Comparing regions of the Epstein-Barr virus ZEBRA protein which function as transcriptional activating sequences in Saccharomyces cerevisiae and in B cells.

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JOlURNAI OFVIROLOGY, Dec. 1993,p. 7472-7481 Vol.67,No. 12 0022-538X/93/1 27472-10$02.00/0

Comparing Regions

of the

Epstein-Barr Virus ZEBRA Protein

Which

Function

as

Transcriptional Activating

Sequences in

Saccharomyces cerevisiae

and in

B

Cells

GEORGE MILLER,'* HOWARD HIMMELFARB,2 LEE HESTON,' JILL COUNTRYMAN,' LYNDLE

GRADOVILLE,'

RAYMOND BAUMANN,' TIANHUAI CHI,3AND MICHAEL

CAREY3

Departments of Pediatrics, Epidemiology andPublic Health, and Molecutlar Biophysics and Biochemistry, Yale UniversitySchoolof Medicine, New Haven, Connecticut06510-8064'; Department of BiochemistryandMoleclular

Biology, HarvardUniversity, Cambridge, Massachlusetts 021152; and Department of Biological Chemistry, Universityof California, LosAngeles, School of Medicine, Los Angeles, California 900243

Received 15 July 1993/Accepted 13 September 1993

The ZEBRA protein activates expression of Epstein-Barr virus early-lytic-cycle genes in human B

lymphocytes. Here it is shown that ZEBRA also behaves as a sequence-specific transcriptional activator in

Saccharomycescerevisiae. Deletional mutagenesis defined three regions of ZEBRA thatparticipateinactivation in S.cerevisiae. Theseregions are designated YI (amino acids [aal 1 to25), YII (aa 51 to 102), and YIII (aa228 to245). Two of the three regions of the native ZEBRA protein act togethertomediateactivation when assayed on ZEBRA binding sites. However, when fused to the DNA bindingdomain of GAL4 andassayed on GAL4

binding sites, regionsYII and YIII were each sufficient to confer activation in S. cerevisiae.Regionsof ZEBRA which affected activation in S. cerevisiae were also required in human B lymphocytes. The amino-terminal region of ZEBRA (aa 1 to 98) was required for activation both in S. cerevisiae and in human B cells; deletion of thecarboxy-terminal 18 aa also significantly reduced activation in both cell types. Thus, the behavior of ZEBRAin human B cells and S. cerevisiae suggests that the protein contains universal activation motifs that interact with conserved components of the transcription machinery. However, certain deletion mutants of

ZEBRAcontaining mutations in the N-terminal region exhibited discordant behaviors in S. cerevisiae and in Bcells. For example, deletion ofZEBRAaa26 to 51 impairedactivation to a great extent in B cells but had little or no effect in S. cerevisiae. Thediscordant mutants may reflectinteractions with a variable domain of a conserved componentorunique interactions with specialized components of the basaltranscriptionapparatus in different cells.

ZEBRA protein initiates a cascade of events leading to

expressionofEpstein-Barr virus(EBV) lytic-cyclegenes(1, 7, 32). ZEBRA activates transcription by binding to ZEBRA responseelements (ZREs)foundin the promotersof itstarget EBVlytic-cyclegenes(5,9,28,47,49). Some ZREs are related

tomammalianAP-1 recognitionsequences; however, ZEBRA

also recognizes sites which are distinct from those bound by AP-1. Like other eukaryotic activators, ZEBRA is a modular

protein (37),andits domainstructuregenerallycorrespondsto

its exon composition. An activating region is present in the

N-terminal portion (approximatelyamino acids[aa] 1 to 100), and the DNArecognitionanddimerization domains are found in the C-terminal portion (aa 167 to 245) (10, 11, 15, 26, 36, 47). ZEBRAstimulates transcription in many different

mam-malian cell types and in vitro in HeLa cell extracts (4, 26). Thus, activation oftranscription by ZEBRA requires neither

B-cell-specific nor EBV-specific factors. These results suggest that ZEBRA interacts with one or more conserved compo-nentsof the mammalian basal transcription apparatus.

Some mammalian cellular and viral transcriptional

activa-tors function in Saccharomyces cerevisiae cells, and certain yeastactivatorsfunction in mammalian cells(10, 12, 23, 24, 25,

31, 33, 34,42, 45,5()). In one approach to demonstrate that a mammalian activating region functions in S. cerevisiae, the

activator,oraportion thereof,is fused to aheterologous DNA

bindingdomain. For example,c-fosfused to the DNA binding

Corresponding

autlhor.

domain present inaa 1 to 87 of the bacterial LexA repressor

protein will stimulatetranscription inS. cerevisiaeofreporter genes bearing nearby LexA-binding sites (25). Similarly, the

activating domain of the mammalian herpes simplex virus protein VP16, when fused to the DNA binding domain con-tained inaa 1 to 147ofthe yeastGAL4protein, will activate transcriptionfrom GAL4 sites in S. cerevisiae (2,6). When the

mammalian activator isitselfa DNAbinding protein, such as

the bovine papillomavirus E2 protein, it can be shown to stimulate transcription in S. cerevisiae from reporter genes which bear E2 cognatebinding sites (24, 34). The activity of the bovine papillomavirus E2proteincan bemeasured in suchan

assaybecause E2 binding sites are not recognized by ayeast factor.

Among the long-range objectives of such experiments in S.

cerevisiaeis todefineconserved cellularproteinswhichinteract with the mammalian activator and to analyze the mode of activation of downstream target genes. Experiments in S. cerevisiae may help to distinguish transcriptional regulatory regions which are directly and solely acted upon by the activator from those thatrequire complexinteractions between groups ofproteins boundto the same promoter. The experi-mentspresentedhere haveexploredthecapacityof ZEBRAto

behave asanactivator in S. cerevisiae.Theseexperimentsshow that ZEBRA is able to activate transcription in S. cerevisiae when bound to promoters by its own DNA binding domain. Portions of ZEBRA also function as activating regions in S. cerevisiae when fused to the DNA binding domain of GAL4. Byuse of deletion mutants, the regionsof ZEBRA necessary

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FUNCTIONS OF ZEBRA REGIONS IN YEAST AND B CELLS 7473

for activation oftranscription in S. cerev'isiae and in human B

lymphocyteswerecompared. One region ofZEBRAwhich is

dispensable for activation of transcription in S. cerevisiae is required in human B cells.

MATERIALS AND METHODS

Yeast strains andmedia. Activationby intact ZEBRA and by mutants with internal, N-terminal, C-terminal, and point mutations thereofwasassayed inyeaststrain W3031A(MATa

cal1-100his3-11,15 leii2-3,112 trpl-l uira3-1 ade2-1) obtained from Rodney Rothstein, Columbia University College of Phy-siciansandSurgeons. Thisstrain is GAL4+; GAL4 is required

to activate the GAL] promoter from which ZEBRA was expressed (22). Activation by chimeric proteins containing the

DNAbinding domain of GAL4 fusedto ZEBRA orportions of ZEBRA was assayed in several yeast strains. Strain

YT6::171 (MATotgal4-542 gal80-538 ura3-52 his3-200 ade2-101 adel lys2-801 trpl-901 arol leui2-3,112 mell Met) bears an

integrated reporter which contains a portion of the GAL] promoter, UASG, fused tolacZ (51). Strain P56::171 is

con-genicto YT6::171 but bears the GALlIP (potentiator)

muta-tion, whichconvertsweakactivators, fusedtotheDNAbinding domain of GAL4, into strong activators (18). Strain JPY9

(MATot ura3-52 his3A200 leu2Al trplA63 lys2Al385gal4A11)

was derived from FY250 provided by Fred Winston,

Depart-ment ofGenetics, Harvard Medical School. Media were

pre-pared asdescribedby Sherman etal. (43).

Reporters. The reporter plasmid pCZA, shown in Fig. LA,

contains a minimal CYC] promoter, including the TATA

element, fused to the ,B-galactosidase gene (28). It also con-tains the selectable gene URA3 and a yeast chromosomal origin ofreplication

(ARS1)

and a centromere (CEN4).

Oli-gomers containing different numbers (1, 3, 5, or 7) of ZIIIB sites wereinsertedinto anXbaI sitein apolylinkerupstreamof the CYCI-lacZ fusion carried on this plasmid (4). The se-quence of the 14-bp oligonucleotide used to make these reporters is 5' GGCAYTGCTAATGT 3'. (The ZIIIB site is underlined.) A reporter which lacks ZEBRA binding sites

contained a 714-bp EcoRI-to-NcoI fragment of EBV DNA

which encompassesthe repeated elementin oriPinserted into the EcoRI site of the polylinker. The reporter 3GAL/CZ

containsthreeoligomerized 17-merGAL4binding sitesinthe pCZA polylinker (6).

Activators. The plasmid vector for the activators was

pBMI25 carrying aLEU2 marker, a gift from James Wang,

Harvard University (Fig. IB). In pBMI25, an 850-bp EcoRI-to-BamnHI fragment contains the galactose-inducible GAL1-GALIO promoter (22). pBM125 contains anARSI origin of

replication and CEN4. The activator pBM125/Z(1-245) [Z(1-245)] contains a BZLF1 cDNA inserted into the unique BamnHIsite ofpBM125. Inthe activatorZ(1-227)+VP16,aa 1

to227of the ZEBRAcDNAhave been fusedin frame atthe

HincIl sitewith aa 411 to 490 of VP16(1,48); this was cloned

as aBamHI fragment intopBM125. Several internal-deletion

mutants of ZEBRA were cloned into pBM125 as BamHI or

BarmHI-to-SalI fragments. These mutants include Z(1-25,

89-245), which contains a deletion of the sequences spanned by

the two HindIll sites in the BZLFI cDNA, and Z(1-25, 141-245), which contains a deletion between the leftmost

HinidIll

siteand theNhel site (1).

Two mutantswith altered amino acids in the basic domain of

exonII,constructedbyoligonucleotide-mediatedmutagenesis,

were obtained from E. Flemington. They are M178-180, in which KRY has beenchangedtoEEL,andM187-189,inwhich

RKC has been altered to EES. These mutants do not bind

DNA in vitro(47).

N-terminal-deletion mutants. A set ofnested

amino-termi-nal-deletion mutants of ZEBRAwas constructed by the

poly-merase chain reaction (PCR). The primers contained, in

5'-to-3' direction, 5' AGATCTGCCGCCGCCATG 3',

fol-lowedby 15nucleotidescomplementarytothe 5' boundaryof the deletion end point. This primer contains a BglII site, followed by a consensus translation enhancer sequence,

fol-lowedbyatranslation initiator codon.Themutantsbeganat aa 2, 25, 52, 77,99, and 131 of ZEBRA.The primerflankingthe 3'end of theZEBRAgenecontainedaBainHlsiteGGATCC preceded by 15 nucleotides complementary to the DNA en-coding the carboxy terminus ofZEBRA. The PCR products were cleaved withBglII and BamnHI and insertedintopBXG1 cutwithBglII and Barn HI.Theyweresubclonedinto theyeast vector pBMI25, whichwas cutwith BaniHl and Sall. The 3' end of the ZEBRA gene was a BglII-PstI fragment of the BZLF1 cDNAcloned intopBluescript.This strategywasused

in orderto preservethe stopcodon atthe end of the BZLF1

open reading frame. The nucleotide sequence of the

N-terminal-deletion mutants was determined in the yeast

plas-mids by the Sanger method.

GAL4 fusions. PCR primers for GAL4-ZEBRA fusion

proteins were either the same as those used in the ZEBRA

amino-terminal deletionsor new onesconstructedby placinga

BanHI site 5'andeitheraBanlHI siteor aBglll site 3' tothe

codingsequenceof interest.These PCRproductswerecleaved with Bglll and BainHI and inserted into the BamtiHI site of pBXGl, which contains the simian virus 40 enhancerdriving

expression of DNAencodingaa I to 147ofGAL4

[GAL4(1-147)] (41). This created an in-frame fusion with the DNA

binding domain of GAL4. The GAL4-ZEBRA fusions were then subclonedinto twoyeast expression vectors,pMA424and

pGG29. pMA424 is a

2[Lm

plasmid in which GAL4(1-147) is

drivenbytheADHI promoter(30). pGG29 isanARSJ CEN3

plasmid which contains GAL4(1-147) driven by the GAL4

promoter,agiftfromGraceGill,DepartmentofBiochemistry, University of California at Berkeley. The junction between

GAL4(1-147) and ZEBRAwassequenced.

,-Galactosidase assays. S. cerevisiac wastransformed by a lithiumacetatemethodwith5

[LI

ofEscherichlia coliminilysate plasmid DNA plus 50

pLg

ofcalfthymus DNA as the carrier

(21).Yeastcellswereplated onto anitrocellulose filterplaced

on the appropriate synthetic complete (SC) yeast medium

drop-out platescontaining2%glucose.Afterthecolonieswere visible(afterabout 48 h), thefiltersweretransferred toX-Gal

(5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside)

indicator

medium drop-out plates containing 2% galactose (43). The

extentofactivation ofLacZwasscoredqualitatively by inspec-tion of the X-Gal plates. Individual representative colonies

weresubculturedfrom theX-Galplatcto aliquidSCdrop-out

culturecontaining2Cc glucose.Aftcrovernight incubation,the cultures were washed once with SC and rcsuspcnded in

drop-out medium containing 2Cc galactosc. Thcsc cultures

were further incubated overnight and were harvested for

0-galactosidase

assay. The cells were fractured

by

vortexing

with glass beads. The lcvel of

P-galactosidase

activity was

measuredasdescribed elsewhere(18),and theproteincontent wasdeterminedbyusingaBio-Radproteindetermination kit.

1-Galactosidase activitywascalculated as 1,000 x the optical

densityat420nm/time (minutes) x theoptical densityat595

nm x volumc (milliliters).

Human B-cell transfections. Transcriptional activation

by

ZEBRA and ZEBRA deletion mutants was

assayed

in the

EBV-negative human B-cell line BJAB. Theinternal-deletion V()L. 67, 1993

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7474 MILLER ET AL.

(ZIIIB)n

VI

,

I

XbaI 43bp 15bp 129bp

MCS CYC 1

Activator (pBM125)

(BZLFI)

850bp

BamHI

Sail

pBR LEU2

C.

ZEBRA

Clone

Z(1-245)

Z(1-227)+VP16

exonI

_ _

I IC

exonI exonm

I I ,

cw I

0

I II .c

I I

167aa a 43aa|

activation DNA dimerizaton

recogrution

0 40 80 120 160 200

Amino Acids

ZEBRA VP16

240 280 320

D.

0

0 (c c

D

CD 0

Co

cc% 0 cscc$

m

Reporter CZA Zl/CZ Z3/CZ Z5/CZ Z7/CZ

Stimulation 1.0 0.4 0.5 1.0 4.3 19.4 1.0 32.4168.4 1.0 11.7 89.2 1.0 3.9 19.5

Reporter (CZA)

TATA

I_

A.

LAC Z

B.

GALl

/10

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FIG. 1. Activation of transcription by ZEBRA in S. cerevisiae. (A) Reporter plasmids. Different numbers of oligomerized ZIIIB sites [(ZIIIB)n],towhich ZEBRAbinds,werecloned intoanXbaIsite in amultiple-cloningsite(MCS)in the vector pCZA (29). As a control, a 700-bp fragmentcontaining oriP, to which ZEBRA does not bind, was placed in the MCS inpCZA.The reportercontains a 144-bp minimal promoter from the yeast CYCI gene upstream oflacZ. (B) Activator plasmids. Derivatives of a cDNA of the BZLF1 gene encoding ZEBRA were cloned asBamHIorBamHI-SalI fragments downstream of the 850-bpGALJ-GALIO(GAL1/10)promoter intheplasmid pBM125. (C) Two activator plasmids which contain ZEBRA. The top line shows the approximate location of the activating, DNA recognition, and dimerization regions of ZEBRA. Restriction endonucleaserecognition sites used for preparation of internal-deletion mutants (see Fig. 2B) and ZEBRA-VP 16 fusions are shown. Z(1-245) is an intactBZLF1cDNA.Z(1-227)+VP16 has the carboxy-terminal 18 aa ofZEBRAremoved andreplaced with aa 411 to 490 of VP16(1). (D)Datafromarepresentative experiment. Yeast strainW3031A was cotransfected with one activatorplasmidand one reporter

plasmid. TheactivatorwasZ(1-245)orZ(1-227)+VP16 or, as anegative control, thepBM125 vector.The reporters were derivatives ofpCZA containingno ZIIIBelementsorone,three,five,or seven ZIIIBelements upstream of the minimalCYC]promoterfused to lacZ. Stimulation is calculated as the ratio of,-galactosidase unitsexpressedfromareporter after exposure to an activatortothe number of 3-galactosidase units expressedfrom the same reporter in the presence of thevectorpBM125.

and C-terminal-deletion mutant activators were cloned in pHD1O13, which contains the cytomegalovirus promoter-en-hancer (8). The N-terminal-deletion mutant activators were

cloned downstream of the simian virus 40promoter-enhancer

in pBXG1, a gift from P. Broad (41). The reporter was Z3E4CAT, in which three oligomerized ZIIIB sites were inserted upstream of the minimal adenovirus E4 promoter, fused to chloramphenicol acetyltransferase (CAT) (4, 16). Cellswere transfected by electroporation with 10 ,ug of acti-vator and 10 ,ugof reporter. CAT assays were performed with

30 ,ul of cellextractprepared48hafter transfection. Proteins were detectedby immunoblotting with polyvalent rabbit

anti-sera to ZEBRA.

ones,nolonger activatedtranscription from reporters contain-ingoligomerized ZREs (Fig. 2B and data not shown).

Fig. ID shows that ZEBRA was a weak activator in S.

cerevisiae.Addition of the VP16 activation domainto ZEBRA in the mutant Z(1-227)+VP16 created an activator fivefold more potent than wild-type ZEBRA. As is characteristic of many weakeukaryotic activators,ZEBRAactivated

transcrip-tion inasynergistic fashion (4).In S. cerevisiae, ZEBRAonly weakly activated (4-fold) transcription from a reporter with

one ZIIIB site and maximally stimulated (32-fold in the

experimentillustrated) transcription from reporters containing three tandemZIIIBsites. The stimulation oftranscription on

RESULTS

Experimental design. The capacity ofZEBRA to activate transcription in S. cerevisiae was examined in two types of experiments. In one type, activators were expressed from plasmid constructs encoding the native DNA binding and

dimerization domains of ZEBRA itself (aa 167 to 227)

to-getherwith variousportions of the remainder ofthe ZEBRA

protein (Fig. 1C). In these experiments, transcription was measured from reporter genes containing one, three, five, or

seven oligomerized ZIIIB ZREs upstream of a CYCI-lacZ fusion.Inthe othersetofexperiments, the activators consisted of ZEBRA, or portions thereof, fused to the DNA binding

domain (aa 1 to 147) of the yeast activator, GAL4. Reporter genes contained GAL4 binding sites upstream of a minimal promoter (CYC1 or GALI) fused to lacZ. In both types of experiments,

3-galactosidase

activity served as a measure of transcription. The ZEBRAdeletion mutants were also exam-ined for their capacity to activate transcription in an

EBV-negativehumanB-lymphocyte cell line. The reporter used in B cells contained three oligomerized ZIIIB sites upstream of a minimaladenovirusE4gene promoter fusedto theCAT gene (4, 16). CAT activity servedas a measureoftranscription.

ZEBRAis a sequence-specific activator inS. cerevisiae. As

shown inFig. 1D,ZEBRAactivated transcription in S.

cerevi-siaefrom reporter genesbearingone ormoreZREs.ZEBRA failed to activate transcription from a reporter gene lacking

ZEBRA binding sites (pCZA) or from a reporter gene

con-taining20copies ofa30-bp repeat from theEBVlatentorigin ofreplication (oriP)towhich theEBVprotein EBNA-1binds

but to which ZEBRA does not bind (data not shown). The

capacity of ZEBRA to activate transcription from reporters bearing single or multiple ZIIIB ZREs was eliminated by mutations which destroyed the ability of ZEBRA to bind to DNA. MutantsZ(M178-180) andZ(M187-189),in which basic

amino acids required forDNAbindingarechanged toacidic

A

Z(2-245)

Z(25-245)

Z(51 -245)

Z(77-245)

Z(88-245) Z(99-245) Z(131-245)

B

Z(1-245)

Z(1-25,89-245)

Z(1-25,141-245)

Z(1-25,141-227) Z(1-227)

Z(M178-180)

Z(M187-189)

ZEBRA Amino Acids

25 50 75100125 150 175 200

P-GalactosidaseUnits

0137 500 10oo 1500

Stimulation Index

2000

11.6

5.4

5.6

2.8

2.5

0.9

1.3

Reporter:Z3/CZ

ZEBRA AminoAcids 5-GatactosidaseUnits Stimulation

o 25 5075100125150175200225250 0137 500 1000 1500 2000 Index

13.7

11.7 10.7

0.9

2.3

Reporter:__________________Z3C0.3

____________________________ ~~~~~~~0.2

Reponter:Z3/CZ

FIG. 2. Activation oftranscriptionbyZEBRAN-terminal-deletion mutants(A)and internal-deletion andpointmutants(B) in S. cerevi-siae.Thedataareaverages forduplicateexperimentswith eachsetof mutants. The experiments were conducted in yeast strain W3031A. Thevertical lines represent the level of,B-galactosidase activity (137 units) as a measure of background transcription from the Z3/CZ

reporter (aderivative ofpCZA containing threeoligomerizedZIIIB sites) cotransfected with thepBM125vector.Thestimulation index is the ratio of,B-galactosidase units measured in yeast cellscarryingan

activatortothose measured in yeast cellscarryingthevectorpBM125.

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7476 MILLER ET AL.

three adjacentsiteswas8-fold thaton one site;this is 2.5-fold synergy, comparing three sites withone site.

The background level of transcription measured in the

absence of an activator increased when reporters contained five or seven oligomerized ZIIIB sites (Fig. ID). This high

background level of transcription, which was not observed from the reporter bearing 20 copies of the 30-bp repeat elements fromoriP(datanotshown),ispresumablyduetothe action of a yeast factor which recognizes the oligomerized

ZIIIB sites and whose activity required a high degree of synergy.

An N-terminal and a C-terminal region of ZEBRA are

required for activation oftranscription in S. cerevisiae. The deletion mutants illustrated in Fig. 2 defined two separate

regionsofZEBRAproteinwhichwererequiredforactivation of transcription in S. cerevisiae. One region was the amino-terminal 98 aa, and the otherwasthe carboxy-terminal 18 aa. A nested set of deletion mutantsof increasing size from the amino terminus showedaprogressivedecrease inactivity (Fig. 2A). Therewasgreater-than-50% reduction ofactivity

follow-ing removal of the N-terminal 24 aa. Removal of the

N-terminal76aacauseda76% reduction ofactivity,andremoval of the N-terminal 98or 130aa eliminated thecapacityofthe

protein to activate transcription. Deletion of the second

re-quired

region,locatedinthecarboxy-terminal 18aminoacids,

in themutantZ(1-227) reduced thecapacityoftheproteinto

activateto less than20%of thewild-type activity (Fig.2B and data not shown). A reduction of activity comparable to that

measured onreporters withthreeoligomerizedZIIIB siteswas

observed on reporters containing five or seven oligomerized

ZIIIB sites (data not shown). Immunoprecipitation

experi-ments showed that Z(1-227)wassynthesizedatthe samelevel as Z(1-245) (datanot shown).

We observed a modest decrease in activation potency in internal-deletion mutants from which aa 26 to 140 were

removed. Thus the mutantZ(1-25, 89-245)was 85% as active asthe wild type, and the mutantZ(1-25, 141-245) was78%as active as the wild type (Fig. 2B). If, however, these internal deletionswerecombined with deletion of the C-terminal18aa, as in the mutant Z(1-26, 141-227), the protein was inactive. These data suggested that the N-terminal 25 aa and the C-terminal 18 aa of ZEBRAwereboth

required

to create a functional activator in S. cerevisiae. The

required

C-terminal

activating

regionofZEBRA could bereplaced by VP16,asin

the mutant

Z(1-227)+VP16

(Fig.

ID).

Intact ZEBRA andportionsof the ZEBRAproteinfused to

the DNAbindingdomain of GAL4 behave asactivators in S. cerevisiae. In preliminary experiments, the entire BZLF1

cDNA encoding ZEBRA was fused to GAL4(1-147) and

expressedfrom the strong constitutiveADHIpromoteronthe

plasmid

pMA424, which contains a high-copy-number yeast

2pLm-plasmid

origin of replication. High-level expression of

ZEBRAunder theseconditions wastoxic toseveralstrainsof S. cerevisiae,asevident bythe appearance ofsmallcolonieson

SC

glucose

plates.These smallcoloniesdevelopedadarkblue

colorwhen transferredtoX-Galplates,indicative ofhigh-level transcriptionalactivation. Strain JPY9wasleast affected among several strains tested. In this strain, GAL4(1-147)-Z(1-245)

activatedtranscription about 14-fold from GAL4binding sites and about 24-fold from ZEBRAbindingsites (Fig. 3A). Thus

theintactZEBRA

protein

fusedtotheDNAbindingdomainof

GALA

behavedina mannerconsistent withtheprotein

contain-ing

two different DNA binding domains. Two constructs

con-taining

the N-terminal activating region of ZEBRA, namely

GAL4(1-147)-Z(1-198)

and

GAL4(1-147)-Z(1-93),

both

acti-vatedtranscriptioninthisassay.Since these twoconstructslack

A

Zt

0

en

Im C)O

00

2000r

1500k

1000k

I

.1.M

3Z3/CZ _ 3GAUCZ

GAL4(1-147) GAL4(1-147) GAL4(1-147) GAL4(1-147) GAL4(1-147)GAL4(1-147) GAL4(1-147) GAL4(1-147)

Activator + + + + + + +

AH Z(1-245) Z(1-198) Z(1-93) Z(228-245) Z(51-103) Z(1-227)

Stimulation 1.0 1.0 65.2 4.3 13.723.8 128 2.0 47.53.7 5.0 2.6 1.8 1.7 1.00.6

B

Z(l98)-1

Z(l-93)

Z(2-51)

ZEBRAAmino Acids FusedtoGal4(1-147)

25 50 75 100 125 150 175 200 225250

Z(2-251

Z(25-51Z Z(51-103) Z(51-77) Z(77-103) Z(99-135) Z(198-245)

Z(228-245)

Galactosidase Units(log10) Stimulation 1.0 2.0 3.0 4.0 Index

4290

= ~~~~~~~~~~~~~~~~~~~~1 972

328

: ~~~~~~~~~~~~~~~~~~~~~~1

~~~~~~~~~~~~~~~~~~~~1

E

~~~~~~~~~~~~~~~~~~~~1

w ~~~~~~~~~110

2 7 16 62

Reporter: 3GAUCZ

FIG. 3. Activation of transcription by ZEBRA or portions of ZEBRAfusedtotheDNAbindingdomainof GAL4.(A) Comparison

ofactivationonGAL4(shaded bars)and ZEBRA(openbars)binding sites. The dataarepooledfromfiveexperiments.JPY9(gal4AJJ)yeast cellswerecotransfected withaGAL4-derived activator andareporter. The two reporters were derivatives of pCZA containing three

oli-gomerized GAL4 binding sites (3GAL/CZ) or three oligomerized ZIIIBsites(Z3/CZ). The activatorsconsistedof ZEBRAorportions

of ZEBRA thatwerefusedtothe DNAbindingdomain of GAL4(aa

1 to147)in theplasmidpMA424(30).AH(amphipathichelix)isan artificial activatorwe used tocompare the relative potenciesof the ZEBRA-derivedactivators(14).Stimulation is the ratio of

3-galacto-sidase units measured in yeast cells carrying an activator to the

background level on 3GAL/CZ orZ3/CZ induced by GAL4(1-147)

alone.Thisbackgroundlevelwas24unitson 3GAL/CZand 10 units

on Z3/CZ. (B) Activation by GAL4(1-147)-ZEBRA fusions in the GALllP strain. Eachplasmid containinga fusion between

GAL4(1-147)andZEBRA,orportionsofZEBRA,wastransformedinto strain

P56::171, which carries an integrated reporter consisting of UASG upstreamof lacZ(18,51).Thestimulation indexrepresentsthe ratio of

13-galactosidase activitymeasured with the GAL4-ZEBRA fusion to the background level of,B-galactosidase activity measured following

transfection of a plasmid containing GAL4(1-147) with no added

activating domain.Thisvalue,4units, is indicatedby avertical line. The dataarefrom a representative experiment.Similar resultswere

obtained in sixreplicateexperiments.

the ZEBRA DNAbinding domain, theywereonly active when measured on GAL4 binding sites. Weak activation, about

fivefold above background level, was also observed when the C-terminal 18aawerefusedtoGAL4. Noactivationwasseen

whenZ(51-103) orZ(1-227)was fusedtoGAL4. None ofthe

constructs containing subregions of ZEBRA were toxic in S.

cerevisiae.

Theresultswiththe GAL4-ZEBRAfusionswereconsistent withthe analysis with deletionmutants. The N-terminal93aa constitutedamoderatelystrongautonomousactivating region J VlIROL.

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when fused to a heterologous DNA binding domain and assayed in S. cerevisiae, whereas the C-terminal 18 aa consti-tuted aweak activating region onits own.

Activation byGAL4-ZEBRA fusions in a potentiatoryeast strain. Because ZEBRAisaweak activator in S. cerevisiae, the intact protein and subdomains ofZEBRA fusedtotheDNA

binding domain of GAL4 were tested for their capacity to

activate transcription in a yeast strain containing a mutant

GAL 1I allele thatconverts weak activating regions fused to

GAL4(1-147) intostrongactivating regions (18).This mutant GALl1 allele is called GALlIP (potentiator).Thepurposeof theseexperiments was todefinesubregionsofthe N-terminal domain ofZEBRA which might contribute to the activating

response. None of these subregions was an activator when fused toGAL4 and expressed in wild-type yeaststrains orin yeast strains in whichGALl1 was deleted (Fig. 3A and data not shown). To alleviate toxicity due to overexpression, and

thereby to increase the sensitivity of the assay for activation domains of ZEBRA, the level ofexpression of the chimeric

proteins was decreased in the GALlIP strain. Thus the

GAL4-ZEBRA fusionswereplaceddownstream of the GAL4

promoter,which is lesspowerfulthan theADH1 promoter, in

plasmids with low-copy-numberARS-CENorigins of

replica-tion. The reporter, containing a UASG bearing four GAL4

bindingsites fused tolacZ,wasinserted into the chromosome of the GALllP yeast strain (18). The potency of GAL4(1-147)-Z(1-245) increased dramatically in the GALliP yeast

strain. When isogenic yeast strains with and without the

potentiatormutationwere compared,the intactZEBRA

pro-tein fused to GAL4 was about 100-fold more active in the

potentiatorstrain (datanotshown). Figure3Bshows that two constructs which contained the entire N-terminal activating

domain, namely, GAL4(1-147)-Z(1-198) and

GAL4(1-147)-Z(1-93), both behaved as potent activators in the GALl1P

strain. Of various subfragments of the N-terminal region, Z(51-103)wasthemostactive; however,itwas not asactiveas the intactprotein orfusions containingthe entire N-terminal

region.Activationbythe carboxyterminus of ZEBRA,aa228

to245, increased about 10-fold when assessed in the

potenti-atorstraincomparedwithwild-typeS. cerevisiae.Although the N-terminal 25 aa were shown to be important in progressive amino-terminal deletionanalysis (Fig. 2A),they did not

con-stitute an autonomous activation domain when fused to

GAL4(1-147) and testedin the GALllPstrain(Fig. 3B).

ActivationbyZEBRA andits deletion mutants in human B cells. In human B cells, a major activation domain was contained in the amino-terminal 76 aa (Fig. 4). There was

progressive loss ofactivityas this regionwas truncated: dele-tion of the N-terminal 25aareducedactivity by 40%; deletion of the N-terminal 50 aa reduced activity by more than 90%.

Z(77-245), Z(99-245),andZ(131-245)wereinactive inBcells.

All of the N-terminal-deletion mutants were expressed in

mammalian cells to similar extents (data not shown). The

progressivedecrease inactivityassociated with the N-terminal deletionssuggestedthataa26to50werecritical for activation inBcells. Theirimportancewasalsoevident from the behavior of internal-deletion mutants. The removal of these amino

acids,asin the mutantsZ(1-25, 89-245) andZ(1-25, 141-245),

markedly reduced activity; the larger internal deletion caused

agreaterloss ofactivitythan the smallerone. Deletion of the

C-terminal 18 aa reduced activity in B cells by 60to 80% in

various

experiments.

Inrelatedexperiments, GAL4(1-147)-Z(1-93)and

GAL4(1-147)-Z(228-245)wereeach foundtofunctionas anactivatorin

human B-cell lines when tested on reporters containing five

oligomerizedGAL4bindingsites upstreamof CAT(data not

Z(2-245) Z(25-245) Z(51-245) Z(77-245) Z(99-245) Z(131-245) Z(1-245) Z(l1-25,89-245) Z(l1-25,141-245)

ZEBRA Amino Acids

25 50 75 100125 150 175 2002252500

Stimulation Index

50 100 150

100

61

100 13

20 Reporter: Z3 E4CAT

FIG. 4. Activation of transcription by ZEBRA and ZEBRA dele-tion mutants in human Bcells. BJAB cells wereelectroporated with 10 ,ug of an activator plasmid and 10 ,ug of a reporter plasmid. The activators consisted of a nested set of N-terminal-deletion mutantsof

ZEBRA cloned behind the simian virus 40 promoter in the vector

pBXG1 or of wild-type ZEBRA and ZEBRA internal-deletion mu-tantsclonedbehind the cytomegalovirus immediate-early promoter in theplasmidpHD1O13.The double horizontal line indicates that these mutants were in different vectors and contained different translation initiation sequences. The reporter, Z3E4CAT, contains three

oli-gomerizedZIIIBsites upstream ofaminimal adenovirusE4promoter fused toCAT (4). Cell extracts prepared 48 h afterelectroporation

wereassayed for CAT activity. Shown are pooled data from duplicate

experiments. The stimulation of transcription by pBXGI/Z(2-245),

190-fold, is considered 100% in the N-terminal-deletion series. The stimulation oftranscription bypHD1O13/Z(1-245),70-fold, is consid-ered 100% for the internal-deletion series.

shown). Thus, the two regions ofZEBRA shown to contain important activatingsequencesby deletion analysiswere also

autonomous activating sequences when fused to a

heterolo-gous DNAbinding domain.

Comparingbehaviors of deletion mutants in B cells and in S. cerevisiae. Figure 5 compares activities of the mutants on reporterswiththree oligomerizedZIIIB sites. InbothB cells

and S. cerevisiae, the extreme N terminuswas important for activity; deletion ofaa I to 25 had similar effects in the two typesofcells, abouta40- to50% loss of activity. InFig.5,this region is designated YI and BI for yeast cells and B cells, respectively. Moreover, the remaining N-terminal98 aa were

required for activity in bothyeastand Bcells,since themutant

Z(99-245) was inactive in both types of cells. Progressive

N-terminal and internal deletions implicated aa 26 to 50, a

region designated BIT,asbeing crucial in Bcells. This region could be removed without altering activity in S. cerevisiae.

Progressive N-terminal deletions implicated aa 50 to 98, a

regioncalledYII,asimportantin S. cerevisiae.Furthermore,as

shown in Fig. 3B, YII could function as an autonomous

activating region in S. cerevisiae when fused to GAL4. This region also served a function in B cells, since the mutant

Z(51-245)wasstill active while Z(77-245)was inactive. Dele-tionof thecarboxy-terminal 18 aaof ZEBRA,aregion called

BIV andYIII in B cells andS. cerevisiae, respectively, led to marked reduction in activityin both systems.

DISCUSSION

Regions of ZEBRA that participate in activation in S. cerevisiae.Threeregions ofZEBRAproteinaffect thecapacity

of theproteintoactivatetranscriptionin S. cerevisiae. None of the regions is required for DNA binding in vitro (36, 47).

Deletion of region YI, containing the N-terminal 25 aa,

reduced the capacity ofZEBRA to activate transcription by

-- -- - --- ---I -- .. - --- --- ----

.--Ma..M..

W///IIIIIIIIIIIIIA

I

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7478 MILLER ET AL.

A

Z(2-245)

Z(25-245) Z(25-245)

Z(77-245)

Z(99-245) Z(l31-245) Z(l1-245) Z(1-25,89-245) Z(1-25,141-245) Z(1-227)

ZEBRA AminoAcids

0 25 50 75 100 125 150 175200 225250

Reporter:

RelativeStimulation BCells

100

61

100 13 3 20 Z3E4CAT

Yeast

100

47

48

24

8

11

100

85

78

17

Z3/CZ

yeast YIII

YI YII

-Emz= B-cells

BIV

BI BII Bll

1 50 100 1 50 200 245 amino acids

Functions in

Z Requiredin Yeast with

a.a. Domain Yeast Bcells GAL4(1-147) Charge P

1-25 YI/BI +

26-50 BII

-51-102 YII/BIII +

228-245 YIII/BIV +

Q

-(3) 4/25 2/25

- -(1) 4/25 3/25

+ + -(3) 9/52 6/52

[image:7.612.62.303.85.385.2]

+ + -(2) 2/18 0/18

FIG. 5. Comparison of ZEBRAactivating regionswhich function in S. cerevisiac and in B cells.(A) Data for deletionmutants.Aseries ofdeletionmutantsweretested foractivityinS.cerevisiae(Fig. 2)and

in human B cells(Fig. 4).The relative stimulation is derived from the data shown in the above-mentioned two figures. For the N-terminal-deletion series,theactivityofconstructZ(2-245)is considered 100%.

For the internal and carboxy-terminal deletions, the activity of an

intact BZLF1 cDNA is considered 100%. (B) ZEBRA activating

regionsinS. cerevisiae and B cells. In the column headed"Required," + signifies >40% reduction upon deletion. Region YII is required only ifregion YI or YIII isnot present. Under the column headed

"Functions," + signifiesthat transcription is stimulatedinthe poten-tiatoryeaststrain (Fig. 3B).Under thecolumn headed"Charge,"the

number is thenetchargeof theregion.Undercolumns P andQarethe

number of prolines and glutamines, respectively, divided by the

number of residues in theregion.

more than 40%; however, this region did not serve as an autonomous activating domain when fused to GAL4(1-147).

Region YI mayplayanaccessoryfunction in activation,either

through interaction with otheractivation domains of ZEBRA or by contacting cellular proteins. It may also affect protein folding. Region YII, aa 51 to 102, was also shown to be

important in S. cerevisiae through analysis of progressive

N-terminal deletions (Fig. 2A). Furthermore, YII functioned

as anautonomous activation domain when fusedto

GAL4(1-147) and assayed inthe GALllPstrain (Fig. 3B).Deletion of

region YIII, residues 228to245,caused the greatest reduction inactivation (Fig. 2B). Substitution of regionYIII withVP16, asin themutantZ(1-227)-VP16,createdanactivator thatwas manytimes morepowerful than wild-type ZEBRA. Since the

activation domain of VP16 itself does not bind DNA, this

finding suggests that removal of the C-terminal 18 aa from

ZEBRA did notsignificantly affect DNAbinding in vivo. The

mutant

Z(1-227)

bindsDNA in vitroto anextent

comparable

to DNA

binding by

wild-type

ZEBRA

(36,

47).

However,

certain ZEBRA mutantswith deletions in the

carboxy-termi-nal 18 aa are deficient in

binding

DNA invitro under condi-tions of

high

salt concentration or low temperature

(36,

47).

Therefore we cannot exclude a contribution of this

region

to

DNA

binding

invivo.

Nonetheless,

region

YIIIwas an

auton-omous activator when fused to

GAL4(1-147)

and

assayed

in the GALlIP strain

(Fig. 3B).

Onemodelto accountfortheresults of deletional mutagen-esis istoconsider these three

regions

ofZEBRAas

dependent

activating

sequences, each of which

requires

the presence of

another

activating region.

When

only

oneof the three

regions

is

retained,

the mutant is inactive.

Thus,

mutant

Z(99-245),

which has

regions

YI and YII

deleted,

and mutant

Z(1-25,

141-227),

which has

regions

YII and YIII

deleted,

are both

unabletoactivate

transcription

(Fig.

2).

Whentwoof the three

regions

are present, the mutant exhibits a reduced level of

activity. Thus,

the mutantwith

only

region

YI

deleted,

Z(26-245),

would be consideredtoactivateas aresult of

regions

YII

and YIII.Themutantwith

region

YIIdeleted,

Z(1-25,

89-245),

activates as the result of YI and

YIII,

and the mutant with

region

YIII

deleted,

Z(1-227),

activates

through

the

activity

of

YI and YII. The residual

activity

of each functional deletion mutant can be accounted for

by

a

hierarchy

of

importance

of

each set of

activating

sequences: YIII > YI > YII. The least

active deletion mutant contains YI and YII. The most active deletion mutantcontains YIII and YI

(Fig.

2B and

5B).

The GAL4-ZEBRA fusions were consistent with the idea that two groups of

activating

sequences were

required

for

ZEBRA to activate

transcription

in S. cerevisiae. A chimeric mutant

containing

regions

YIand

YII,

GAL4(1-147)-Z(1-93),

functionedas an activator in

wild-type

yeast strains.

However,

neither

region

YI nor YII alone functioned as an

activating

sequence when fused to GAL4 and

assayed

in

wild-type

S.

cerevisiae.

Nonetheless,

region

YIIwas functional when fused

to

GAL4(1-147)

and

expressed

in GAL11P yeast cells

(Fig.

3B).

In this yeast

strain,

GAL4(1-147) by

itselfbehaves as a

weak activator

(18).

It is

possible

that the

activity

ofZEBRA YII was revealed in the GALlIP strain

through

synergywith

the

cryptic

activating

sequencesin

GAL4(1-147).

Whatever the

mechanism of action of the GALl1 P

mutation,

it serves as a

useful tool for the detection of weak activation domains

by

increasing

their

capacity

to activate

transcription.

A

paradoxical

resultwasthat thechimeric

protein

GAL4(1-147)-Z(1-227),

which contains ZEBRA

regions

YI and

YlI,

didnotactivateeither in

wild-type

or

potentiator

yeaststrains.

While this result may be

explained by

a

problem

in

protein

folding,

another

interpretation

is thataninternal

portion

of the

ZEBRA

protein

may exert an

inhibitory

effect on

transcrip-tional activation.

Inhibitory

sequences inthe central

region

of the GAL4

protein

have

recently

been characterized

(44,

46).

GAL4,

like

ZEBRA,

hasactivationdomainsatthe amino and

carboxy

termini, separated

by

a

large

internal domain which

contains the

inhibitory

region.

Whatisthe natureofthese

dependent

activationdomains?

They

are all

weakly acidic; perhaps

the accumulation ofnet

negative charge

turnsthe whole

protein

intoan activator

(12,

37).

However,the

hypothesis

that

negative charge

by

itselfisa

major

determinantofactivation potency seems

unlikely,

since

deletion of

YIII,

with a

charge

of

-2,

has a

major

effect on

activation potency but deletion of

YIl,

with a

charge

of

-3,

haslittle or noeffect

(Fig.

5B). Regions

YI and YIIare both

relatively

proline

rich

(16%).

YIIis

moderately

glutamine

rich

(11%) (Fig.

5B). Region

YIII contains a consensus site for

cdc2

kinase,

suggesting, perhaps,

that

phosphorylation,

and the

B

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resulting increase in net negative charge, may contribute to

ZEBRA'scapacityto behaveas an activator. Point

mutagen-esis is needed to define the requirements for these different motifsin transcriptional activation (20).

Parallels in activating behavior of ZEBRA in S. cerevisiae and in human Bcells. In S. cerevisiae and in human B cells,

ZEBRAbehavesas asequence-specific activator of transcrip-tion; it activates transcription ofreporter genesbearing ZREs,

and it does not activate reporter genes lacking such elements

orthose containing binding sites for anotherEBV regulatory

protein, such as EBNA-1. The genesresponsive toZEBRA's action in S. cerevisiae may bear synthetic promoters with oligomerized ZREs,ortheymay contain natural promotersof viralgenes,suchasBMRF-1 and BZLF1,whichare activated by ZEBRA as it switches latent EBV to lytic replication in humanBcells (6a). ZEBRA's action isthrough directcontact

withDNA,both in S. cerevisiaeand in humanBcells.ZEBRA mutants, suchasZ(M178-180), which donotbindDNAfailto

activate synthetic promoters (Fig. 2B) and certain natural

promoters, such as BMRF-1, in S. cerevisiae and in B cells (data not shown). In fact, transcriptional activation of oli-gomerized ZIIIB sites in S. cerevisiae in the presence of the

DNA binding mutants is lower than background level (Fig. 2B). These mutants might leadto less-than-background-level activationthrough formation of heterodimers with endogenous

yeastproteins which bind ZIIIB sites. This mightprevent the

yeast proteins from bindingDNA oractivating transcription,

orboth.

ZEBRAisaweak activatorin S.cerevisiae,asit is in human

B cells. In S. cerevisiae, the activation potencyof ZEBRA is

about one-fifth that of GAL4 region II, the strong carboxy-terminalactivating domain ofGAL4(datanotshown). Fusion of the powerful activation domain of herpes simplex virus

VP16 to ZEBRAincreases its activationpotency6- to20-fold in human B cells and about 5-fold in S. cerevisiae (1). As is particularly characteristic of weak activators, transcriptional activity by ZEBRA is synergistic. Model promoters with a

singlebinding siteareactivatedpoorly in humanBcellsandin

S. cerevisiae. Promoters with two or more binding sites are

activated strongly in both systems. The same magnitude of

synergyis observedin vivo in human Bcells,invitroin a HeLa cell extract, and in S. cerevisiae (4).

Conservation of ZEBRA'sactivating regions inS. cerevisiae and in human B cells. The overall functional architecture of

ZEBRA protein is preserved in S. cerevisiaeand in humanB

cells. Acomplex activation domain consisting of several sub-regions is contained in the N-terminal 98 aa; deletion of this entire region severely impairs activation both in S. cerevisiae and in mammalian cells (Fig. 2A and 4) (11, 15, 36). This regionrepresentsanautonomousdomain sufficienttoactivate transcriptionwhen fused toGAL4and expressedinS.

cerevi-siaeorin mammaliancells(Fig. 3) (reference 11 and data not

shown). Furthermore,we show herefor the first time that the carboxy-terminal 18 aa contribute to ZEBRA's ability to behave as an activator in EBV-negative B cells and in S.

cerevisiae and also constituteanautonomousactivating region.

Experimentsthatexamine the capacity of activators to work in heterologous systems have led to the hypothesis that some activators are "universal" and others are "specialized" (38). Universalactivators, suchasthe acidicC-terminal80aaofthe powerful herpes simplex virus activatorVP16 orthe

C-termi-nal 114 aa of the S. cerevisiae activator GAL4, operate in a wide variety of eukaryotic cells, including those of yeasts,

insects, and mammals (2, 6, 23,40). Furthermore, somepoint mutations ofGAL4increaseordecrease its activationpotency in the samedirection in both yeast and mammalian cells(12).

Specialized activators, such as the glutamine-rich domain of the Sp-1 transcriptionfactor, work in mammalian cells butnot

inyeastcells(13). Universal activators are thoughtto contact some component of the transcription machinery which is

highlyconservedthrough evolution (13, 17). Inthe sense that

auniversal activatorcanfunction in bothyeastand mammalian cells, ZEBRAfits the definition.

However, certain mutants with deletions in ZEBRA's

N-terminalactivating regionbehavedinadiscordant fashionin S.

cerevisiae and in human B cells. Forexample, removal of the

N-terminal 50 aa reduced ZEBRA's activity to 7% of the

wild-type level inBcells butonlyto48% of thewild-typelevel in S. cerevisiae. Moreover, in the progressive N-terminal-deletionseries,removalofZEBRAaa26to50 causedamajor

reduction ofactivityinBcells buthadnoeffect in S. cerevisiae.

Furthermore, the internal deletion ofaa 26to88causedonly

minimal loss ofactivity in S. cerevisiac and profound diminu-tion in Bcells. These resultsindicate thataregioncontaining

aa 26 to 50isan essentialcomponent of ZEBRA's activating

domainin human Bcells butnot in S. cerevisiae. Others have

also implicated this region as important for activation by

ZEBRA in other mammalian cells such as HeLa and

EBV-negative Burkittlymphomacells(11, 15). Inrelatedwork,two ofus(T.C. andM.C.)have found thataa 26to51 functionas an autonomousactivatingdomain when fusedtoGAL4(1-147)

and assayed in mammalianepithelial (Cos-1) cells. The

ques-tion is whether thosemutations which have agreatereffecton

the activity of ZEBRA in mammalian cells than in yeast

disqualify the protein as a universal activator. One possible

cellular target for ZEBRA's action is the TATA-binding protein,TBP(35).Thecarboxy-terminal 180aaof thisprotein

are highly conserved through evolution (3, 13, 17, 19, 35). It

has been shown that ZEBRAcan interact directly with TBP

and that amino acids in ZEBRA's N-terminal region are

required for this interaction (27). A likely hypothesis is that some of these amino acids also interact with yeast TBP and

account forZEBRA's universal behavior. Those amino acids ofZEBRAwhich are requiredin mammalian cells butnot in

S. cerevisiae may reflect interactions between ZEBRA and nonconservedregionsintheamino terminus ofTBPwhichare

specificfor mammalian cellsor interactions betweenZEBRA and otherproteins, suchasTBP-associatedfactors,which are not thoughttobeconserved in evolution (39). Thus,ZEBRA

hasattributes of bothauniversal anda specializedactivator.

ACKNOWLEDGMENTS

WearegratefultoMark Ptashnefor advice and encouragement.We thank Peter Broad,Paul Farrell, ErikFlemington, Grace Gill, Clark

Huang, JunMa, Anita Mikeljohn, Susan Oleski, Maryellen

Polvino-Bodnar, Ivan Sadowski, Sam Speck, and James Wang for gifts of

plasmids.Wegive specialthankstoJoePearlbergfor yeaststrains. This work was supported by grants from the American Cancer Society(MV-547andVM-24M)toM.C. and G.M. and from theNIH (CA12055andGM32308)toG.M. andM.P.

The work was initiated when George Miller was an American Cancer Society facultyscholarinthe laboratoryofMarkPtashne.

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