice with freshmedium,and refed withDMEM-0.5% FCS. After 12 to 16 h, 100 ,ul of culture mediumwastaken foranHGHassay.(Itisimportanttotake HGH sampleswithin 12to 16 h after transfection,because t

andT/tcommonmodestly repressedthe RSV LTRpromoter/

enhancerafterlonger incubation).Thecellswerethen treated in threedifferentways.For theexperimentsdescribedinFig. 3, 5, and6,cellsweremaintainedinDMEM-0.5%FCS for 24 h and then stimulated with 20% FCS for8 h. For the experi-mentsdescribed inFig.4 andTable 1,cellswereincubated in DMEM-10% FCS for 32 h. For theexperimentsdescribed in Fig. 7, cells were maintained inDMEM-0.5% FCS for 24 h

and then stimulated with 20% FCS for 30 min. After these treatments, cells were harvested for chloramphenicol

acetyl-transferase (CAT)orSi nuclease protection assay.

HGH, CAT,and Si nucleaseprotectionassays.HGHlevels

in the culture medium were measured with a solid-phase,

two-site radioimmunoassay kit under the conditions

recom-mended by the manufacturer (Nichols Institute Diagnostics).

Thisassaycandetectaslittleas0.2ngof HGHperml andis

linearin therangeof 0.2to50 ng/ml. CAT activityassays were

performedasdescribedpreviously (45), using equivalent

quan-tities of extract protein. The products of the CAT reaction

were chromatographed and quantitated by automated

scan-ning (Betascope). To quantitate the levels of mRNA, total

cellular RNA was extracted with aguanidinium

thiocyanate-phenol mixtureas describedpreviously (8). S1 nuclease

pro-tection assays were performed as described previously (45),

using 25 ,ugof whole cell RNA anda5'-end-labeled,synthetic

100-nucleotide (nt)-long probe extending from nt 21 of the

CATgene to -19 of the human c-Fos gene. Intactfos-CAT

mRNAwould beexpectedtoprotectan81-ntsegmentofthis

probe. The nuclease-resistant fragments werefractionated in

an8% sequencing gel.

Celllabelingwith[35S]methionineandanti-T/t immunopre-cipitation.Cellswerelabeled andlysed,andtheextractswere

immunoprecipitated with the anti-T/t monoclonal antibody pAB419asdescribedpreviously (4, 6).

RESULTS

Transrepressionof the humanc-fos promoterby SV40t. To study the effect of t on the human c-fos promoter, we per-formed transient cotransfection assays. A plasmid encoding

intact t was cotransfected into CV-1P cells together with a

plasmid, pFC700, which contains a copy of the human c-fos promoter (nt -710 to +42 relative to the start site of tran-scription) positioned immediately upstream of the bacterial

CATgene.Thet-encoding plasmid(pRSV-t/cDNA)contained

an RSV LTRpositioned upstream of the t coding sequence (Fig. 1). A plasmid, pRSV-HGH, which contains an RSV LTR-driven HGHgenewas also cotransfected as aninternal HhidM

BamHI

BamHI

t

T/t common

T

VOL.68, 1994

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c

0

E

x 8 z -a

> > > >

a: c: cc cc

0C 0C 0

A

S..

_ _ 4..

4_

1 2 3 4 5

tcommon T

524 404 62 17 202

10.0 11.8 7.7 8.4 16.5

B 30

25

T/tcommon

-1 2 3 4

FIG. 2. Immunoprecipitation of SV40 T, T/t-common, or t polypeptide following transfection of CV-1P cells with a T-, T/t-common-, ort-encoding plasmid. Sevenmicrograms ofpRSV-TEX, pRSV-T/tcommon, pRSV-t/cDNA, or pRSV.3-BglII was transfected into CV-1P cells in 35-mm-diameter plates. Cellswere labeled and lysed, and the extracts were immunoprecipitated with the anti-T monoclonal antibody pAB419 as described previously (4, 6). The positionsofT, T/tcommon, andtareindicated.

control for transfection efficiency. Additionalplasmidswhich

were individually cotransfected withpFC700werethose (Fig. 1) which contain an RSV LTR linked to the T/t-common codingsequence

(pRSV-T/tcommon)

andtoanintactTcDNA

sequence (pRSV-TEX). All of these t-, T/t-common-, or

T-encoding plasmidsdirected thesynthesisofabundant prod-uct,innearly equivalentamounts,andof thepredicted molec-ular size inCV-1Pcells(Fig. 2).

As shown in Fig. 3, synthesis of t and the T/t-common peptide (residues1to82)ledtosignificant repressionofCAT synthesis.TherepressionofCATsynthesis bycotransfectionof

pRSV-T/tcommon required synthesis

ofthe truncatedt

product,

sincepRSV-T/tcommonAPromoter, aplasmidcontaining onlythe

proteincoding unit, hadnorepressive effect (Fig. 3A, lane 2).

Amaximum of 8-to10-foldrepression of pfos-CAT(pFC700) by t and 20- to 30-fold repression by T/t common were

repeatedlyseeninmorethan 10experiments.Thadadistinct

but lessprofound effect inthis assaythan eitherof the other

two proteins (Fig. 3).

To rule out the possibility that the repression of CAT

synthesis from pFC700 by t and T/t common was due to a

peculiar property of the fusion gene, a different c-fos-CAT

fusion plasmid, FC4 (13), was cotransfected with the t- or

T/t-common-encoding plasmid. Similar repression of CAT synthesisfromFC4wasobserved (56). Wealsonoted that the

repression of the c-fos promoter by t and T/t common was

z

0

(nU)

CR CL

IL

0.

Lu

20

15

-10

5-

0-T

t T/t com T

FIG. 3. (A) Repression ofthe humanc-fospromoterbytheSV40 tand T/t-commonpolypeptides.TwomicrogramsofpFC700and0.3 ,ugofpRSV-HGHwerecotransfectedinto CV-1P cells with 10,ug of the following plasmids: pRSV.3-BglII (lane 1), pRSV-T/ tCommonAPromoter (lane 2), pRSV-t/cDNA (lane 3), pRSV-T/tcommon (lane 4),andpRSV-TEX (lane 5). (B)Comparisonof foldrepression by the SV40 t, T/t-common (T/t com), and T polypeptides. Two microgramsofpFC700and 0.3,ugofpRSV-HGHwerecotransfected into CV-1P cellswith 10 jigofpRSV.3-BglII, t/cDNA,

pRSV-TItCommonworpRSV-TEX.CATactivitywasmeasured,and thedegree

ofrepression (ratioof CATactivityinthepresenceofvectorplasmid [pRSV.3-BglII] overthat inthepresenceof theeffectorplasmid [see above])wascalculated. Thesameexperimentwasperformed12times. From thesedata,a meanof foldrepressionfor eachpotential effector andastandarddeviation ofeachmeanvalueweredetermined.

more dramatic in young than in old CV-1P cells (56). This result indicated that the cellular factors which mediatet and

T/t-common repressoreffect might havebeen altered orlost

duringcellaging.Therepressionof thec-fospromoterbytand

T/tcommon wasalso observed inHeLaandBALBc/3T3 clone

A31 cells in similar cotransfectionexperiments (56).

tdoes notrepressall RNApolII promoters.Todetermine

whethert-orT/t-common-mediated repressionwasspecificto thec-fospromoterorduetoglobal repression ofgene

expres-sion, a plasmid containing the SV40late promoter linked to CAT (pL16-CAT)wascotransfected with the twot-encoding plasmids.No effects oftorT/tcommon onSV40latepromoter

wereobserved(Fig. 4A).Previously,Loekenetal.(27)showed

Eftector protein

CATactivity (pmol mg min)

HGH (ng ml)

.in It, iwt"

II

.1M.

,;

-4

T

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TRANSREPRESSION BY SV40 SMALL t ANTIGEN 6183

pFC700 pL16-CAT

B

b

_.

* .

lb

4 ~

~

.

1 2 3 4 5 6

- t T/tcom

645 77 25 41

Effector

t T/tcom protein

CAT activity 40 36 (pmol/mgImin)

8.5 6.8 7.2 6.4 5.0 5.2

HGH

(ng/mI)

pFC700 pEC1 13-CAT

-4 - ,

lb.. _S

a,

I

2 3 4

1260 329 112 579

9.7 7.4 10.2 7.8

FIG. 4. Specificity of transrepression ofRNA polII promoters byt. (A) Effects oft andT/tcommon (com) on the SV40 late promoter,

pL16-CAT. Two micrograms of thereporterplasmids, pFC700orpL16-CAT,and 0.3 jigof pRSV-HGHwerecotransfected into CV-1P cellswith

10,ug of pRSV.3-BglII (lanes 1 and 4), pRSV-t/cDNA (lanes 2 and 5),orpRSV-T/tcommon (lanes3and 6). (B) Effect oftonthe adenovirus E2A

promoter(pEC113-CAT). Two micrograms of thereporterplasmids, pFC700orpEC113-CAT, and0.3,ug ofpRSV-HGHwerecotransfectedinto

CV-1 cells with 8 ,ug of either pRSV.3-BglII (lanes 1, 3)orpRSV-t/cDNA (lanes 2 and 4).

thattcould transactivate the adenovirusE2Apromoterinthe

CV-1 cells. Thus,we examined whether tcould transactivate

E2Apromoter(pEC113-CAT) in thesamecellsinwhich itled

toinhibition ofpfos-CAT expression. As shown in Fig. 4B, this

wasindeed thecase.These resultssuggest that the repression

ofc-fospromoterbytandT/tcommonisnotlikely duetoan

effecton CATmRNAprocessingor on CAT itself and that t

possesses discrete transactivation and transrepression

func-tions whichcanbothbe expressed in thesamecell.

Repression of thec-fos promoteris tconcentration depen-dent andrequirestheN-terminalregionoft.Tofurther study

the interaction oftandT/tcommonwiththec-fospromoter, increasing amounts of pRSV-t/cDNA or

pRSV-T/tcommon

were cotransfected with pFC700 in ratios of 1:1, 2:1, 3:1, 4:1,

and 5:1 (Fig. 5).Inhibition ofgeneexpressiondirectedbythe

c-fospromoter was dependenton t andT/t-common

concen-tration, and 2.8- and7.5-foldrepression bytandT/tcommon,

respectively,could beobservedevenataslowas a1:1 ratio of

effector plasmidto reporterplasmid.

Wenext examinedwhether functional tprotein is required

for repression and which regions of t are important for

repression. Specifically,weconstructedseveral in-frame

dele-tion mutants of the t cistron (A34-39, A40-45, A46-51, and

A65-70) and one deletion-substitution mutant(sb59-64). The mutations were introduced into the T/t-common region of t cDNA. In sb59-64, residues 59to 64 oftwere substituted in framebythesequence NAAIRS(28). Uponcotransfection of

each of these mutants with pfos-CAT (pFC700) into CV-1P

cells, onlythe A65-70mutantretained itsabilitytorepressc-fos promoter (Fig. 6). The results of pulse-chase experiments

showed thatA34-39,A40-45,and A46-51mutantproteinswere

relatively unstable,while A65-70 and sb59-64proteinswere as

stable as wild-type t protein (29). The fact that the stable mutant,sb59-64,couldnotrepresspfos-CAT (comparelanes1 and 9 in Fig. 6) implies that synthesis of wild-type t or

T/t-common peptide is needed for the repression of c-fos expression. Moreover, a significant part of the sequences needed forc-fos repression mightlieNterminaltoresidue65,

because the stable mutant,A65-70,was asactiveaswild-typet.

100

pRSV-t/cDNA

0 pRSV-TItCommon

6o

-EL ii

60

2

20

40

0 2 4 6 8 10

[pRSV-VcDNA]or[pRSV-T/t Common] (go)

FIG. 5. Concentration-dependent repressionof thec-fos promoter

by t and T/t common. Two micrograms of pFC700 and 0.3 jig of

pRSV-HGH were cotransfected into CV-1P cells with increasing

amounts of pRSV-t/cDNA or pRSV-T/tcommon. pRSV.3-BglII was

addedtokeep the totalamount ofinput DNAconstant(12.3 jigof

DNAper35-mm-diameterplate).

A

Effector protein CATactivity (pmol/mg/min) HGH

(ng/ml)

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CATactivity (pmol/mg/min) HGH (ng/mi)

1 2 3 4 5 6 7 8 9

108 267 33 19 135 206 169 45 104

8.2 9.0 7.2 7.0 8.6 6.3 8.0 7.8 8.0

FIG. 6. Effects of wild-type and mutant t proteins on the c-fos promoter. Construction of the mutant t plasmids is described in Materials and Methods. Two micrograms of pFC700 and 0.3 ,ugof pRSV-HGHwerecotransfected into CV-1Pcells with 10,ugofvarious tplasmids, asindicated above thelanes.

t leads to reduced c-fos-CAT mRNA abundance. To deter-mine whether the inhibitory effect of t and T/t common on

c-fos-CAT

wasreflectedinareducedabundance of

c-fos-CAT

mRNA, whole cell RNA was prepared from CV-1P cells

cotransfected with pFC700 and either the backbone vector

(pRSV.3-BglII)or aneffector plasmid encodingt,T/tcommon,

or anin-frame deletion mutant oft. A 100-ntsyntheticprobe

extendingfromnt 21 of theCATgene tont -19 ofc-foswas

usedinanS1nucleaseprotectionassay.Asshown in Fig. 7, the abundance of

c-fos-CAT

mRNAwas reduced dramaticallyin

the presence oft orT/t common, although the residual RNA

retained the same 5' end(s) as the unaffected product.

Fur-8 nt 1 41

1 2 3 4 5

Effector

Protein

HGH

(ngImI)

t T.t

com

t tA40-45

6.0 4.8 5.4 4.2 4.5

FIG. 7. S1 nucleaseprotection assay of c-fos-CAT mRNA. Two

microgramsofpFC700and0.3 ,ugofpRSV-HGHwerecotransfected intoCV-1Pcellswith 10 ,ugofthefollowing plasmids: pRSV.3-BglII (lane 1), pRSV-t/cDNA (lane2and4), pRSV-TItcommon(lane 3),or

pSV-tA40-45(lane 5).Thepositionof the 81-ntfragment protected by

c-fos-CATmRNAisindicated.Com,Common.

TABLE 1. RepressionofAP-1 activityonTRE-dependentCAT geneexpressionbytandT/t-commonpolypeptidea CAT fusiongene Effectorprotein CATactivity Repression HGH

construct (pmol/mg/min) factor (ng/ml)

pcoll-517/+63 21.0 9.0

t 2.1 10.0 6.8

T/tcommon 1.4 15.0 8.0

pcoll-73/+63 7.4 8.6

t 1.3 5.7 6.8

T/tcommon 0.9 8.2 7.8

plxcollTRE tata 7.1 10.2

t 0.9 7.9 7.6

T/tcommon 1.0 7.1 10.1

p5xcoll TREtata 23.4 8.0

t 3.7 6.3 6.2

T/tcommon 4.0 5.9 6.8

ptata 0.23 9.5

t 0.21 1.1 7.0

T/tcommon 0.22 1.0 8.6

aTransfection, HGH, and CAT assays were performed as described in Materials andMethods. Two micrograms of the CAT fusionplasmid and 0.3 ,ug

ofpRSV-HGHwerecotransfected with 10 ,ug ofpRSV.3-BglII,pRSV-t/cDNA,

orpRSVWT/tCommon.

thermore, although wild-type t could efficiently impede the accumulation of

c-fos-CAT

mRNA,a tdeletion mutant (tA40-45), lacking six amino acids of its T/t-common region was

inactive in thisassay.Thus,tandtheT/t-common peptideboth blocked the accumulation of

c-fos-CAT

mRNA. Taken

to-gether with the results showing that t had wholly different effects on different promoters driving CAT synthesis in the

same cell(Fig. 4), one canconclude from these data thatthe observed repression effects oft andT/t-common peptide are mostlikely manifestat the level oftranscription.

Repression of AP-1transcriptional

activity.

Sincethe prod-uctofc-fos genecomplexes withamemberof the Junfamily to form the AP-1 transcription factor (9, 39), we examined the effects oftand T/tcommon on AP-1 transcriptional activity.

Since AP-1 can activategenescontaining theTRE,inpartby directbinding to this element,weusedsynthetic TRE-contain-ing promoters cloned upstream of the CAT gene to measure AP-1 transcriptional activity. Either one or five copies of a

human collagenase promoter TRE (collTRE) were inserted upstream of TATA within a TATA-CAT plasmid to give

plxcollTRE tata orp5xcollTRE tata(24, 36).These reporter plasmids were cotransfected into CV-1P cells with either a backbone vectorplasmid (pRSV.3-BglII) or an effector plas-mid(pRSV-t/cDNAor

pRSV-T/tcommon).

As shown in Table 1, CATsynthesis directed by coll TRE was significantly inhib-itedbytorT/t common,while that from the basic TATA-CAT

plasmidwas notaffected. This result indicated that t and T/t

common can block the appearance of AP-1 transcriptional activity. This conclusion was also supported by the observation thattandT/t common could inhibit the function of a human collagenase promoter (pcoll-517/+63 or pcoll-73/+63) which

contains asingle copyof theTREin its natural position (Table 1). The fact that t and T/t common can block AP-1 activity is

consistent with these proteins repressing endogenous c-fos

geneexpression.

DISCUSSION

SV40t canactivatetranscriptionfrom certainpol II and pol

IIIpromoters(27).Here, we showed that t can also inhibit the

activity of the human c-fos promoter and diminish AP-1 abundance. Thesetwoeffectsarepotentiallyrelated, although

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TRANSREPRESSION BY SV40 SMALL t ANTIGEN 6185

definitivemeasurementsof genomic c-fos promoter activity in the absence and presence of t will be needed to establish a clear linkage between them. Both t andT/t common, which contains only the N-terminal 82 amino acids of t, displayed

c-fos transrepression activity, and the truncated fragment

appearedtoinhibit the action of the c-fos promoter even better than t.

Repression of the c-fos promoter by t orT/tcommon is not

a result of the sequestration of limiting quantities of one or moretranscription factors by the RSV promoter present in the transfected plasmids but requires the synthesis of t or

T/t-common polypeptide. This assertion was supported by the

following findings: (i) cotransfection of pRSV.3-BglII, which containsonly the RSV LTR, did not repress the c-fos promoter

(comparelanes 1 and 2 inFig.3A)(56); (ii) clear repression of thec-fospromoter wasnoted even at low inputs of the t orT/t

common-encoding plasmids (Fig. 5); and (iii) the c-fos

pro-moter wasnotrepressed by several mutant t-encoding plasmids

(Fig. 6).

Repression of thec-fospromoterby t andT/tcommon was

observedinbothserum-inducedand cyclingCV-1Pcells (Fig. 4 and 7) (56). That t may prevent serum-stimulated c-fos

induction has been reported previously (34). However, this t

effect was not observed in all promoters. In addition to the

c-fos,

collagenase, adenovirus E2A, and SV40 late promoters

(see Results), we also examined the effect of t on several cellular and viral promoters.Preliminary resultsindicated that

t can repressJun-B, mousemetallothionein-I, herpes simplex virus thymidine kinase, proliferating cell nuclear antigen, and

humanimmunodeficiencyvirus type 1 LTR promoters but had

no effect on thecytomegalovirus major immediate-early

pro-moter-enhancer (56). By contrast, t stimulated the adenovirus E2A promoter(Fig. 4B) (27). Therefore,tcanrepresscertain cellular and viral promoters and activate others. Thus,

t-mediated repression is not due to a general shutoff of the

transcription machinery.

The molecular mechanism underlying the repressor effects of t and T/t common on promoter activity is not known. Because t lacks detectable DNA-binding activity (38, 52), it

probably does not carry out its transrepression function

throughadirectDNA-bindingmechanism. In this respect, it is worthnoting thatt canassociate withprotein phosphatase 2A and inhibit the enzymatic activity of the latter (46, 59). By

modifyingtheenzymatic activityofprotein phosphatase 2A,t

might inactivate one or more transcription factors, and this

could, in theory, lead to transcriptional repression of some

promoters.Otherpossibilities exist, including specific

interac-tionsoftwithone or moretranscriptionfactors. In this respect, it is worthnotingthattheamino-terminalregionofT,

includ-ing the T/t-common region, can interact with the

TATA-binding protein

(19a).

To

study

the mechanism

by

which t represses the

c-fos

promoter, we have initiated an effort to locate the cis-acting elements within the

c-fos

promoter

re-sponsible for the t repressor effect.

Preliminary

results

(56)

indicated that multiple sequence elements in the

c-fos

pro-moter could mediate repression by t

independently.

The promoterregionfrom-710to -363 didnotcontain sequence elementsresponsivetot, but theregionfrom -225to-61 did. This region includes the primary cis-acting sequenceswhich contribute to the basal promoter

activity

of the gene

(43).

Thus,it ispossiblethattmediates

c-fos repression by altering

the action of elements which control basal promoter

activity.

Consistent with this

prediction,

we also found that the basal

transcription activity of the

c-fos

promoterwas

repressed by

t

(56).

T/t common was a more effective

c-fos

repressor than t,

while T was much less active in this regard (Fig. 3A, 4A, 5, and 6). Among the various explanations for these quantitative differences is the possibility that the observed repression function resides in the N-terminal T/t-common region of t and T, and the unique regions of t and T modulate its action differently. Consistent with the first part of this conclusion was the observation that a stable t mutant (sb59-64), which con-tains substitution mutations at T/t-common region, was nearly inactive in repressing the c-fos promoter (Fig. 6). Several other stable t mutants which contain substitution mutations within the N-terminal region have been constructed, and they too weredefective in transcriptional repression (56).

The transrepression and transactivation functions of t are partially separable. In this regard, although T/t common was a relatively efficient repressor, it, unlike t, appeared to lack transactivation function (27). That t possesses both transcrip-tional activation and repression functions which are, at least in part,separable is also true for the adenovirus ElA protein (26, 47). Recently the ElA protein was shown to repress AP-1

activity, and down regulation by ElA may participate in the transforming process (36). That inhibition of transcription factor function byanoncogene maybe linked to its transform-ing activity has also been reported for v-rel. v-rel, but not nontransforming v-rel mutants, inhibits NF-KB function,

indi-cating that the transforming activity ofv-relmight be associ-ated with its inhibitoryaction on NF-KB function (1).

Transcriptionalrepression by ElA can be closely correlated withitstransforming activity (26, 47, 53, 55, 58). t can enhance the transforming activity of T (5, 32). Indeed, the T/t-common region, which has clear transcription repression activity (Fig.

3A, 4A, 5, 6, and 7), is essential to T transforming function (28,

32). Moreover, the presence oft can supportTtransforming action when the segment from residues 1 to 82 of the latter has been deleted (32). However, this region of t did not, alone,

possess transformation helper activity (2, 32). This result

indicates that if the t transcription repression function is linked toits transformation helper function, it is not sufficient for the expression of the latter activity.Inkeeping with this suggestion, mutations in theunique C-terminal region oftdo inhibit the transformation helper activity of t, indicating that the C-terminalregion oftis alsorequired for the expression of the latteractivity(23).

The N-terminal regionofElA, includingconserved region

1, has been shown to be important for both transcriptional repression and transformation functions of ElA(26,47,53,55,

58). Our present data and previous reports (28, 32, 58)

suggested that the N-terminal region oftisalsoimportant for the similarfunctionsoft.Moreover,theN-terminalregion of

t reveals some structural homology to that of ElA (14, 58). Bindingofa300-kDaproteintoElA iscloselyrelatedtoElA

transcriptional repression and transformation function and

depends upon the integrity of the ElA N-terminal segment

(53, 55).Giventhisfinding,it will beinterestingto determine whether p300 or an analogousprotein binds to t and, if so, whether it is involved in thetrepressionfunction.

Finally, why does a tumor-promoting

protein

repress the

expression of a

growth-promoting

proto-oncogene such as

c-fos? Althoughthereisnoprovenanswer tothis

question,

itis worthnoting that whateverbiologicalvalue suchaneffect may

have,it isprobablylinkedtothe survival ofSV40 in its natural

host, in which it is not a tumor virus. This virus

persists

effectivelywithoutelicitingdisease in

healthy primates,

and the related BKand JC viruses do the same in humans. Conceiv-ably, one role for t, which does not seem tobe essential for viral DNA replication in

cycling

cells,

is to promote viral

persistenceincertain otherwise

permissive

cells whichareout VOL. 68, 1994

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of cycle. One might expectthatthe initiation of cellular DNA replicationatthe handsofamitogenlike Tcould belethal to a cell which can otherwise support autonomous viral DNA replication.Intheabsence of the latter,which wouldrequire a

nonreplicating cell to exit

Go

and enter S and to activate

certain immediate early genes such as c-fos in the process, a resting host cell could, in theory, sustain the persistence ofa viral replicon in a stable form.

ACKNOWLEDGMENTS

We thank Ron Prywes for supplying pFC700; Mary Loeken for supplying pEC113-CAT and pL16-CAT; Hans van Dam and Peter Herrlich forprovidingpcoll-517/+63,pcoll-73/+63, plxcoll TRE tata, p5xcoll TRE tata, and ptata; and James A. DeCaprio for critically reading themanuscript.

Thiswork wassupported by a grant from theNational Institutes of Health.

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