The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription.

0022-538X/90/062457-10$02.00/0

Copyright © 1990, American Society for Microbiology

The Adenovirus ElI Early Promoter Has Multiple

EIA-Sensitive

Elements, Two of Which Function Cooperatively in

Basal and

Virus-Induced Transcription

CHITRAF. MANOHAR, JON KRATOCHVIL,t ANDBAYAR THIMMAPAYA*

Departmentof Microbiology and Immunology, Northwestern UniversityMedical School, 303 EastChicago Avenue, Chicago, Illinois 60611

Received 8 November 1989/Accepted 6 February 1990

Themechanism by which the adenovirus-encoded nuclearoncogene EIA activates transcriptionofseveral viral and hostpromotersisanimportantissue in the regulation of eucaryoticgeneexpression and virus-host

cell interactions. Identffication of cis-acting elements of the promoters and the cognate host transcription factors that are targets for EIA action is crucial for our understanding of the EIA-mediated control of coordinately regulatedgenes.The adenovirusEIIearlypromoterhasacomplex architecture and containstwo overlapping promoters with start sites at +1 (major promoter) and -26 (minor promoter). The major promoter responds strongly to virus-encoded trans activators EIA and EIV and contains four elements: a TAGA motif analogoustotheTATAbox,twoEIIFsitespresentinaninverted orientation, andanATF/CREB site. To determine precisely the roles played by these cis-acting elements in both basal and virus-induced transcriptionwhen thepromoter is situated in its natural context,weinvestigatedthephenotypeofaseries of linkerscanpromotersubstitutionmutantsinserted into theviral chromosome. Promoterconstructsharboring linkerscanmutationsineach elementwererebuiltintoanovel EIA- adenovirus vector,andtranscriptional activitywasmonitoredinvirus-infected cells.Inthe absenceof virus-encodedtransactivators, basalactivity in vivowasdependentonallfour cis-acting elements. Surprisingly, apromoter mutantwithonlyoneofthetwo ETTF sites intact could not promote transcription in vivo, suggesting that the two ETTF sites function cooperativelyeveninbasaltranscription. Promotersharboringmutations in either of these twoEIIF sites also failed tobindtoaninfection-specific form of EIIFingel shiftassaysandcompeted onlyveryweaklyfor EIIF bindingwith thewild-type promoterfragment. The dramatic cooperativity shown bythe twoinverted EIIF sites of the ETT promoter both in vivo and in vitro could reflect simultaneous contact of both sites by the transcription factorEIIF.Furthermore, promotermutantswith mutations in theTAGA motif,thetwoETTF sites, and the singleATF site all failed torespondtovirus-encodedtrans activators. Whereasrecent results demonstrate that EIIF activity can be modulated independently by EIV, leading to transactivation of this promoter, ourresultsand those published previously strongly indicatethat thethree different transcription

factors that bind toTAGA,EIIF,and ATFmotifs of the ETT earlypromoterareall targets forEIA regulation

invivo.Thus,strongtransactivation of the EII earlypromoterthrough these multiple EIA-sensitive elements andindependently bytherecently discovered EIV pathwaysuggeststhat theEIIearlypromoterisstringently regulated in virus-infected cells. Suchastringent regulation of thispromoteris consistent withthevitalroles played by the threegeneproductsof thistranscription unit inthevirallytic cycle.

Oneimportant step in theregulation of cellular andviral gene expression isat the levelof initiation oftranscription. Inmost cases, this is determinedby the type of cis-acting elements and the way they are organized in a given pro-moter, the host transcription factors that bind to these sequences, and the virus- orhost-encoded trans-acting fac-tors that stimulate

transcription

ofthese promoters. Early promoters of human adenovirus provide excellent model systemsin which to study thistype of transcriptional

regu-lation. Inhumancellsinfected with adenovirustype 2 (Ad2) orAdS, six early polymerase

IT

promoters arecoordinately regulated, and their efficient

transcription

is dependent on the289-amino-acid viral EIAprotein (reviewedin reference

53). Most ifnotall upstream DNA sequence elements that are required for basal EIA-independent transcription of these promoters have been defined(reviewedinreference5), along with several host transcription factors that bind to

* Correspondingauthor.

tPresent address: Abbott Laboratories, North Chicago, IL 60064.

them (reviewed in reference 30). Yet despite intensive

ef-forts, no uniform mechanism to explain how either these

well-defined promoters ortheEIA-sensitive polymerase III promoters(25, 71) arestimulated hasemerged.

Forthe pastseveral years,ourlaboratoryhasfocusedon the structure and mechanism of activation of one of the

EIA-activated promoters, the ETT earlypromoter. The EII

unit is one ofthe most important of the early regions for successful completion ofvirus infection. It encodes a 72-kilodalton(kDa)DNA-binding protein (DBP),140-kDa DNA

polymerase, andthe 80-kDa precursorterminal

protein,

all of whicharevitalfor viral DNAreplication (66).Inaddition,

several otherimportantmetabolic roles areassociated with DBP, such as mRNA stability (2), repression of EIV

tran-scription (54), host range function (33), assembly of virus

particles(55), andtransformation

(15).

A

stringent

transcrip-tional control mechanism to ensure

synthesis

of

optimum

amountsof these crucial

proteins

duringthe

growth

cycle

is

expected. TheEIIearly promoteris also sensitiveto trans-activationby simian virus 40large-Tandsmall-t

antigens

in additiontoadenovirus EIA

proteins (46-48).

This promoter 2457

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is complex and contains two overlapping promoters with start sites at + 1 (major promoter) and -26 (minor promoter) (49). By linker scan (LS) mutagenesis studies, weand others (52, 72, 73) have shown that the major EII early promoter contains two cis-acting sequence elements: aTAGA motif between -22 and -30, analogous to theTATAbox, and an ATF-binding site located upstream between -68 and -77. This element is also found in the adenovirus EIII and EIV promoters and in a number of hostpromoters thatrespond to cyclic AMP (8, 11-13, 21, 26, 41, 42, 45). A host transcrip-tion factor, ATF, that binds to this sequence has been identified (28, 37, 41, 42, 63, 70). This factor has been purified (19, 29; C. F. Manohar et al., unpublished results), and its binding activity does not change aftervirus infection (28, 63, 70). Gel shift experiments have shown that a host protein designated EIIF binds to a third sequence element,

TTTCGCGC, duplicated between -35 and -68. A host 55-kDa polypeptide binds to this sequence and promotes transcription in vitro (69). The promoter-binding activity of this factor increases severalfold aftervirusinfection(34, 64, 70), and recent results show that this dramatic increase in binding activity is due to amodification ofEIIFinduced by one of the EIVpolypeptides (10, 20, 22, 25a, 60). Although the effect of EIV polypeptide on EIIF has recently been extensively analyzed at thebiochemicallevel, thefunctional

significance of these twocopiesof thesequence elementand genetic evidence fortheirrole in EII early promoter activa-tion havenot beenclearly established. Inaddition, transfec-tion studies have not provided unambiguous evidence for any sequenceelements in theElI earlypromoterthat can act as targets for EIA activation. In LS mutagenesis studies,

everymutantpromoter that was analyzed fortranscription

wasstimulated by EIAgeneproducts intransfection assays

(48,52, 72). Ontheother hand,certain deletionmutagenesis

studies haveimplicated, at least inpart, a role for the EIIF sites in EIA transactivation ofthe EII early promoter (27, 73), buttheroles ofother sequence elements in EIA trans-activation have notbeen resolved. Thisraises the question

as towhethertranscriptional regulation studied in transient

transfection assaysfaithfully reproduces the regulation that goes on in virus-infected cells when the promoters are present in their natural context.

Toaddress thisquestion, we havereturned a series of LS substitution mutants tothe viralchromosome, using anovel

EIA- adenovirus vector, and analyzed their transcription

with or without coinfection with a wild-type (WT) virus to provide EIA polypeptides. Results presentedhere andthose

publishedpreviously lead us to conclude that the EII early

promoter in virus-infected cells is regulated by three dif-ferent transcription factors interacting with four separate

DNAmotifs, all ofwhich appear to beinfluenced byEIA.

MATERIALS AND METHODS

Sources for HeLa suspension cultures, WT adenovirus

and thed1321 variant, plasmidsthatcontainthe WTAdS

EIl

early promoter fused to chloramphenicol acetyltransferase (CAT)-coding sequences(EIIA-early WTCAT),and the LS mutant derivatives of this plasmidwere as described previ-ously (52, 63,64).Aplasmidthatcontainedtheright terminal

portion from 78.5 (XbaI)to 100 map units of the adenovirus sequences (pA5-130), which was used to rebuild the pro-moter constructs into virus, was described in a previous report (6). Mutant d1321 contains the same deletionasdoes

d1312(6, 31).

The promoter mutants were first rebuilt into the EIII

region ofpA5-130. The viralportion oftheplasmidwasthen

ligated witha0- to78.5-map-unitXbaIfragmentof d1321 as described before (see Fig. 2; 6). Human 293 cells were

transfected with theligated DNA sample, and virus stocks werepropagated. Thevirusstocks were plaquepurified and

titeredon293 cells. The mutationswereconfirmedagainby analyzing mutantviralDNAas described previously (6).

Nuclear extracts fromvirus-infected cellswereprepared

byinfectingHeLacellsuspensionculturesat25PFU per cell

for 8 h in the presence of

1-p-D-arabinofuranosylcytosine

(araC) at 25

pg/ml

(9,

63).

For CAT assays, virus infection wascarriedoutfortheperiods indicatedinthe figurelegends in the presence of araC (25 ,ug/ml). Protein concentrations were determined by the method of Bradford (7). Gel shift assays were performed as reported previously (64), with salmon sperm DNA as the nonspecific competitor. This

nonspecific competitor DNA allowed detection of EIIF

factorspecifically (64, 70).

Primer extension was carried out according topublished protocols(1).Briefly, 15 pug ofpoly(A)+ RNAs was annealed with a 5'-end-labeled CATprimer (from +110 to +136)

(105

cpm)in30,ul ofabuffercontaining80%formamide, 40.0 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH6.4), 400.0 mM NaCl, and 1.0 mMEDTA overnight at 30°C. The nucleic acids were ethanol precipitated and suspended in a

buffercontaining50 mM Trishydrochloride(pH 8.0), 40 mM NaCl, 0.5 mM EDTA, 5 mMMgCl2, 3 mM dithiothreitol, 40

,M deoxynucleoside triphosphates, and 1.25 [L of RNasin. Avian myeloblastosisvirus reverse transcriptase (20 to 40 U) was then added, and the sample was incubated for 3 h at

42°C. At the end of incubation, reaction was stopped by

addingRNase A(40pug/ml)and EDTA (20 mM). The nucleic acids were extracted with phenol, ethanol precipitated, and resolved on a6% DNA-sequencing gel. B-Actin mRNA was quantitated under identical conditions with 5.0 ,ug of

poly(A)+ RNA, using a 27-nucleotide (nt) primer comple-mentaryto +78 to +105 of the human,B-actin gene.

RESULTS

Strategy to analyze theEIlearly promoter inthe context of viral chromosome. Figure 1 shows the four transcriptional control elements that have been identified for the ElI early promoterwith respect to the major start site by this labora-tory and others (52, 63, 64, 70, 72). We previously con-structed and characterized intransfection assays a setof 15 LS substitution mutants of the EII early promoter fused to the CAT gene (52). Figure 1B shows the locations of these mutations with respect to different transcriptional control elements. We have now returned these LS mutants into the viral chromosome, using a novel EIA-deficient adenovirus vector system (6). In this vector system, the ElI promoter mutants were cloned into thenonessential EIIIregion of the virus, with the orientation of transcription of the newly introduced ElI promoter constructs identical to that of the resident ElI gene (Fig. 2). The original EII gene, which provides functions vital for viral DNAreplication, remained intact. Since the promoter sequences are fused to the re-porter CAT gene, the mutant promoters can be analyzed unambiguously in a sensitive CAT assay. The virus vector contained an EIA deletion identical of that ofd1312, so the virus could be propagated efficiently in the adenovirus-transformed human 293 cell line by trans complementation. Mutant viruses that harbor the LS substitution mutations in the ElI promoter can be analyzed for EIA-dependent and EIA-independent transcription by infecting HeLa cells with and without, respectively, the WT virus.

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-76 -69 -67 -60 -29 -23

TGACGTAG TTTCGCGC TTAAGA

-~---m

rAN EIIF -- 1 TG

-50 _4'.

CGCGCTTT

-45 -36

ATF EIIF(D)

-74/-85 -52/-63 -55/-66 ATF/EIIF(D)

-63/-73 -65/-75

EIIF (P) -31/-41 -35/-46 -40/-50

TAGA -19/-29 -25/-34

FIG. 1. Transcriptional control elements of the Ad5 EII early promoter, theircognate transcription factors, and the LS substitution mutations that mutate these defined control elements. (A) ElI earlypromoterstructure.Locations ofasequenceanalogousto the TATA

sequence(TAGA motif), twoEIIFsitespresentinaninverted orientation, andasingle ATF site and thetranscription factors that bindto thesesequences areshown. Althoughnotidentified, it is presumed thatatranscriptionfactor bindstotheTTAAGAsequence.(B)Locations

of theLSsubstitutionmutations in relationtothedifferent cis-acting elements. EIIF-P, Proximal EIIF site; EIIF-D,distal EIIF site. Further details of thesemutantscanbeobtained from Murthyetal. (52).

Basaltranscriptional activity of themutant promotersand the cooperativity between the two EIIF sites for function in vivo. To determine the contribution of each cis-acting ele-mentofthe ElI earlypromoterfor basaltranscription, HeLa cells were infected with adenovirusmutants containing the WTandmutant derivatives ofthe EII-early CATconstruct (Ad5EII-EWT-CAT)at20 PFUpercellandinthepresence

ofaraC. Eight hours after infection, cell lysates were pre-pared and CAT activity present in the lysates was deter-mined as described previously (6, 17). The CAT activity

detected incellsinfectedwithmutantviruses harboring the EII-early CAT constructs was solely due to transcription driven from the EII early promoter. Because, as shown previously (6) andinthis study(Table 1; Fig. 3A, lane 1; data notshown), amutantvirus (Ad5 0-CAT) in whicha promot-erless CAT construct was cloned into the EIII region in a backgroundidentical to that ofthe adenovirus mutant car-rying the WT EII-early CAT construct did not show any

ClO I

100

pA5-130

Xboll

3

Xl

78~5 83

Oto78-5 from virus, 78-5tolOOfromplasmid,

ligate, tronsfect HindHll BomHl EcoRI Xbal Xhol

o79,5g5 75-9 785 83 100

EIAA5

PIIA-F (%AT

-4 ^~-4 C,11A-C7k*A

EIIA

FIG. 2. StrategyforrebuildingtheEIIAearlypromotermutants

intoanEIA-Ad5 vector(see Materials and Methods for details). Restriction sites are positioned on the Ad5 map units. Symbols: X,chimericgene; _, deletionpresentin the EIAgeneof variant d1312(31). EIIA,TheoriginalElI geneof the virus.

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detectable CATactivityat8 h postinfection in the presence orabsenceofEIA geneproducts. This result indicatesthat the DNAsequences upstream of thenewEIIearly promoter didnotcontribute topromoteractivity fortuitously. Figure

TABLE 1. CATexpression in adenovirus mutantsharboring ElI earlypromotermutationsa

CATexpression

Product Fold

Mutation formedb %

Activity

induction (+EIA/-EIA) LS+ LS + LS+ LS+

d1321 WT d1321 WT

Mock 0 0 0

AdS0-CAT 0 0 0

WT 0.176 2.99 100.0 100.0 17.0

-19/-29(TAGA) 0.008 0.011 4.5 0.37 1.4 -25/-34(TAGA) 0.008 0.009 4.5 0.30 1.2 -31/-41 (EIIF-P) 0.007 0.008 4.0 0.27 1.2 -35/-46(EIIF-P) 0.009 0.009 5.1 0.30 1.0 -40/-50(EIIF-P) 0.009 0.009 5.1 0.30 1.0 -49/-59 (?) 0.171 1.887 97.0 63.1 11.0 -52/-63(EIIF-D) 0.01 0.056 5.6 1.87 5.6 -55/-66 (EIIF-D) 0.01 0.059 5.6 1.97 5.8 -63/-73(EIIF+ATF) 0.012 0.078 6.8 2.6 6.5 -65/-75(EIIF+ATF) 0.015 0.073 8.5 2.4 4.5 -74/-85(ATF) 0.034 0.223 19.3 7.5 6.6

-82/-92 0.181 1.51 103 50.5 8.3

aThecis-acting elements affected by the mutationsareshown in parenthe-ses.EIIF-P,ProximalEIIFsite; EIIF-D,distalEIIF site.Dataobtained from theexperiment shown inFig.3 arepresented.HeLa cells(60-mm-diameter dishes;2x 106cells)wereinfectedwithAdSmutantscontainingthevarious CAT constructs withWTvirus(+EIA)ord1321(-EIA)at20 PFUper cell in thepresence ofaraC(25p.g/ml)and harvestedat8 hpostinfection; thecell lysateswereprepared,and theirproteinconcentrationsweredeterminedby

the methodof Bradford(7). CAT activitywasassayedasdescribed previ-ously, (17),usingequalamounts ofprotein. Assayswere performedunder conditions in which theconversion of[54C]chloramphenicol toits products

was less than30% of thetotal substrate added. Whennecessary, the cell lysateswerediluted toobtainvalueswithin this range.

INanomolesofacetylatedchloramphenicolper106cells.SeelegendtoFig.

3forexperimental details.

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LS-74/-85

FIG. 3. Expression of the WT andthe LS derivatives of the ElI early promoter in the context of the viral chromosome in the absenceorpresenceofEIAgeneproducts. SeefootnoteatoTable 1forexperimental details. (A) CAT expressionof the WT and the LSmutants of the ElI earlypromoterin the absence of EIA. Ad5 O-CAT isan EIA- AdS variant that contains a promoterless CAT constructintheEIII region (6). (B)CATexpressionin thepresence of EIAproducts. Mutant viruseswerecoinfectedwithaWT virus. In EIA- experiments, mutant viruses were coinfected with the EIA- virus dl321. However, no difference in the pattern of CAT expressionwasobserved whetherinfectionwascarriedoutaloneor

with EIA-virus d1321. The autoradiograms shown in panelsAand Bwereobtainedby exposing the thin-layer chromatography plates for different periods of time. For quantitation, see Table 1. (C) Schematic representation ofthe mutations in the various LS

mu-tantswithrespecttodifferenttranscriptionalcontrol elements. 3Ashows CAT activities for the WT and LS mutantsat8 h postinfection in HeLa cells infected with mutant viruses; Fig. 3C showsaschematicrepresentationof the LSmutants withrespecttothecis-actingelements of thepromoter.CAT activitywasquantitated by directly countingtheradioactive spots inthe thin-layer chromatography plate. Inourearlier study, we found that the levels of hexon mRNA (internal control)didnotchangebetweenmutantinfections inasingle experiment (6). Therefore, when using infections it is not

necessary to normalize the CAT activity to an internal

control. Relative levels of CAT activity of the different promoter mutants are shown in Table 1. A 20- to 25-fold-reduced CAT activity was observed for mutants that alter the TAGAmotif(LS mutants -19/-29 and -25/-34) and the proximal (LS mutants -31/-41, -35/-46, and -40/-50) and distal (LS mutants -52/-63 and -55/-66) EIIFsites.MutantsLS-74/-85, whichmutatesexclusively the ATF site, was fivefold defective. Two LS mutants (-63/-73and -65/-75)that abutboth distalEIIFand ATF sites were also severely defective (20-fold). Thus, efficient basaltranscriptionof themajorElI earlypromoterinvivois dependent on all four identified cis-actingelements. Unex-pectedly, there was a dramatic cooperativity between the

twoEIIFsites forpromoter function.

Integrity

of bothEIIF siteswasvital for

transcription.

For

example,

inmutantsLS

-31/-41,

-35/-46,

and

-40/-50, only

the

proximal

EIIF

site was

mutated;

the distal EIIF site was

intact,

yet the

mutants were defective

by

20-fold.

Similarly,

LS mutants

-52/-63 and -55/-66 exhibited a 20-fold-reduced

activity

although

the

proximal

EIIFsite in thesemutantswasintact.

Thus,

mutations in either of the two EIIF sites reduced

transcription

dramatically,

andonesite did notcompensate

forthe other. This result

strongly

suggestsa

high degree

of

cooperativity

between thetwoEIIF sitesinbasal

transcrip-tion.

Simultaneous

requirement

of both EIIF sites for efficient

EIIF

binding

invitro.TheobservationthattheElI promoter

required

synergistic

action ofthe two EIIF sites for basal

transcription

prompted

usto

investigate

theformation of the

EIIF

complexes

in vitro more

closely.

We showed earlier

that the nuclearextracts

prepared

from virus-infected cells at7 h

postinfection

formed

DNA-protein

complexes

specific

for thetwoEIIFsites located between -35and -68

(64).

It wasalsoclearin thesestudiesthatEIIF failedtobindtoLS mutants that mutated

specifically

either the

proximal

or

distal EIIF

site, indicating

that a

single

EIIF site

probably

was notfunctional. These

experiments

were

repeated

with twoLSmutantsthatmutated thetwoEIIFsites

individually.

A DNA

fragment

from-17to-96wasendlabeledandused in

gel

shift assays with nuclear extracts

prepared

from virus-infected cells. As shown

previously,

when the WT promoterDNA

fragment

wasusedas a

probe,

three slower-moving bands

representing

three different DNA

protein

complexes

were observed

(Fig. 4A,

lanes 2 and

12).

The

major

band

(band

I)

wasdueto

EIIF,

sinceitwas

competed

against by

an

oligonucleotide carrying

the

EIIF

site of the

EIA promoter andanunlabeledWTpromoter

fragment

but not

by

an

oligonucleotide

carrying

an ATF site

(Fig. 4A,

lanes 3to

6;

64). By

methylation

interferencestudiesand

by

useofaseriesofLSmutantsacrossthepromotersequence, weshowedearlierthatband IIIwas

nonspecific (64)

whereas band I made

specific DNA-protein

contacts with the two

TTTCGCGC motifsbetween -35 and -68. Inourextensive

study,

the appearance or the

intensity

ofband II was not

consistent,

and in many

experiments

it could notbe

abol-ished with

oligonucleotides

that carry the

EIA-EIIF

or the ATF sequence

(Fig. 4A,

lanes 9to

11).

The

intensity

ofthis

band also remained

unchanged

when

gel

shift

experiments

werecarriedoutwithaWTpromoter

fragment

and

increas-ing

concentrations of nuclearextract

(C.

F.Manohar and B.

Thimmapaya,

unpublished results). Thus,

we believe that

this band is a

nonspecific

band. Consistent with earlier

results,

mutantsthat alter the

proximal (LS -35/-46)

orthe

distal EIIF

(LS -55/-66)

site

(Fig. 4B)

failed to form the

EIIF

complex,

similartoresults for the WT promoter

(Fig.

4A,

lanes 7 and

8; 64). However,

it is

possible

that these

promoter

mutants with

single

EIIF sites may bind to EIIF

withamuchreduced

efficiency.

These

single-site

complexes

may

migrate along

with one of the

nonspecific bands;

this

has notbeen examined further.

A

simple

competition experiment

was

performed

to

deter-minewhetherpromotermutantsinwhich

only

oneof thetwo

EIIF siteswasmutatedwas

biologically

active withrespect

toin vitro factor

binding.

Gel shift assays were carriedout withlabeled WT

probe

in the presence ofdifferent

concen-trations ofunlabeled DNA

fragments

corresponding

to LS mutants -35/-46 or -55/-66. LS mutants -35/-46 and

-55/-66containmutations in the

proximal

anddistal

EIIF

sites, respectively

(Fig.

4B).

These unlabeled promoter

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WT , -551-66 WT WT

2 3 4 5 6 7 8 9 10

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4

6

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z

80

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LS-55/-66 X i z

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FOLD EXCESS

FIG. 4. Simultaneous requirement ofthe two EIIFsitesforEIIFbindingfrom infectedcell extracts.Gel shiftexperimentswereperformed withsalmon sperm DNA (2.0 ,ug per assay) as the competitor and with 88-base-pair end-labeled DNAfragments (fromBssHII at -17to Hindlllat-96; 64)from theWT promoter orits LS derivativesasprobes. About 0.2 ngofthe3'-end-labeledprobes withorwithoutvarious specific competitor DNAs were incubated with 10

jig

of protein obtained from nuclear extracts preparedfrom virus-infected cells (see MaterialsandMethods). I, II,and III denote the three different DNA-protein complexes detectedinour assays and arediscussed in thetext. Inexperiments shown inpanelC,complexIIwasdetected as a faint band, and therefore itsposition isnotindicated. (A) EIIFcomplexes formed in gel shiftassayswith theWTand thetwoLSmutantprobes -35/-46 and -55/-66.Competitor oligonucleotides: EIIF,5'-GATC GTTTGGCCATTTTCGCGGGAAAACTG-3' (nucleotidesequencefrom-234 to -217ofthe EIApromoter); ATF,5'-GATCCTAAAAAA TGACGTAACGGTTAAAGTC-3'(nucleotide sequence from -57 to -31 of the EIV promoter).Oligonucleotideswereusedasduplexeswith theappropriate complementary sequence. Lanes: 1,probe withoutprotein;2 to 6, assayswithWTprobeand withEIIF or ATF sequences ascompetitors; 7 and 8, assays with LS -35/-46 and -55/-66 probes, respectively, and with no specific competitor DNAs; 9 to 11, competition byEIIF or ATF sequencesforthe DNA protein complexes detected when theLS -55/-66probewasused; 12, control. (B) Diagram showing the mutationspresentin probes LS -35/-46andLS -55/-66 withrespectto two EIIFsites.(C) Competition byWTand mutantpromoterfragments derived from LS -35/-46andLS -55/-66on EIIFbinding.Assayconditionswere asdescribed above. Lane 12, Competition by a 90-base-pair pBR322 DNA fragment. (D) Graph showing the competition effects ofWT(0) and LS -35/-46 (0) promotersforEIIFbinding. EIIFcomplexes detectedinthe gel showninpanelCwereexcisedand counted and thenplottedaspercentage WTprobebindingversusfold molarexcessof the unlabeled DNAfragments.

fragments contained one of the two EIIF sites intact yet

failedtocompeteefficiently withthe WT promoter (Fig. 4C, lanes 5 to 11;Fig.4D). Forexample, at a 5-fold molar excess

ofthe unlabeled WT promoter fragment EIIF binding was

nearly abolished,whereasat a7.5-foldmolar excessofDNA

fragments derived from mutant LS -35/-46 or -55/-66, EIIFbinding was notsignificantly affected (Fig. 4C,lanes 5 and 9;Fig.4D). (For reasons that we cannotexplain,the WT promoter also competed with the nonspecific band in this experiment [Fig. 4C, lanes 3 and 4].) Competition was detected only when the mutant promoters were used at a 35-fold molarexcess(Fig. 4C,lanes 6 and10;Fig.4D). EIIF

bindingwasnotabolishedeven at a105-fold molarexcessof mutant DNA fragments; 25% ofWT binding was still ob-served at this concentration (Fig. 4C, lane 8; Fig. 4D),

indicatingthat asingle EIIF site binds to EIIF witha very low affinity. This observation combined with the fact that these mutants inwhich only one of the EIIF sites is intact retained less than5%of the basaltranscription (Fig. 3A and

Table 1) indicate that the ElI early promoter shows a simultaneous requirement for both the sites to function in

vivoandin vitro. These resultsareconsistentwith therecent results ofHardy et al., who showed the dramatic

cooper-ativity ofthetwo EIIF sites for EIIF

binding

in vitro

(22).

Roleofcis-actingelements inthe induction oftranscription

by virus-encoded trans activators. One ofthe aims of this

studywas to determine

precisely

the contributions ofeach

cis-acting element oftheElI

early

promoterwhen the EIA protein was provided in cells through the natural infection process rather than the transfection approach. Since our mutantslack EIAcoding sequences, we could evaluate the effect of EIAby coinfecting the mutants with a WT virus. This analysis has become somewhat

complicated by

the recent finding that in virus-infected

cells,

the

cooperative

interactionof EIIF with two EIIF sites is facilitated

by

the EIVE6/7polypeptide(25a).Ourvector was

designed

before this newobservation and contains an intact EIV.

Coinfec-tion with WTAd5 or

d1356,

a viable variant that lacks the

Ii

.1

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[image:5.612.145.473.79.376.2]

E6/7 polypeptide, will influence the EIT early promoter activity through EIIF. However, this effect is confined to only EIIF. This and other previously published results (see Discussion) have allowed us to evaluate the EIA effect on the different cis elements of thepromoter.

[image:6.612.321.553.77.509.2]

HeLa cells were infected in the presence of araC with mutantviruses harboring CAT constructs alongwith WT or d1321, the parental virus used to construct these mutants (6). At 8 h postinfection, cell lysates were prepared and CAT activity was determined. Figure 3B shows CAT activities detected for various mutants in the presence of WT virus. Table 1shows a comparison of promoteractivities ofvarious mutants for basal and virus-induced transcription. As ex-pected, theWT promoter was induced 17-fold in the exper-iment shown in Fig. 3B. Surprisingly, mutant promoters with mutations in the TAGA box (LS mutants -19/-29 and -25/-34) and the proximal EIIF site (LS mutants -31/-41, -35/-46, and -40/-50) were not induced by virus-encoded transactivators in any of ourexperiments(Fig. 3B andTable 1). Transcription of mutants with mutations in the distal EIIF site (LS mutants -52/-63 and -55/-66) and the ATF site (LS -74/-85) were induced five- to sixfold. These data were highly reproducible in numerous experiments. There was no difference in the pattern of expression when WT virus wasreplaced by anAdS mutant that synthesized only the largeEIA protein(50) (data not shown). One mutant with a substitution mutation located between the two EIIF sites showed normal phenotype with respect to both basal and virus-induced transcription (Fig. 3B and Table 1). Wedid not see an overexpression of CAT in this mutant as we did in transfection assays (52).

As stated earlier, the ElI early promoter contains dual overlapping promoters with a minor start site at -26. The promoteractivity in this study was quantitated onthe basis

of expression of a reporter gene. It was therefore possible that a five-to sixfold stimulation oftranscription by the ATF and thedistal EIIF site mutantsin WTcoinfectionswasdue to CAT activity resulting from RNAs transcribed from the -26 startsite; the distalEIIF site and the ATF site could be as important as theTAGA and theproximal EIIF motifs for themajor promoter in virus-inducedtranscription. Toclarify this point, we carried out primer extension analysis of CAT mRNAstranscribed from Ad5 mutants that contained muta-tions in the TAGA motif, the two EIIF sites, and the ATF site. HeLa cells were infected with mutant CAT viruses containing mutations in individual control elements and coinfected with WTAdS or the EIA- parental virus, d1321. Poly(A)+ RNAs were prepared and annealed with a 27-nt-longprimer complementary to +110 to +136(with respect to the major start site) of the CAT gene. Theprimers were then extended with reverse transcriptase as described in Materials and Methods. This strategy can distinguish be-tween the mRNAs transcribed from the two overlapping promoters. The mRNAs transcribed from the major pro-moter should generate a 136-nt-long product, whereas the mRNAstranscribed from the minor promoter should gener-ate a product 162 nt long. As an internal control,

P-actin

mRNA was also quantitated from the same RNA samples,

using an appropriate primer (Fig. SC) (see Materials and Methods for details). Basal transcription from the major promoterwas dramatically reduced (7- to 10-fold, as judged from densitometric analysis of the autoradiogram) for the four mutants that individually perturb the TAGA motif, the two inverted EIIF sites, and the single ATF site (Fig. 5A). When coinfected with WT Ad5, transcription of the major promoter of the WT CAT gene was induced by 10-fold,

cr-LAJ F

< I-.

WT Ad - +

123

C}

-A_<

(-

-I-4 5 CtDL

IeI

6r)=

6 7

D

-to.=

I

8 9

Il) 02

I0lI}

10 11 213

192 -184

(MINOR)

(MAJOR) 4m

4p

124: 123

104-2 3 4 5 6 78 9 1011

234-213-

-192 184

-- 4p --. -- --.

'Am

124 _

123 *,

104- ..

©

104-<-z26 (MINOR)

.MA+R (MAJOR)

i 234 56 7 810119

""00"loom0-W- "

¶B-A

CTIN

FIG. 5. Quantitationof CATmRNAstranscribedfrom themajor

andminor ElI early promoters in the presence orabsence ofWT AdS infections. HeLa cellswereinfected withAdSmutants(20PFU per cell) containing aWT promoter-CATconstruct orthemutant

derivativeswith WTAd5ord1321(20PFU percell)inthe presence of araC. Poly(A)+ RNAs were prepared at 8 h postinfection and annealed to aCATprimer(from +110 to +136)or ,-actinprimer

(from +78to+105).Theprimerswerethenextended withreversed transcriptase and electrophoresed as described in Materials and Methods. A5'-end-labeledHaeIIIdigestofpBR322DNAwasused as markers.(A)Effect of LS mutations onbasaland virus-induced transcription from the major start site; (B) longerexposure of the samegeltoshow theeffect of mutationsonthe minorstartsite; (C)

quantitationofP-actinmRNAs.

whereas themutantpromoters

uniformly

failedto

respond

to the virus-encoded trans activator EIA or EIV. Identical results were obtained in two

independent

experiments;

the stimulation of

transcription

of ATF and the distal EIIF mutants for the

major

promoter was not more than 1.5-fold

(quantitation

basedondensitometer

scanning).

We therefore

conclude that afive- to sixfold increase in CAT

activity

for thesemutantsinthepresenceofWTAdS

(Fig.

3B and Table

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http://jvi.asm.org/

1)most

likely

wasduetomRNAstranscribed from the minor

promoter.Indeed, although basal transcription from the -26

site was somewhat reduced for these mutants, the virus-induced transcriptionwasunaffected (Fig. 5B). The

P-actin

mRNA levels remained unchanged in all ofthese samples.

These results suggest that the TAGAmotifand the ATFsites

areequally sensitivetoEIAregulation.WithregardtoEIIF,

although

ourresultscannotclearly distinguish between the

EIAandEIVeffectsontheEIIFsites, previously published

results clearly indicate thatthese sites are sensitive to EIA

regulation (see Discussion). Thus, we conclude that all

cis-acting

elements ofthe ElI early promoterand the

cog-natehosttranscriptionfactorsaretargetsforEIAregulation.

DISCUSSION

This is thefirst time that the control elements ofthe ElI

early

promoterhave been

analyzed

inthecontextoftheviral

chromosome. This

analysis

has shed newlight onthe

tran-scriptional

regulation of this widely used promoter. By

evaluating

the contributions ofeachtranscriptional control

element in both basal and EIA-mediated activation when

these elementsare

organized

in the milieu of

nucleoprotein

complex

andintheirnatural

environment,

wehavebeen able

to

(i) provide genetic

evidence forthe

requirement

of thetwo

inverted EIIF sites for both basal and virus-induced

tran-scription

and

(ii)

show that the three

previously

identified

transcription

factorsthatinteractwith theTAGA

motif,

the

two EIIF

sites,

and the ATF site are all targets for EIA

action.

Ourresults

clearly

demonstrate that the

integrity

of both EIIFsites is critical forthe promotertofunctionwhether it is in the presenceorabsence of virus-encoded trans activa-tors. Promoters with mutations in either the proximal or distal EIIF site fail to function in basal and virus-induced

transcription (Fig.

3 and5;Table 1). The EIIFcomplexcan

barely

be detected with mutant promoters

carrying

muta-tionsineither the

proximal

orthedistalEIIF

site,

and such mutants are very inefficient in

competing

for EIIF

binding

with the WT promoter (Fig. 4). The requirement for both sitesin theElI promotercanbe

explained by

the

cooperative

interaction ofEIIF with the two EIIF sites. The two EIIF

sites

individually

may bind to EIIF only very weakly,

whereas the two sites in an inverted orientation cooperate

dramatically, resulting

inefficient factor

binding.

Therecent

demonstration

by Hardy

and Shenk (22) that EIIF binds

cooperatively

tothe EIIF sites of the EIIpromoterin vitro

supports this contention. Our results

complement

these

biochemical observations and

provide

genetic evidence for therole of

cooperative binding

of EIIFforpromoterfunction

in vivo.

Recently,

it has been shown that the virus-encoded EIV

polypeptide

canfacilitatethe

cooperative

interaction of EIIF

withthetwoEIIF sites oftheElI earlypromoter(10, 20, 21).

Whether EIA also can promotethe

cooperative

interaction

ofEIIF with thepromoteris notknownatpresent. InadditiontotheAdSElI earlypromoter,EIIFhasbeen

implicated

in

transcription

of AdSEIA (35) and c-myc (23,

67)

promoters. Two EIIF sitesare present in eachofthese promoters. It seems

likely

that the two EIIF sites may cooperatefor

transcription

in thesecases too. The distance between the two EIIF

sites,

their sequence context, and their orientation

probably

allcontributetothis

phenomenon.

The

cooperation

of the two EIIF sites in

transcriptional

activation of the ElI promoter in basal or virus-induced

transcription

that we observed has some similarities with

thatreported for the bovine papillomavirus ElI-dependent enhancer elements: twocopies of the bovine papillomavirus enhancer element cooperate in EII-dependent transcrip-tionalactivation;asingle sitecanfunction only very weakly

(65).

The behavior of the EIIF-binding sites of the EIA pro-moter is considerably different from that of the ElI early promoter. The oligonucleotides that carry either of these

sites cancompete efficientlywiththe WT ElI promoter for EIIF binding, whereas the single EIIF site of the ElI promoteris veryinefficient(Fig. 4A; 64). A single EIIF site from EIA promoter is sufficient to activate transcription

fromaheterologouspromoter(34). The different affinities of thetwoclasses ofEIIFsites for EIIFbindingmaybe dueto

their sequence context. The flanking sequences of a

cis-actingelement can have aremarkable effect withregard to

factorbinding. Forexample,the 17 EIIprotein-bindingsites of the bovine papillomavirusgenome showawide range of

binding affinitiestoElI protein, dependingon the sequence context(43).

The strategy used here to investigate the EII promoter

mutations involved insertion ofmutantpromotersas

dupli-cate copies in the viral genome. There is a possibility that thesemutantpromoters competewiththe residentpromoter

forthe

limiting transcription

factors. Ourresults show that

the WTCAT construct is

efficiently

transcribed and

trans-activatedintheEIIIregion.We haveanalyzedthe WT and mutant promoters under identical genetic backgrounds.

Therefore,

itis

unlikely

that such a competition will

influ-encethe finalnatureof the results obtained and the interpre-tation.

Asecondmajorfinding of this study isthat three

promot-er-specific

motifs participate in EIA enhancement. This

conclusion is based on the

following

observations. Our results clearly show that promoters with mutations in the

TAGAsequence,thetwoEIIFsites,and the ATFsitefailto

respond to virus-encoded trans activators. Previously, in

transfection assays

using

cloned genes, it was shown that

only the EIIF sites were involved in the EIV-mediated

transactivation(16). Recentbiochemical studies confirmthis observation and show that(i) EIVmodifiesEIIFtogenerate

an

infection-specific

form ofEIIF(20)and

(ii)

this

modifica-tion is mediated by direct physical association ofthe EIV

polypeptide

(Huang and

Hearing,

in press). Given these

observations,

it is reasonable to conclude that mutants

containingmutations in the TAGA and ATFmotifof theEII

early promoter failto respond to virus-encoded trans acti-vators because these sites are targets for EIA regulation. Ourresultsdonot

distinguish

between theeffectsofEIAand EIVonEIIF. However,Zajchowskietal. (73)showed that intransfection assays,adeletionmutantof theElI promoter in which only the two EIIF sites were deleted failed to

respond to EIA. Inother

studies,

5' deletion mutants with

deletions

extending

into the distal EIIF sites failed to

re-spond to EIA,

suggesting

that EIIF at least in part is

involved in EIA transactivation

(27, 52).

Moreover, in an adenovirus-transformed cell line that containsaconditional lethal mutation

affecting

the EIA large

protein,

at nonper-missive temperature, EIV

expression

was notaffected buta

large decrease in EIIF

activity

was

observed,

strongly

suggestingaroleforEIAinEIIFactivation

independent

of EIV (3). Thus, it is almost certain that

immediately

after

infection,before the EIV

polypeptide

is

made,

theElI

early

promoteris activated by EIA

through

these

multiple

EIA-sensitive elements.

In the promoter mutant in which the TAGA motif is

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mutated, the otherEIA/EIV-responsive elements, EIIFand ATF, are intact. Therefore, the promoter should have re-sponded to EIA stimulation at least on a small scale. Similarly, proximal EIIF site mutants contain TAGA and ATF sites intactyetarevirtually nonresponsivetoEIA.This showsthatotherEIA-responsiveelements failto exerttheir EIA-related phenotype either cumulatively or individually when any one of the EIA-responsive elements is unable to function. One element alonecannotconferEIA responsive-nesstothepromoterin thiscase.These resultsarestrikingly

different from those obtained in stable and transient trans-fection assays. Mutations in these elements reduced basal transcription but did not affect EIA-induced transcription (48, 52, 72). Although we do notknow the specific reasons

for these differences, transfectionassays,whether stableor transient, do not reproduce in the infected cells the regula-tory pattern that is in effect in the viral chromosome. In stable transfection assays, a mutant with only the TAGA

sequence was sufficient forbasal and EIA-stimulated tran-scription (32), and LS mutations that affected basal tran-scription didnotaffectEIA-mediatedtransactivation. When measured after transfection, mutations in the TAGA motif, EIIF sites,orthe ATF sitewere all inducedby EIAtonear normal levels (48, 52, 72). Similarresults werealso obtained for theEIA induction of the AdS EIII promoter(12, 38).

Although a number of laboratories have attempted to define the mechanismbywhichEIAstimulatestranscription of RNApolymeraseIIorIIIpromoters,sofarnoconsensus

has developed. It is, however, clear that not all transcrip-tional control elementsare sensitivetoEIA regulation (57). In virusexperiments,the TATA motif has beenimplicatedin EIA transactivation of the AdS EIB promoter (56, 57, 68). TATA box was also implicated in the case of the rabbit

P-globin

(18) and human HSP70 (62) promoters in EIA stimulation. For theEIVpromoter, theCREB/ATFfactor is believedto be the target for EIA (13, 14, 36). Our studies suggestthat all threetranscriptionfactorsregulatingtheEII earlypromoteraretargetsforEIA. It isnotclearatpresent whether the recently postulated EIA-sensitive TFIID that interacts withEIA-sensitiveTATA elements(39, 40) isalso responsible for transcriptional activation of promoters con-taining analogous sequences.Whatever factor interacts with the TAGAsequence,ourresults indicatethat it isatargetfor EIAaction.

What kind of mechanismcanbeenvisagedfor the activa-tion of theEII earlypromoterby EIA?Twohypotheseshave beenputforwardto definethe mechanism ofEIA transacti-vation. One is based on thephosphorylation of EIA-sensi-tivetranscriptionfactors leading toan enhanced bindingof theseproteinstotheirtarget promoters. Recent results show that in virus-infected cells, several transcription factors, includingEIIF, arelikely tobe modified atthe posttransla-tional level (4, 24, 58). It is conceivable that the three transcription factors that are specific to the EIl early pro-moterareallmodifiedby EIAinvirus-infectedcells. Each of thesemodifiedfactors mayenhance the transcriptionalrate by binding directly either to DNA or to the multiprotein transcriptional complexes. The other mechanism postulates that EIA protein functions directly in the vicinity of the promoterandincreases transcription by bindingtothe DNA directly or by binding to the DNA-transcription complex (44). If this were the mechanism, the EIA protein would facilitate the interaction of themultiple EIA-sensitive tran-scription factors with their target sites on the EIl early

promoter. Our results are consistent with either of these

mechanisms.

This is the first instance in which

multiple

transcriptional

control elements andtranscriptionfactors have been

impli-cated in a

single

promoter in EIA transactivation.

Excep-tionally stringent

control of this promoter is

perhaps

to be

expected considering

the

importance

of the gene

products

that it controls. The 72-kDa

DBP,

the 140-kDa DNA

poly-merase,and the80-kDaterminal

protein

areall vital for viral

DNA

replication

(66).

The 72-kDaDBP alone has

multiple

functions in virus

replication (2, 15, 33, 54, 55).

Transacti-vation of the ElI

early

promoter

by

the different

EIA-sensitive

transcription

factors combined witha novel

addi-tionalmechanism of transactivation

by

the EIV gene

(16, 20,

22, 25a,

60)

guarantees an efficient

transcription

of this

promoter when EIA becomes available in the cell and ensures an

adequate

supply

of these

proteins

in the

lytic

cycle

of the virus.

ACKNOWLEDGMENTS

WethankNoel Bouck and Richard Scarpullafor criticalreading

ofthemanuscript,ManoharR. Furtado forhelpin virus construc-tionexperiments, and Prithi Rajan forproofreading. The

oligonu-cleotides wereprovided by theNorthwestern

University

Biotech-nologyFacility.

Thisworkwassupported byPublic HealthServicegrant A120156 from the National Institutes of Health. B.T. was an established

investigatoroftheAmericanHeartAssociation duringthe

perfor-manceof this work.

LITERATURE CITED

1. Ausubel, F. M.,R. Brent, R. E.Kingston, D. D. Moore,J. A. Smith,J.G.Seidman,and K.Struhl. 1987.Current

protocols

in molecular biology. Greene Publishing Associates and John Wiley&Sons,NewYork.

2. Babich, A.,andJ.R. Nevins.1981. ThestabilityofearlymRNA is controlled by the viral 72 kD DNA

binding protein.

Cell 26:371-379.

3. Babiss, L. E. 1989. The cellulartranscription factor E2F re-quires viral ElA and E4 gene products for increased DNA

bindingactivityand functionstostimulateadenovirusE2A gene

expression. J. Virol. 63:2709-2717.

4. Bagchi,S., P.Raychaudhuri,andJ.R. Nevins. 1989.

Phospho-rylation-dependentactivation oftheadenovirus inducibleE2F

transcriptionfactorinacell-freesystem.Proc.Natl. Acad.Sci. USA86:4352-4356.

5. Berk, A. J. 1986. Adenovirus promotersandEIA transactiva-tion. Annu. Rev.Genet. 20:45-79.

6. Bhat, G.,L. SivaRaman,S. Murthy, P. Domer, andB. Thim-mappaya. 1987. In vivo identification of multiple promoter domains of adenovirus EIIA-late promoter. EMBOJ. 6:2045-2052.

7. Bradford, M. M. 1976. A rapid and sensitive method forthe quantitation of microgram quantities of protein utilizing the principleofprotein-dye binding.Anal. Biochem. 72:248-254. 8. Deutsch,P.J.,J.P.

Hoeffier,

J.L.Jameson,andJ.F.Habener.

1988. Cyclic AMP and phorbol ester-stimulated transcription

mediatedby similarDNAelementsthatbinddistinct

proteins.

Proc.Natl. Acad. Sci. USA85:7922-7926.

9. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a

soluble extractfrom isolated mammaliannuclei. Nucleic Acids Res. 11:1475-1489.

10. Durr-Jansen,P.,H.Boeuf,andC.Kedinger. 1989.Cooperative bindingoftwoE2Fmoleculesto an

Ela-responsive

promoteris

triggered by the adenovirus Ela, but not by a cellular Ela

activity. EMBO J. 8:3365-3370.

11. Engel, D. A., S. Hardy, and T. Shenk. 1988. cAMP acts in synergy withEIAproteintoactivatetranscriptionofthe aden-ovirusearlygenes E4 andElA.Genes Dev. 2:1517-1528. 12. Garcia, J.,F. Wu,and R. Gaynor. 1987. Upstream regulatory

regionsrequiredtostabilizebindingtothe TATA sequence inan

on November 10, 2019 by guest

http://jvi.asm.org/

adenovirusearlypromoter.Nucleic AcidsRes.15:8367-9385. 13. Gilardi, P., and M. Perricaudet. 1984. The E4 transcriptional

unitof Ad2: farupstream sequences arerequired for its trans-activation byEIA.Nucleic Acids. Res. 12:7877-7888. 14. Gilardi, P., and M. Perricaudet. 1986. The E4 promoter of

adenovirus type 2 contains an ElAdependent cis-acting ele-ment. NucleicAcids Res. 14:9035-9049.

15. Ginsberg, H.S.,M.J. Ensinger, R. S. Kaufman, A. J. Mayer, andU. Lundholm.1974. Cell transformation:astudy of regula-tion with types 5 and 12 adenovirus temperature-sensitive mutants. ColdSpring Harbor Symp. Quant. Biol. 39:419-426. 16. Goding, C., P.Jalinot, D.Zajchowski, H. Boeuf, andC.

Ked-inger. 1985. Sequence-specific trans-activation of the adenovi-rus Ella early promoter by the viral EIV transcription unit. EMBOJ.6:1523-1528.

17. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinantgenomes whichexpress chloramphenicol acetyl-transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. 18. Green,M. R.,R.Treisman,and T. Maniatis. 1983.

Transcrip-tional activation of cloned human beta-globin genes by viral immediateearlygeneproducts. Cell 35:137-148.

19. Hai, T.,F. Liu,E. A. Allergretto,M.Karin,and M. R. Green. 1988. Afamilyofimmunologicallyrelatedtranscription factors that includes multiple forms of ATF and AP-1. Genes Dev. 2:1216-1226.

20. Hardy,S.,D.A.Engel,and T.Shenk. 1989.Anadenovirusearly region4 geneproduct is required for induction of the infection-specificform of cellularE2Factivity. GenesDev. 3:1062-1074. 21. Hardy, S., and T. Shenk. 1988. Adenoviral control regions activated byEIAandthe cAMPresponseelementbind thesame factor. Proc. Natl. Acad. Sci. USA85:4171-4175.

22. Hardy, S.,and T.Shenk. 1989. E2Ffrom adenovirus infected cells binds cooperatively to DNA containing two properly oriented andspaced recognition sites. Mol. Cell. Biol. 9:4495-4506.

23. Hiebert, S. W.,M.Lipp,andJ.R.Nevins.1989.ElA dependent trans-activation of the human MYCpromoteris mediatedbythe E2Ffactor. Proc. Natl. Acad. Sci. USA 86:3594-3598. 24. Hoeffler,W.K.,R.Kovelman,and R.G. Roeder.1988.

Activa-tion oftranscription factor IIIC by the adenovirus EIAprotein. Cell 53:907-920.

25. Hoeffler,W.K.,and R.G. Roeder. 1985. EnhancmentofRNA polymeraseIIItranscriptionbythe EIA geneproduct of aden-ovirus. Cell 41:955-963.

25a.Huang,M.,and P.Hearing. 1989.The adenovirusearly region4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a directcomplex. Genes Dev.3:1399-1710.

26. Hurst,H.C.,and N.C.Jones.1987. Theidentification of factors that interact with the EIA-inducible adenovirus E3 promoter. Genes Dev. 1:1132-1146.

27. Imperiale, M. J., R. P. Hart, and J. R. Nevins. 1985. An enhancer element in the adenovirus E2 promoter contains sequences essential forthe uninduced and ElA-induced

tran-scription. Proc. Natl. Acad. Sci. USA82:381-385.

28. Jalinot, P.,B. Devaux, and C. Kedinger. 1987. The abundance and in vitrobinding ofthreecellularproteins interacting with the adenovirus ElIa early promoter are not modified by the Ela geneproducts. Mol. Cell. Biol. 7:3806-3817.

29. Jalinot, P.,M.Wintzerith, M.Gaire,C.Hauss, J.M.Egly, and C.Kedinger. 1988. Purification andfunctional characterization of a cellular transcription factor that binds to an enhancer elementwithin the adenovirusearly Ellapromoter. Proc.Natl. Acad.Sci. USA85:2484-2488.

30. Jones, N.C.,P.W.J. Rigby,and E. B. Ziff.1988.Trans-acting protein factors and theregulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 2:267-281.

31. Jones, N. C., and T. Shenk. 1979. An adenovirus type 5 early gene function regulates expression ofotherearly viral genes. Proc. Natl. Acad. Sci. USA76:3665-3669.

32. Kingston, R. E., R. J. Kaufman, and P. A. Sharp. 1984. Regulation oftranscription of the adenovirusElI promoterby

Elageneproducts: absence ofsequencespecificity.Mol. Cell. Biol. 4:1970-1977.

33. Klessig, D.F., and T. Grodzicker. 1979. Mutations thatallow human Ad2 andAdStoexpresslategenesinmonkeycellsmap in theviralgeneencodingthe72K DNAbindingprotein. Cell 17:957-966.

34. Kovesdi,I., R.Reichel,andJ.R.Nevins.1986.Identification of acellulartranscriptionfactor involved inEIAtrans-activation. Cell 45:219-228.

35. Kovesdi, I.,R.Reichel,andJ.R.Nevins. 1987. Role of adeno-virus E2 promoterbindingfactor in EIA-mediated coordinate genecontrol.Proc. Natl.Acad.Sci. USA 84:2180-2184. 36. Lee,K. A.W.,and M. R.Green.1987. Acellulartranscription

factorE4F1interacts with Ela-inducible enhancerandmediates constitutive enhancer function in vitro. EMBO J. 6:1345-1353. 37. Lee,K. A.W.,T.-Y.Hai,L.SivaRaman,B.Thimmappaya,H. C.Hurst,N.C.Jones,and M. R.Green.1987. A cellularprotein, activating transcription factor, activates transcription of multi-ple EIA-inducible adenovirus early promoters. Proc. Natl. Acad. Sci. USA 84:8355-8359.

38. Leff, T., J. Corden, R. Elkaim, and P. Sassone-Corsi. 1985. Transcriptional analysis of the adenovirus-5 EIII promoter: absence ofsequence specificity for stimulation by Ela gene products. Nucleic AcidsRes. 13:1209-1221.

39. Leong, K.,and A. J. Berk. 1986. Adenovirus early region 1A proteinincreasesthe numberoftemplatemolecules transcribed incell-freeextracts.Proc. Natl. Acad. Sci. USA 83:5844-5848. 40. Leong, K.,L.Brunet,and A.J.Berk.1988. Factorsresponsible forthehigher transcriptional activityofextractsof adenovirus-infected cells fractionate with the TATA-boxtranscription fac-tor.Mol.Cell. Biol.8:1765-1774.

41. Leza, M. A., and P. Hearing. 1988. A cellular transcription factor bindstoadenovirusearly regionpromoters andto acyclic AMP responseelement.J. Virol.62:3003-3013.

42. Leza,M.A.,and P.Hearing. 1989.Independent cyclicAMPand EIAinduction of adenovirusearlyregion4expression.J. Virol. 63:3057-3064.

43. Li, R., J. Knight,G.Bream,A.Stenlund,and M. Botchan.1989. Specific recognition nucleotidesandtheirDNAcontext deter-mine theaffinityofE2proteinfor17bindingsites in the BPV-1 genome.Genes Dev.3:510-526.

44. Lillie, J. W.,and M. R.Green. 1989.Transcriptionactivationby theadenovirusElaprotein. Nature(London)338:39-44. 45. Lin, Y.-S., and M. R. Green. 1988. Interaction ofa common

cellulartranscriptionfactor, ATF, withregulatoryelementsin both Ela- and cyclic AMP-inducible promoters. Proc. Natl. Acad. Sci. USA 85:3396-3400.

46. Loeken, M., I. Bikel, D. M. Livingston, and J. Brady. 1988. Trans activation ofRNA polymerase II and III promotersby SV40 smalltantigen.Cell55:1171-1177.

47. Loeken, M.,G.Khoury,andJ. Brady.1986. Stimulation of the adenovirus E2 promoter by simian virus T antigen or EIA occursbydifferentmechanisms. Mol. Cell.Biol. 6:2020-2026. 48. Loeken,M. R.,andJ. Brady. 1989. The adenovirusenhancer. Analysisofregulatorysequences andchangesinbinding activ-ity ofATFand EIIF following adenovirus infection. J. Biol. Chem.264:6572-6579.

49. Mathis,D.J.,R.Elkaim, C.Kedinger,P.Sassone-Corsi,and P. Chambon.1981.Specific bindingoftheadenovirustype-2early and late E2 transcription units. Proc. Natl. Acad. Sci. USA 6:2684-2694.

50. Montel,C.,G.Courtois,C.Eng,and A.J.Berk.1984.Complete transformationbyadenovirus 2requiresboth EIAproteins.Cell 36:951-961.

51. Montminy, M. R., and L. M. Bilezikjian. 1987. Binding ofa nuclear protein to the cyclic AMP response element of the somatostatingene. Nature(London)328:175-178.

52. Murthy, S. C. S., G. P. Bhat, and B. Thimmappaya. 1985. Adenovirus EIIA early promoter: transcriptional control ele-ments and induction by the viral pre-early EIA gene, which appears to be sequence independent. Proc. Natl. Acad. Sci. USA82:2230-2234.

53. Nevins, J.R.1987.Regulationofearlyadenovirusgene

on November 10, 2019 by guest

http://jvi.asm.org/

sion.Microbiol. Rev. 51:419-430.

54. Nevins, J. R., and J. J. Jensen-Winkler. 1980. Regulation of earlyadenovirus transcription: a protein product of early region 2specificallyrepressesregion4transcription.Proc.Natl. Acad. Sci. USA77:1893-1897.

55. Nicolas, J. C., P. Sarnow, M. Girard, and A. J. Levine. 1983. Host range temperature conditionalmutants in the adenovirus DNAbinding protein are defective in the assembly of infectious virus. Virology 126:228-239.

56. Parks, C. L., S. Banerjee, and D. J. Spector. 1988.Organization ofthe transcriptionalcontrol region oftheEIb gene of adeno-virus type 5. J. Virol. 62:54-67.

57. Pei, R., and A. J. Berk. 1989. Multiple transcription factor binding sites mediate adenovirusEIAtransactivation. J.Virol. 63:3499-3506.

58. Raychaudhuri, P., 5. Bagchi, and J. R. Nevins. 1989. DNA binding activity of the adenovirus-induced E4F transcription factor is regulatedbyphosphorylation. GenesDev. 3:620-627. 59. Reichel, R., I. Kovesdi, and J. R. Nevins. 1988. Activation ofa

pre-existing cellular factor as a basis for adenovirus E1A-mediated transcription control. Proc. Natl. Acad. Sci. USA 85:387-390.

60. Reichel, R., S. D. Neill, I.Kovesdi, M. C. Simon, P. Raychaud-huri, and J. R. Nevins. 1989. The adenovirus E4 gene, in additiontothe ElA gene, is importantfor trans-activationof E2 transcription andfor E2F activation. J. Virol. 63:3646-3650. 61. Sassone-Corsi, P. 1988. Cyclic-AMP inductionofearly

adenovi-rus promoters involves sequences required for EIA trans-activation. Proc. Natl. Acad. Sci.USA85:7192-7196. 62. Simon, M. C., T. M. Fisch, B. J. Benecke, J. R. Nevins, and N.

Heintz. 1988. Definition ofmultiple, functionally distinctTATA elements,oneofwhich isatargetinthehsp70promoterforEIA regulation. Cell 52:723-729.

63. SivaRaman, L., S. Subramanian, and B.Thimmappaya. 1987. Identification ofafactorinHeLacells specificfor an upstream transcriptionalcontrol sequenceofanEIA-inducible adenovirus promoterand itsrelative abundanceininfected and uninfected

cells.Proc.Natl. Acad. Sci. USA83:5914-5918.

64. SivaRaman L., and B. Thimmappaya. 1987. Two promoter specific host factors interact with adjacent sequences in an EIA-inducible adenovirus promoter. Proc. Natl. Acad. Sci. USA 84:6112-6116.

65. Spalholz, B. A., J. C. Byrne, and P. M. Howley. 1988. Evidence forcooperativity between E2bindingsites in E2 trans-regula-tion ofbovinepapillomavirustype 1. J.Virol. 62:3143-3150. 66. Stillman B. W., J. B. Lewis, L. T. Chow, M. B. Mathews, and

J. E. Smart. 1981.Identification of thegene and mRNA for the adenovirus terminalproteinprecursor.Cell 23:497-508. 67. Thalmeier, K., H. Synovzik, R.Mertz., E.-L. Winnacker, and

M.Lipp.1989.NuclearfactorE2Fmediates basictranscription andtrans-activationbyEIA of the human myc promoter. Genes Dev. 3:527-536.

68. Wu, L., D. S. E. Rosser, M. C. Schmidt, and A. J. Berk.1987. A TATAboximplicatedin EIAtranscriptional activation of a simple adenovirus2promoter. Nature(London)326:512-515. 69. Yee, A. S.,P. Raychaudhuri,L.Jakoi,andJ.R.Nevins. 1989.

The adenovirus-inducible factor E2F stimulates transcription afterspecificDNAbinding.Mol. Cell. Biol. 9:578-585. 70. Yee, A. S., R. Reichel, I. Kovesdi, and J. R. Nevins. 1987.

Promoterinteraction of the ElA-inducibleE2Fanditspotential rolein the formationofamulti-component complex.EMBO J. 6:2061-2068.

71. Yoshinaga, S., N. Dean, M. Han, and A. J. Berk. 1986. Adeno-virus stimulation of transcription by RNA polymerase III: evidencefor anElA-dependentincrease intranscriptionfactor IIIC concentration. EMBOJ. 5:343-354.

72. Zajchowski, D. A., H. Boeuf, and C. Kedinger. 1985. The adenovirus-2early Ellatranscription unitpossessestwo over-lapping promoters with different sequence requirements for ETa-dependent stimulation. EMBOJ. 4:1293-1300.

73. Zajchowski, D. A., H. Boeuf, and C. Kedinger. 1987. Ela inducibility of the adenoviralearlyE2apromoterisdetermined by specific combinations ofsequenceelements. Gene

58:243-256.

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