Identification of critical cis elements involved in mediating Epstein-Barr virus nuclear antigen 2-dependent activity of an enhancer located upstream of the viral BamHI C promoter.

JOURNAL OFVIROLOGY, May1992,P. 2846-2852 0022-538X/92/052846-07$02.00/0

Copyright ©1992,American SocietyforMicrobiology

Identification of Critical

cis

Elements

Involved in

Mediating

Epstein-Barr Virus Nuclear Antigen

2-Dependent

Activity

of

an

Enhancer

Located

Upstream

of the

Viral BamHI C Promoter

XIANW. JINAND SAMUEL H. SPECK*

Division ofTumorVirology, Dana-Farber CancerInstitute, andDepartment of Pathology, HarvardMedicalSchool, 44Binney Street, Boston, Massachusetts 02115

Received 27 November 1991/Accepted 11February 1992

The sixgenes encoding the Epstein-Barr virus nuclear antigens (EBNAs) aretranscribed fromoneof two promoters,BamHICpromoter(Cp)orBamHl Wpromoter(Wp), locatednearthe left end of the viralgenome.

During theestablishment of viral latency in B lymphocytes, Wp is used exclusively beforeaswitchtoCpusage.

We andothers havepreviously identifiedanenhancer in theregionupstreamofCp which requiresEBNA 2 for

activity(M. Woisetschlaeger, X. W. Jin, C. N. Yandava, L. A. Furmanski, J.L.Strominger, andS. H.Speck, Proc. Natl.Acad. Sci. USA88:3942-3946, 1991; N. S. Sung, S.Kenney, D. Gutsch, and J. S. Pagano, J.Virol. 65:2164-2169, 1991). Infection ofB lymphocytes with a mutant virus lacking the EBNA 2 gene results in prolonged usage ofWp and failure to switch to Cp usage, indicating that EBNA 2 transactivation of the

enhancer upstream ofCpmaybe critical forpromoterswitching.Inthis study,wehavedefinedthe minimal EBNA2-dependent enhancer by using a series ofdeletion mutants. The results ofsite-directed mutagenesis revealed that therearethree regions ofthe enhancer thatareimportant for activity,two of which appearto bind B-lymphocyte-specific factors.

Epstein-Barr virus (EBV) is a lymphotropic human

her-pesvirus which is the etiologic agentof infectious

mononu-cleosis, aself-limiting lymphoproliferative disorder. In

addi-tion, EBV is associated with two human cancers, African

Burkitt's lymphoma and nasopharyngeal carcinoma. Infec-tion of human B lymphocytes with EBV predominantly establishes a latent infection with littleor novirus produc-tion and concomitant growth transformation of the infected cells (immortalization).

In latently infected growth-transformed B lymphocytes, six EBV nuclear antigens (EBNAs) and three viral

mem-brane proteins (latent membrane proteins [LMPs]) are

known to be expressed (for reviews, see references 4, 12, and26). The viralgenesencoding these antigensare

distrib-utedthroughout the viralgenome. However, transcription of theseantigens is driven bypromotersthatareclusterednear

the terminal repeatsofthe viralgenome. Two viral promot-ers have been identified (BamHI C promoter [Cp] and BamHI W promoter [Wp]) that are involved in driving

transcription of the six EBNAgenes(Fig. 1). The activities

ofCpandWp are mutually exclusive in all clonal cell lines

which have been examined (34). Wp is exclusively used during the initial stagesofinfection and thenaswitchtoCp

usage occurs(35).

Severallines ofevidence indicate that EBNA 2 isessential for growth transformation of B lymphocytes and that it is involved in modulating the activity of several viral and cellular promoters (1, 5, 9, 11, 13, 21, 30, 31). EBNA 2 has been shown to transactivate Cp (28, 33) as well as the

promotersforthegenesencoding LMP 1, LMP 2a, andLMP

2b (1, 5, 32, 37). Characterization of the mechanisms by which EBNA 2 transactivates EBV latent promoters is essentialtounderstanding establishment and maintenance of latency and is likely to provide important insights into the

*Correspondingauthor.

roleof EBNA 2 plays in the processoflymphocyte immor-talization.

We and others have previously identified a region

up-stream of Cp (-429 to -245 bp) that contains an EBNA 2-dependent enhancer (28,33). Furthermore,wehaveshown that infection of B lymphocytes with a nonimmortalizing

strain of EBV lacking the EBNA 2 gene resulted in Wp activity andafailuretoswitchtoCp activity (33). The latter observationprovided evidence that EBNA 2 is required for viral promoter switching. In this study, we further dissect

the EBNA 2-dependent enhancer associated with Cp to evaluate the functional roles of various cis elementswithin thisregion thatare involved inmediating EBNA 2 transac-tivation.

MATERIALSANDMETHODS

Cellculture, transfection, and CATassays. DG75 cells (an

EBV-negative Burkitt's lymphoma cell line), Jurkat cells (a T-cell line), and HeLa cells (an epithelial cell line) were

grown at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum as previously described(25).

DNA transfections were carried out by using DEAE-dextranasdescribedpreviously (16) with modifications. The cells (5 x 106 to 1 x

107)

were spun down at 1,000 x g,

washed once with phosphate-buffered saline (PBS) and

resuspended in500,ulof RPMI 1640 medium withoutserum

(GIBCO). Cellswere then transferred to sterile tubes

con-taining2,ug of the relevantCsCl-purified plasmid DNA, and the final concentration of DEAE-dextran was 500 ,ug/ml.

Cellswere incubated at37°Cfor30 min and then 250 ,ulof 20% dimethyl sulfoxide (in RPMI 1640 medium without serum) was added for 2 min. After the dimethyl sulfoxide shock,cellsweretransferredtoa15-ml conical tube with 10

ml ofRPMI 1640 medium andcentrifuged at1,000 x gfor 5 min. The cellpelletwas thenresuspended in 10 ml of RPMI 2846

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EBNA4

C1C2 WOW1W2 W1W2 W1W2

IEBNA2 OTHER

EBNAs

W1W2

X

/

2\V/T

40 60

k(0

400 -380 -360

I

-340

MGATTATG_A_C GTCGAGTGCTAT CaAA

GTGcorm m

DNA" I proetonbd

REG0NSca _rDI

domI go

domah"

dom ain

TGCA 0.07

domain

CTGCA CGCG

0.12 0.26

domisnI

domainIl

domainIN'

doman o0go*

FIG. 1. Summary of transcription initiating from Cp and Wp, activities of the GTG core enhancer mutants and competition with

oligonucleotides containing specific domains within the EBNA 2-dependent enhancer. Thecommon5' leaderpresentin all the transcripts encodingthevarious EBNAs isillustrated in thetophalfof the figure. Alsoshown is thesequenceof the regionupstreamof Cp containing

anEBNA2-dependent enhancer. The specific nucleotide alterations introduced in the GTGcoremutantsareindicated,andthe regions which

lostprotection from DNase I digestion with these mutationsare indicated beloweachmutant. EBNA2 transactivation ofeach GTGcore

mutantisgivenrelativetotheunmutated enhanceractivity. In addition, the regions included in each competitor oligonucleotideareindicated

and the results of thebinding competitions shown in Fig. 5aresummarized.

1640 medium with 10% fetal bovine serum and cultured at 370C.

Transfectedcellswereharvested at72 h posttransfection,

spun down at 1,000 x g, washed once with PBS, and

resuspendedin 100 ,ulof0.25 M Trischloride (pH 7.5).The cell suspension was then lysed by three rounds of snap

freeze-thawing, and the debriswasremoved by

centrifuga-tion. The chloramphenicol acetyltransferase (CAT) assays wereperformed aspreviously described (8) and quantitated

by aBlotanalyzer (Betagen Corp., Waltham, Mass.).

Plasmid construction and site-directed mutagenesis. Plas-midscarryingdeletions in the EBNA2-dependent enhancer regionweregeneratedby cloning polymerasechain reaction

(PCR)-amplifiedenhancersequencesdownstream (XhoI and

Sacl sites) of a CAT reporter construct containing the enhancerless simian virus 40 (SV40) early promoter. The PCRs were carried out as described previously (18) with

slightmodifications. Pairs ofsingle-stranded synthetic oligo-nucleotides(30-mers) homologoustoregionsintheenhancer

were used as primers to generate each deletion by PCR amplification. The 5' primerscontained anXhoI restriction

site at their 5' ends, while the 3' primers contained aSacI

restriction site at their 3' ends. Twenty cycles of PCR amplificationwerecarried out.

The plasmids containing mutations within the EBNA 2-dependent enhancerwere generated by cloning the

PstI-Sacl fragment containing the SV40 promoter, CAT gene,

and the enhancer(from -415 to -321bp) intoaBlueScript vector(Stratagene), and then by generating single-stranded plasmid and performing site-directed mutagenesis as

previ-ously described (6). All mutations introducedwere

charac-terizedby DNAsequencing (22).

Preparation ofnuclearextracts and DNase I footprinting. Nuclear extracts were prepared essentially as previously

described (3). Briefly, 1 liter of cells were harvested and washed withPBS.All subsequentstepswerecarriedouton

ice. The cells were suspended in 5 volumes of prechilled

buffer A (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid] [pH7.9],1.5 mMMgCl2, 10 mM

KCI,

0.5 mMdithiothreitol)andhomogenizedinaDounce homog-enizer, and the crude nuclear fraction was recovered by centrifugation at 10,000 x g for 10 min. The nuclei were

suspended in buffer C (20 mM HEPES [pH 7.9], 25% [vol/vol] glycerol, 0.42 M KCI, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol)andhomogenized again inaDounce homoge-nizer. The extractwas then stirredgentlyfor30min at4°C and clarifiedby centrifugationat25,000xgfor 30min.The supernatantwasdialyzed againsttwochangesof50 volumes of buffer D(20mM HEPES[pH 7.9],20%

[vol/voll

glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol) for 5 h and centrifuged at

25,000 xgfor 20min.Thesupernatantwasrecovered,and

oriP

" . . .

w

BamHI

Fragments

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2848 JIN AND SPECK

protein concentration was determined

by

Bio-Rad protein assay. Aliquotswere storedat -70°C.

DNase Ifootprintingwas carried out asdescribed previ-ously

(14).

Binding reactions were

performed

in a solution containing 10 mM Tris

(pH 7.9),

0.5 mM EDTA, 0.5 mM

dithiothreitol,

2.5%

glycerol,

2%

polyvinylethanol

with 1 ,ug of poly(dI-dC) in the reaction buffer for 20 min at room temperature. DNaseI wasthenadded andincubatedatroom temperaturefor 30s.

Digestion

was

stopped by

theaddition of 150 ,ul of stop buffer (8 M urea,

0.5%

sodium dodecyl sulfate, 5 mM EDTA). The samples were extracted two times with phenol, two times with

phenol-chloroform,

one time with chloroform; the samples were then precipitated with ethanol and run on a8% acrylamide denaturing gel.

For thecompetition

studies,

the

following oligonucleotide

pairswereannealed and

ligated:

domain I, 5'-CTAGACAC

GCCGTGGGAAAAG-3',

5'-GATCCTll1TCCCACGGCGT

GT-3', domain II,

5'-CTAGAGGTTCAGTGCGTCGAG-3',

5'-GATCCTCGACGCACTGAACCT-3',

domain III, 5'-CT AGACGTCGAGTGCTA

TCTG-3',

5'-GATCCAGATAGC ACTCGACGT-3'; and domain III', 5'-CTAGATCGAGTGC

TATCTTTGGAACAG-3',

5'-GATCCTGTITCCAAAGATAG

CACTCGAT-3'.

RESULTS

TheminimalEBNA2-dependent enhancer element mapsto theregion protectedfromDNase Idigestion byB-cellnuclear extracts. An EBNA 2-dependent enhancer has been previ-ously mapped to a184-bp region(-429to-245bp) upstream of Cp

(33).

To fine map the minimal sequence element(s) required for enhancer activity, a number of deletion con-structsweregenerated and cloned downstream of the CAT gene in a reporter construct driven by the minimal SV40 early promoter(Fig. 2). Characterization ofEBNA 2-depen-dent enhancer activity of the various deletion constructs revealed that theregion from -380to -331 bp is essential for enhancer

activity

[SVpCAT(-380/-331)

in

Fig.

2]. This

region

corresponds to the region protected from DNase I digestion by nuclear extracts prepared fromBlymphocytes

(33).

It is important to note that our previous DNase I

footprinting

results indicated that there was no striking difference in the protection pattern observed with extracts prepared from an EBV-negative Burkitt's lymphoma cell line and two EBV-positive Burkitt's lymphoma cell lines

expressing

EBNA 2

(33).

This result suggested that EBNA 2 isnot

directly interacting

with an enhancerelement.

Inclusion of upstream sequences between -415 and -380

bp

resulted in an increase in enhancer activity, indicating the presence of a positive regulatory element(s) in this region. However, these upstream sequences alone did not exhibit any detectable EBNA 2-dependentenhancer activity

[SVpCAT(-429/-370)

in Fig. 2]. In contrast, inclusion of downstream sequences to -245bp appeared tosignificantly inhibit enhancer activity [e.g., compare activities of

SVp-CAT(-429/-310)

and

SVpCAT(-429/-245)

in Fig. 2]. In-terestingly, ourprevious DNase I footprinting studies dem-onstrated that nuclear extract prepared form the EBV-positive Burkitt's

lymphoma

cell line clone 13 gave a protection pattern in the region from -330 to -310 bp that was distinct from that observed with nuclear extracts

pre-pared

from other B-cell lines (indicated in Fig. 2) (33).

Activity

studies in clone 13 cells, whose resident EBV genome has a deletion that spans the EBNA 2 gene and therefore does not express EBNA 2, revealed that the enhancer(in the absence of cotransfection with an EBNA 2

SVP7-

I CAT

-380 -330 -310

'777i

b4p arFtp4 -245bp

*.___ -264bp

-286bp

-310bp

:.370bp

-415bp

. -321bp

A40 bp -264bp

-286bp

-310bp

-i80bp

-310bp

:421

bp

30bp j10bp

L...2.1bp

-34,bp -245bp

-283bp

SVpCATCONTRL

0 10 20 30 40 50

RELATIVE ACTIVITY

FIG. 2. Deletion analysis of the region

containing

the EBNA

2-dependent

enhancer. CATconstructswerecotransfected into the DG75 cell line with either acontrol plasmid (solid

bar)

orwith an

EBNA 2

expression

vector

(stippled bar).

Activities are given relativetothe

activity

observed with theparent

SVpCAT

reporter

plasmid

cotransfected with control

plasmid.

Allactivitiesrepresent the averages ofat least five

independent

experiments. CI-13 Ftpt, clone 13

footprint.

expression

vector)

significantly

suppressed

activity

of the minimal SV40 minimal

early

promoter

(33).

This result also

suggested

the presence cis elements in this

region

of the viral genome that may bind cellular factors that function to suppress enhancer function.

Three domains within the

protected

region

are

important

for EBNA

2-dependent

enhancer

activity.

Since the

region

protected

by

nuclear extracts

prepared

from

B-lymphocyte

cell lines was

large,

we

suspected

that several cellular factorswere involved in

binding

to theenhancer.

Thus,

to

identify

functional domains

binding

cellular

transcription

factors within the minimal enhancer

element,

a series of site-directed mutationswereintroduced into the

region

from -415 to -321 bp and

assayed

for EBNA

2-dependent

enhancer

activity (Fig.

3).

Each mutation introduced substi-tutions of 3 to 6 nucleotides. Two domains

appeared

to be critical for enhancer

activity.

Mutations in the

region

from -379to -368

bp

and mutations in the

region

approximately

-357to-350

bp dramatically

reduced

activity

(see

activities of

-3701-375, -3721-368, -3571-353,

and

-3541-350

mu-tants in

Fig.

3).

In addition, the

-343/-339

mutant also

significantly

reduced EBNA 2-dependent enhancer

activity

to

approximately

20% ofthe unmutated enhancer.

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[image:3.612.330.565.73.397.2]

400 -380 -360 -340 -320

AAGATTATCAAGTTGGTGTAAMCACGCCGTGGGMAAAAATXTATGGTTCAGTGCGTCGAGTGCTATCITTGGAAAGTAGAAA

agcta

agc

aa ctt

oriP &Wp

homology

aagt

tcgc

cgcg

gatc

cggc

aagct

g ccg

cgc

aagctt a ac

ggcc

MAJOR B CELL FTPT

WT ;e', >ist! |

-401/-3977 -395/-392

-Mic

-388/-383 -379/-375 i -372l-368 -366/-363 -.

-360/-357 ; -358/-54A E,,

-357l-3533 -354/-350 3

-349,-46-..",1.

-349/-344 <# Fg

-343,-339 z

-336/-333 l

0 0.2 0.40.6 0.8 1 1.2 RELATIVEACTIVITY

FIG. 3. Mutational analysis of the EBNA 2-dependent enhancer. Site-directed mutations were introduced into an SVpCAT reporter constructcontaining the EBNA 2-dependent enhancer (from -415 to -321 bp) cloned downstream of the CAT gene. EBNA 2-dependent enhancer activities aregiven relative to that of the parent construct. The activities are expressed relative to the unmutated promoter and represent theaverages of three independent experiments. WT, wild type; FTPT, footprint.

Comparison of the nucleotide sequences in these regions of the enhancerrevealedalowlevel of homology(indicated by overbars inFig. 1 and 3). Each domaincontains a GTG coresequence andsubsequent mutations targetingthe GTG core in each of these domains also significantly reduced enhanceractivity (see GTGcore mutants inFig. 1), indicat-ing that the region of homology between these sites are important forfactorbinding. It ispossiblethatthese domains bind the same or related cellular transcription factors and that these factors mediate EBNA 2 transactivation. How-ever,itshould be noted thatourprevious DNaseI footprint-ing (33) indicated that the region from approximately -365to -380bp (domain I) binds afactor(s)presentin bothBandT

lymphocytes,

while the other two regions (domains II and III)were not protected byextractfromaT-lymphocyte cell line (Jurkat). We have now extended this observation to extractsprepared from theHeLacell line which alsodidnot protectdomain II or III but didprotect domain I (datanot shown).

Cellularfactors present inan EBV-negative Burkitt's lym-phoma cell line bind independently to the domains in the EBNA 2-dependent enhancer. To determine whether muta-tions thatsignificantly diminishedenhanceractivityresulted inonlyalocalperturbationoffactor bindingorwhether they disrupted bindingtoother sites within theenhancer, DNase Ifootprintingwascarriedoutwiththe mutantsthattargeted the conserved GTG core sequence (refer to Fig. 1 for structuresofGTG core mutants). Nuclearextractprepared from the DG75 cell line (an EBV-negative Burkitt's

lym-phomacellline)wasutilizedinthe

protection

assay employ-ingthewild-type and mutant EBNA2-dependentenhancers (Fig. 4). In addition, as a control, the

-366/-363

mutant which targeted the

A/T-rich

region between domain I and domainII(andhadlittle effectonenhancer

activity)

wasalso analyzed.

Analysis of the protection pattern exhibited by these

mutants demonstrated that there was a direct correlation between mutations that cause asignificant loss in enhancer activity and loss of binding of a cellular factor(s). The

-366/-363

mutation, which had little effect on EBNA 2-dependent enhancer activity, did not appear to signifi-cantly affect the binding of the cellular factors to the en-hancer, although it slightly altered the DNAse cleavage patternboth in the absenceandpresenceofextract.DNase I footprinting of three GTG core mutants (-375/-372,

-354/-350,

and

-344/-341

mutants), all of which exhibit significantly reduced EBNA2-dependent enhanceractivity, revealed loss of protection in the region of -380/-370,

-362/-345,

and

-343/-326,

respectively (Fig. 4; results summarized inFig. 1). The 3' boundary of domain I,aswell as the 5' boundary of domain II, could not be accurately

determined,

because there are no DNAse I sites within the

A/T-rich region

located between these two domains. Nota-bly, with each of the three GTGcore mutantsthere wasonly loss of binding of the cellular factor(s) to the mutated domain, indicating thatbinding of cellular factorstoeach of these domains is independent of binding to the other do-mains within the enhancer. Thus, the loss of

activity

ob-served with each mutant is due to

specific

loss ofbinding onlyatthat siteintheenhancer,

demonstrating

thatefficient transactivationbyEBNA 2 appears torequire the

binding

of all these cellular factors totheenhancer.

Multipledomains within the enhancer binddistinct cellular factors. To assess the possibility that common or closely relatedtranscription factorsbindtothe

regions

ofhomology within the enhancer, DNase I

footprinting

in

conjunction

with binding competition was carried out employing syn-thetic double-stranded

oligonucleotides

specific

foreach of the domains (Fig. 5; refer to

Fig.

1 for

oligonucleotide

structuresandsummaryof

results).

Addition ofanunlabeled multimerized double-stranded

oligonucleotide

containing

ei-therdomain Iordomain II

effectively

competed

for

binding

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2850 JIN AND SPECK

co =

E c c c

r

E E E

-- -- r- -- D.

+- + + - + DG75 EXTRACT

i i~~

0 0co 0

0=0 = 0=_

-o0 0 'OX -OX

r

(D - + -4 + ++ + + DG75EXTRACT + + +

-330 -340 -350 -360

4:

:.:.

30

0*!w

i.~~~~~z.~

-370 - -

---380

-*a

*as--390 ---e _

d -3W-- .

FIG. 4. DNase I footprinting analysis of wild-type and mutant

EBNA 2-dependent enhancers. The DNA fragments used in the protectionassaycontained theupstreamregion oftheCppromoter

from -415 to -321 bp. Footprinting was performed with crude

nuclear extracts prepared from DG75 (an EBV-negative Burkitt's lymphoma cell line). Distinct domains revealed by loss ofprotection with the specific domain mutantsare bracketed on the right-hand

sideof thegel, and the relative positions of the protected region with

respect totranscription initiation from Cp are indicated. The

mu-tants employed are indicated at the top of the gel by the region mutated.The A/Tdomainmutantis the-366/-363mutantshownin Fig. 3. The domain I, lI, and IIImutantsemployedarethe GTGcore

mutantsshown inFig. 1.w.t.,wildtype. +,withDG75extract; -,

in the absence of DG75extract.

of theDG75cellular factor(s)totherespective domain. This result indicatesthat distinct cellular factors bindtodomains I and II, since these oligonucleotides did not efficiently

compete forbinding tothe other sites.

Whenanoligonucleotide spanning the homology region in domain III (domain III oligo [Fig. 1]) was employed as a

competitor in the DNase I footprinting assay, no loss of protectionwas observed. Carefulexamination of the loss of DNase I protection exhibited by the -344/-341 domain III mutant (Fig. 4), revealed that unlike the domain I and domain IImutations the lossofprotection wasasymmetric

withrespect tothe mutation (results summarized in Fig. 1). The domain III competitor oligonucleotide only contained

sequences extending tothecenterof the loss of protection. Therefore, it may not contain the entire sequence required

forbinding the appropriate cellularfactor(s). Indeed, extend-ing the competitor oligonucleotide to contain the down-stream sequences (domain III' oligo [Fig. 1]) resulted in effective competition for binding to domain III (Fig. 5). In addition, this oligonucleotide also effectively competed for binding to domain II but did not compete for binding to

[image:5.612.353.528.76.350.2]

domain I. This suggests that a common or closely related

FIG. 5. Competition for binding of cellularfactorsto individual

domains within the EBNA 2-dependent enhancer. DG75 nuclear

extractwas preincubated with either 160 or320 ng(left and right lanes, respectively, of each oligonucleotide competition) of the

indicatedoligonucleotides for 10 minat roomtemperature,before

labeled fragments were added and DNase I digestion was

per-formed. Thestructuresof thecompetition oligonucleotidesaregiven

in Materialsand Methods andareshownschematicallyinFig. 1. +,

with DG75extract; -, in the absenceof DG75extract.

factormaybind todomainsIIand III. However, the failure

of either the domain IIoligonucleotide orthe short domain

III oligonucleotide to effectively compete for domain III binding raises the possibility that more than one factor is

involved in binding to domain III and thatbinding of these factors to domain III is cooperative. This model would explain the asymmetric loss of protection exhibited by the -344/-341 mutant and the ability of the longer domain III oligonucleotide (domain III' oligo [Fig. 1]) to compete for binding of factors toboth domains II and III.

DISCUSSION

We and othershavepreviously shown thata184-bp region

(-429to -254bp)upstreamofCp functionsas anenhancer in EBNA 2-positive cell lines, butnotin EBNA 2-negative cell lines(28, 33). Recent studies (5, 29, 32, 37) have shown thatupstreamregions of the LMPpromoterscontain EBNA 2-dependent enhancers. Like the EBNA 2-dependent Cp enhancer, a region upstream of the LMP 1 promoter has

been shown tofunction asan EBNA2-dependent enhancer

when cloned ineither orientationupstreamofaheterologous

promoter. Similarly, Wang et al. (31) recently reported a

186-bp fragment (-275to-89bp) from theupstreamregion ofthe cellularCD23promoterthatexhibits EBNA 2-depen-dent enhancer activity which is largely orientation and distance independent.

domainlII

domain11

-330

-340

-350

domain

domairnIII

-360

domairn 11 a i

-370

-380 *

-390_....

-390 f

^*m ,_ft _. -

-domain

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In this study, we have mapped the minimal EBNA 2-de-pendent enhancer associated with Cp to a 50-bp region (from -380 to -331 bp). However, it is clear from the deletion analysis that surrounding sequences influence enhancer ac-tivity (Fig. 2). Sequences upstream of the minimal active enhancer (-415 to -380 bp) had a positive effect on en-hancer activity, while downstream sequences (-330 to -245 bp) had a negative impact on activity. This indicates the presence of both positive and negative regulatory elements in the region surrounding the minimal EBNA 2-responsive enhancer fragment.

Within the minimal Cp EBNA 2-dependent enhancer we have identified three important cis elements involved in binding cellular factors (domains I to III). These domains contain a region of low-level homology centered around a GTG core sequence. Mutations targeting the GTG core sequences all resulted in significant reduction in enhancer activity, directly implicating the homology regions in en-hancer function. DNase I footprinting analyses revealed that mutations in these domains abrogated binding of cellular factors to these sites. Furthermore, binding of cellular fac-tors to each domain appeared to be independent of binding to the other domains.

Competition analyses employing specific oligonucleotides provided evidence that the factor(s) which binds to domain I is distinct from the factors binding to domains II and III, since an oligonucleotide containing domain I did not com-pete for binding to any of the other domains and, conversely, oligonucleotides containing either domain II or domain III did not compete for binding to domain I. A common factor may be involved in binding to both domainII and domainIII, since an oligonucleotide containing domain III (domain III' oligo [Fig. 1])competed effectively for the factor(s) binding to domain II. However, an oligonucleotide containing do-main II did not effectively compete for binding of the factor(s) bound at domain III. The latter result, in conjunc-tion with the observaconjunc-tion that a mutaconjunc-tion indomainIII inthe region of homology between domainsII and III resulted in an asymmetric loss in protection, supports a modelin which two factors bind cooperatively to domain III. One of these factors binding to domain III is predicted to be identical or closely related to the factor binding to domainII.This model predicts that the common factor, in conjunction with the other domain III-binding factor, exhibits a significantly higher affinity for domain III than domain II.

Previously, extensive analysis of the SV40 enhancer has identified two domains, A and B, which areimportantforfull enhancer activity (10). Mutational analysis of domain B revealed the presence of a repeated sequence motif (GTG TGGAAAG) which is important for enhancer activity (36). Similar motifs have also been found in the enhancers of a variety of other viruses. Comparisonofthesesequences has identified a viral enhancer core consensus sequence

TGTGG(A/T)(A/T)AG.

Domain I within the Cp enhancer matches the core consensus sequence at seven of nine positions and may bind the same or a related factor(s). It should be noted, however, that the polyomavirus enhancer contains several sequence motifs resembling those ofSV40 enhancer, yet thepolyomavirus enhancer doesnot compete efficiently with theSV40 enhancer in cell-free transcription, electrophoretic mobility shift, and DNase I footprinting assays (20, 23).

At the present time, the mechanism by which EBNA 2 functions to activate transcription is not clear. Our present studies implicate at least some of the cellular factors in-volved inbindingto the minimal enhancer. Currently, there

is no evidence that EBNA 2 functions by directly binding DNA. Indeed,ourpreviouslyreported DNase I footprinting data employing extracts prepared from EBV-negative and -positive B-cell lines did not reveal any consistent differ-encesin theobservedprotection pattern when EBNA 2 was present or absent (33).

In the absence of specific binding of EBNA 2 to DNA, there are two distinct mechanisms which may explain how EBNA 2 functions: (i)EBNA 2mayindirectly interactwith the enhancer bybindingtoacellularfactor(s) boundto acis element(s)withintheenhancer;or (ii) EBNA 2 may act at a distance by affecting complexes formed between a cellular factor(s) involved in binding to the enhancer and some cellular control protein(s), thereby activating the transcrip-tion factor. There is evidence for both mechanisms among viraltransactivators. Theherpessimplexvirus VP16 protein targetsviralpromotersbydirectinteraction with the cellular transcription factor Oct-1 (7, 27). Similarly, it has been shown that adenovirus Ela protein stimulates transcription oftheviralE4promoter by interacting with promoter-bound ATF-2 (15). Human T-cell leukemia virus type I Tax and hepatitis Bvirus X protein appear to function viaATFand AP-2, respectively (19, 24). Alternatively, it has

recently

been shown that adenovirus Ela protein transactivates the adenovirus E2 promoter in some cells by

liberating

the cellular transcription factorE2Ffrom a complex

containing

either the retinoblastoma gene product or cyclin A

(2,

17).

Thus, for adenovirus Ela protein both mechanisms appear to be important.

ACKNOWLEDGMENTS

We thankmembers of S.H.S. laboratoryfor critical readingof the manuscript.

This work was supported by grant CA43143 from the National Institutes of Health (to S.H.S.), a Leukemia Society of America Scholar Award (toS.H.S.), and a Postdoctoral Fellowshipfrom the Lady TataMemorial Trust (toX.W.J).

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