Human JC virus perfect palindromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells.

JOURNAL OFVIROLOGY, Jan. 1993,p.572-576 0022-538X/93/010572-05$02.00/0

Copyright© 1993,AmericanSocietyforMicrobiology

Human

JC Virus

Perfect

Palindromic Nuclear Factor

1-Binding

Sequences Important

for

Glial

Cell-Specific Expression

in

Differentiating Embryonal Carcinoma Cells

KOTLOU.KUMAR, ALAN PATER, ANDMARY M. PATER*

BasicMedicalSciences, Faculty of Medicine, Memorial University of Newfoundland, St.John's, Newfoundland AlB 3V6, Canada

Received 16 April 1992/Accepted 22 September 1992

The brain cellspecificity of thehuman papovavirus JC viruswasexaminedby site-directedmutagenesis of thenuclearfactor 1 (NF1)motifs within the viralregulatory region. TheNF1 motifsites,located withinthe 98-bp tandem repeats that contain 6-bp perfect inverted palindromic sequences, were important for glial cell-specific expressionofJCvirus indifferentiatedembryonalcarcinoma cells in vivo.TheNF1siteonthe late

sideoftherepeats wasnotimportant, a factconfirmed byinvitro transcription studies. Theseobservations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactionsoffactors inglial cellnuclearextractswith NF1 sites.

Thehuman papovavirus JC virus (JCV) is thought to be theetiologic agentforprogressivemultifocal leukoencepha-lopathy (12, 13). This often-fatal demyelinatingdisease oc-curs in immunocompromised individuals, andJCV-positive lesions caused by this disease have recentlybeenobserved muchmore frequently because of theAIDS epidemic (17). Since JCV exhibits highlystrict host cell specificity (7)and since the activities of its regulatoryelements arelimited to glialcells(15), this virusserves as anespeciallygood model forstudyingbraincell-specificgeneregulation. JCV

regula-toryDNAcontains three nuclearfactor1 (NF1) motifs. The motifonthe lateside of thetwo98-bprepeats(NF1 I)is 3bp long,whereasthetwomotifswithin therepeats(NF1II and NF1 III) eachhave6-bp inverted palindromicrepeats. The identification of these sequences as NF1 sites is based on

sequence homology and DNase I footprinting-competition

(2, 4, 10, 11). A45-kDaprotein presentin humanfetalglial cells butnotinnonglialcells interacts withNF1II andNF1

III (9) and facilitates JCV promoter activities (the late promotermorethan the earlypromoter)innonglial cells(8) and cell extracts (1). Since the cis-acting elements in the central 98-bp repeat interact with a complexarray of pro-teins, it is notclear which oftheprotein-DNA interactions arerequired forexpression of theearlypromoter/enhancer of JCV(JCVE),which ismorestrictlybrainspecific thanthe late promoter/enhancer (17). Here, we have examined in detail thesequences important for glial cell-specific expres-sion ofJCVE. Site-directed mutagenesisand P19embryonal carcinoma cells that can be differentiated into mixtures of oligodendrocytes and astroglial cells (14) were used to ex-amine the roleofNFl-binding sequences. The perfect pal-indromicsequencesNF1IIandIIIbutnotNF1Iwerefound

tobeimportant.

We havepreviously shown that efficient activityof JCVE is restrictedtoglial cells (11).Moreover, DNase I protection

assaysconfirmed thatthreecompletelyprotectedregions,all

containing NFl-binding motifs, were produced specifically for glial cell extracts (11). To determine the role of NF1 motifs in cell-specific expression of JCVE, site-directed

* Correspondingauthor.

mutagenesis of the enhancerregionwasundertaken. Muta-genesis was with a kit from Bio-Rad and the PvuII-to-HindIII fragmentfromnucleotides (nt)270to5112(Fig. 1). Mutations were generated with complementary-strand-ho-mologous oligonucleotides with appropriate base substitu-tions. Oligonucleotides from nt 235 to 206 were used for region I, from nt 165 to 137 for the pJCEcatII and pJCE-catI.IImutations ofregion II,andfromnt 152to129 for the remaining region II and III mutations. The mutationswere confirmedby sequencing, and themutated enhancer (Hin-dIl-to-Smal) fragmentswereinsertedinthecorrect orienta-tioninto theHindIIIsiteofpSVOCAT (Fig. 1; 11). Plasmids were testedfor their functional activities inP19 embryonal carcinoma cells. Undifferentiated (UD) P19 cells were dif-ferentiated into amixture ofglial cells, neurons, and astro-cytesbytreatmentwith retinoic acid (RA)and into cardiac andskeletalmusclecellsbytreatmentwithdimethyl sulfox-ide as described elsewhere (14). Chloramphenicol acetyl-transferase (CAT) assayswere asdescribed elsewhere (5), and 3-galactosidaseplasmidwasused ascontrol.

Mutation ofNF1I-andE4TF1 (11)-bindingsites in DNase I-protected regionI didnotresult insignificantalterations in CAT activity (Fig. 2). However, constructs which contain mutated GCCAnucleotidesontheright sideof theperfectly palindromic NF1 II (Fig. 2; II and 1.11) had 3.6- and 3.1-fold-lower CAT activities in glial cells. Surprisingly, these mutants had more than twofold-higher activity in differentiated musclecells(Fig. 2).Theimportance of com-binations of the three JCVNF1 sequenceswas also exam-inedwith mutations in NF1 IIplus III, I plus III, and all three sites (Fig. 1). These mutants had, respectively, 5.8-, 4.3-,and 7-fold lowerCATactivityinRA-differentiatedglial cells(Fig. 2).

The in vivo effect of NF1 mutations within the 98-bp repeaton JCVEexpression wasconfirmed byin vitro tran-scription assays (Fig. 3). While substantial activity was observed forglialcell extracts, aresidual activitywas also observed for muscle cell extracts (data not shown). No activity was observed in UD cell extract. Template with mutations of the threeNF1sitesgreatlyreduced the levels of transcript. Template containingmutationsin theNF1I motif supported wild-type levels of in vitro transcription and

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early 1 2 109 110 207 late

RTATA

NF1In

y TATA NF1 U

I

I

NF1l

I

5112 14 22 39 51 66 112 120 137 149 164 213 225 270

Plasmid NF1 HE NF1 EI NF1l

TGGCTGCCAGCCA

57 132 57 132

TGGCTGCCAGCCA 155

155 208

TGGAAAGCAGCCA 231

-a ---a - c

134 143

--- -c

134 143

--- -c -a--- -c

gta---a-c

gta---a-c

gta---a-c

gta---a-c

gta---a-c

-aa---a-c 208

-a--- -c

pRUJCEcat

(WT)

[image:2.612.102.529.47.348.2]

---pmRIJCEcat gta---a-c

FIG. 1. Wild-type and mutated NFl motifs in JCVE sequencesin CAT plasmids and DNase I-protected regions. (Top) Diagram of sequencespresentinCATplasmids. Arrows indicatesequencesfor the firstsevenconstructslisted below, and arrowheads indicatesequences

forthe lasttwo constructslistedbelow.Early and late sidesareindicated. TATA boxes and NFl site motifsareindicated and delineated by

darklines.Nucleotide positions aregivenbelow and above the lines. Boxes indicate the 98-bprepeats.(Bottom) List of mutated plasmids, mutatedsequences,and DNaseI-protectedsequencesfor the threeNF1 sites. Mutated NF1 nucleotidesareindicatedby lowercase letters. Wild-type NF1 nucleotidesareindicated in pJCEcat by capital letters and in other plasmids by dashes. Invertedpalindromicsequences are

underlined for pJCEcat. DNase I-protected sequences of Fig. 4 are indicated by overlines and nucleotides numbers. The complete

oligonucleotidesequenceprotectedfor regions NF1 II and NF1 III is5'-AGGGAT.GGCTGCCAGCCAAGCATG-3' andthat forregionNFl I is5'-GGAAGITQAAAGCAGCCAAGGGAA-3'.

pmRII

nRII* RA

pni

1.11.111

1.111

11.111

1.11

II

* DMSO

0 UD 1 2 3 4 5

I*.*

,.

,_r:

-1 2 3

.,w

I...",.

__~ *

am~~~~~~~~~~~Of 4 .:

WI.9

0 10 20 30 40 50 60 70

% CONVERSION

FIG. 2. Chart ofinvivoactivityof JCVmutantNFl sites.Results

arefor CATassaysinUD,dimethylsulfoxide(DMSO)-differentiated,

and RA-differentiated P19 embryonal carcinoma cells. NFl site mutationsareindicatedbytheuniqueletters for theplasmidsofFig.

1. The cellsweretransfected 8 h afterbeing platedwith 10,ugoftest

plasmid and 10 1LgofpUCl9DNA. Values forCATactivitywere

calculated from assays with less than 30% conversion and were

normalized withassaysfor Roussarcomaviruspromoter

3-galacto-sidaseplasmid. Resultsaretheaveragesof threeexperiments.

FIG. 3. In vitrotranscription assays. Assay productswere run on5%acrylamide gelswith molecularweightmarkers.(Left)DNA

templatesweredigestedwithNcoI. Lanes: 1, pJCEcatand75 pLgof extractsfrom P19 UDcells;2 and4, pJCEcatI.II.IIIand 60and 75

j±g,respectively, ofextractsfrom RA-differentiatedcells; 3and5, pJCEcat and 60 and 75 ,ug, respectively, of extracts from

RA-differentiated P19 cells.Tr,550-nttranscript;IC, 286-bpend-labeled

fragment servingas internal control thatwasaddedbeforephenol

extraction.(Right)DNAtemplatesweredigestedwithPvuIH.Lanes:

1, pJCEcat and incubation in the presence of 1 p.g of a-amanitin

RNApolymeraseII inhibitorperml; 2,pJCEcat; 3, pJCEcatI. Tr,

162-nttranscript. In vitro transcription assays were as described

elsewhere(1). 573

34 pJCEcat (WT)

pJCEcat I

34-pJCEcatI

36 45

____________-pJCEcat II

36 45

208

pJCEcat

.1I

pJCEcat

U.D1

pJCEcatil.E.M

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574 NOTESJ.VRL

AG F UD RA

U-m

aso aw. A..

AG F UD RA

e-i

W

a..Ro

aO aUW

a,,*& M o

K-

a-WT Jc Jo"i

JC ii 1*III Jc'.I.I"-i

FIG. 4. DNase I footprinting ofmutant JCV regulatoryelements. NFl sites mutated in the probes are indicated at the bottom and

correspondtothose ofFig.1. Forprobes,nt5112to270of JCVwild-typeand mutatedfragments (Fig. 1)werecloned into thepUC19 XbaI

site,andSall-to-Smnalfragmentswereendlabeledatthe Sail site with[a-312p]dCTP.Assaysweredoneasdescribed elsewhere (6). Lanes:

AG,chemicalcleavageofpurines;F,nonuclearextract;UD,UD-cellextract;RA,RA-differentiated cellextract.Protectedregions I,II,and IIIarebracketed. Protected sequences andbindingmotifs fortranscriptionfactorsareas described in thelegendtoFig. 1.

transcription byRNApolymeraseII(Fig. 3). These in vitro studies substantiate that NFl II and III but not NFl I are importantforefficientglialcell-specific transcriptional

activ-itYOfJCVE.

Next,thepossibilitythatoneof theperfectly palindromic

motifs in NFl II and NFl IIIwould be sufficient for glial cell-specific expression of JCVE was examined. Plasmids with single repeat regions which had either a wild-type

(pRII)ormutated(pmRII)NFl IIwereconstructed(Fig. 1). Wild-typepRIIhadthesameprofileofactivities in the three

P19 cell types as the NFl I-plus-III mutant (Fig. 2). This included a3.5-fold-lower activity, forglialcellsonly, of the

wild-type single repeat compared with the whole enhancer

construct. This lower level is very similar tothe level seen

when one of the two repeat region sites of the whole enhancer construct was mutated. The implication is that NFl Iand the otherdeleted sequences havenomajorrole in

glial cell-specific expression. The mutation of NFl II in

pmRIIfurther reducedCATactivity3.5-foldcomparedwith the effect ofwild-type pRII in glial cells. This result was

consistent with results for mutated completeenhancer

con-structs. Thus,these studies indicate that NFl II andIIIare individuallyandcooperatively important forglialcell

spec-ificity Of JCVE expression. In contrast, in differentiated muscle cells, mutant pmRII showed 2.4-fold more activity

than pRII. Also, pmRII activity was 1.9-fold higher in muscle cells than in glial P19cells, and both results are in

agreementwith observations for thefive mutations of

differ-entrepeatregion NFl siteswithin thecontext ofthe whole enhancer(Fig.2).Thesedatagivecredencetothesuggestion

in apreviousreport thatsequences including this NFl site inhibit nonglial cell expression, which was based on the inhibition by these sequences of the simian virus 40

pro-moterbasal-level activity(16).

'I [

AG F RA UD

a-O

WOW

a

a

q a

as

a.-.,

a a

Is-, is

a a

a

a

73

4.

a

a-S iF

"a

a

a-'0

S

a-C

a-S

Sr

a-I"1

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NOTES 575

A

F

UD

RA

F

UD

RA

B F

UD

RA

=F.

I

=

I

lo == eg _ = m

14

3M- _

FIG. 5. Mobility shiftassays. Assayswere done asdescribed elsewhere (3, 6) but with 5 Figofpoly(dI dC)per assay. Arrowheads indicate freeprobes. Lanesare asinFig.4.(A)Assaysusing theintact JCV enhancerprobe. Probe was asinFig. 4for wildtype.Left and rightpanels, 2.5 and5 ,ugof protein,respectively,per assay. Arrowindicatesthe specificlow-mobilityDNA-proteincomplex forRAlanes. (B) Competition assays with 5 ,ug of protein, end-labeled RII oligonucleotide probe, and 250 ng (200-fold excess) of competitor oligonucleotides. Oligonucleotides usedasprobe(RII)and as competitors after reannealing with complementaryoligonucleotideswere as follows: RI, 5'-AAGGGGAAGTGGAAAGCAGCCAA-3'; RII, 5'-TGGCTGCCAGCCAA-3'; mRII,

5'-GTAU:C]

AQACCAGCA-3'. Lanesforcompetitionassays arelabeled fortherespectiveoligonucleotide competitors.

Sincemutatingone orbothpalindromic NF1 siteswithin the98-bp repeats hadgreatlyreducedJCVE activity in vivo, we examined in vitro DNA-protein interactions with

cell-specificfactors in thecontextofmutated NF1 sites. DNase Iprotectionassayswerecarriedout(6).The NF1 I mutation eliminated protection only for region I (Fig. 4). For the

mutation on the GCCA side of NF1 II, protection was restricted to the central nucleotides of the region thatwas protected for wild-type probe and excluded the GCCA sequences. Inaddition, therewas asimilar effectonDNase I protection ofnonmutated region III, suggesting coopera-tiveinteraction of theproteins bindingtothese sites(Fig.4).

This was consistent with observing less effect on in vivo

activity bymutation of the second of the tworepeatregion

sites thanby mutation of the firstone (Fig. 2).This applies evenwhen the second site is NF1III, the downstreamsite,

which appearsto haveagreatereffect (compare WTandII with .III and L.II.III effects in Fig. 2). Probes containing

mutations inbindingmotifsonboth sides of NFl II and NF1 IIIwere completely unprotected inregions II and III. The effect ofmutatingNF1 I in additiontoNF1II orNF1 II and III was simply additive. Therefore, the effect on in vivo

activity ofNFl-binding site mutations

(Fig. 2)

was corre-lated with alterations in the in vitro interactions with

cell-specific factors as revealed by DNase I protection assays

(Fig. 4).

Tofurtherstudy the mechanism of tissue specificity, the nature ofproteins interactingwith the NF1sites, especially

theperfectlypalindromicNF1site,wasexaminedby

mobil-ityshift assays. Inaddition tocomplexesobserved forUD extracts,alow-mobilityDNA-protein complexwasdetected

onlywithglialcellextractswhenawhole

fragment

of

JCVE

was used asprobe (Fig. SA). The amount of this complex

increased with twofold-higherproteinconcentrationfor RA extracts, while amounts of othercomplexes did notchange foreither UD or RAextracts.DNA-protein binding was also examined bymobility shift assays using an oligonucleotide

for the region II NF1 motif as probe (Fig. SB). A diffuse low-mobility DNA-proteincomplexwas detected onlywith

glial cell extracts. Competition was observed only when wild-type but notregion I or mutated region II oligonucle-otidewas usedascompetitor.

Wehave examined theJCVE sequences important forglial cell-specific expression. The tissue specificities of the per-fectly palindromic NF1 sites NF1 II and III appear to be involved in both the activation ofJCVE expressionin glial

cells and the inhibition ofexpression innonglial cells. P19 cells have proventobeagood system for demonstrating this mode of tissue-specific regulation of gene expression, as they can be differentiated into different cell types with identical genotypes. The results demonstrated that of the three JCV NF1 sequences, only the perfect palindromic binding sites present in NF1 II and III are important in

brain-specific expression ofJCVE. The mechanism for

de-termining

the

specificity

ofgene

expression

is

expected

to involve protein factors. The proteins involved in glial cell

extract-specificDNaseIprotectionandmobilityshift assays (Fig. 4 and 5) might represent such factors. It has been reported that theexpressionofahuman cDNAderived from fetal glial cells and encodingthe 45-kDa protein increased

transcription from the JCV late promoter more than that from the JCV early promoter (8). It is possible that the

expressionofJCVE,which ismorestrictlybraincell

specific

than the late promoter (17;

unpublished

observations),

re-quires additional factors. Our ongoing studies should pro-VOL.67, 1993

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576 NOTES

vide information on the possible involvement of such a factor(s).

We thank S. Atkins for typing the manuscript.

This investigation was supported in part by grants awarded by the Medical Research Council and the National Cancer Institute of Canada (withfunds from theCanadianCancerSociety).

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