Journal of General Virology (2005), 86, 1261–1267 DOI 10.1099vir.0.80620-0 Correspondence
Functional interaction of Oct transcription factors with the family of repeats in Epstein–Barr virus oriP J.Almqvist,1J.Zou,1Y.Linderson,1C.Borestrom,2E.Altiok,3
H.Zetterberg,2L.Rymo,2S.Pettersson1and I.Ernberg1
Correspondence
I.Ernberg
Ingemar.Ernberg@mtc.ki.se 1Microbiology and Tumorbiology Center(MTC),Karolinska Institute,Nobels va¨g16,Box280, S-17177Stockholm,Sweden
2Department of Clinical Chemistry and Transfusion Medicine,Sahlgrenska University Hospital, S-41345Go¨teborg,Sweden
3Acibadem Genetic Diagnostic Center,Libadiye Cad,Bogazici Sitesi,Goztepe,34724Istanbul, Turkey
Received18September2004 Accepted26January2005The family of repeats(FR)is a major upstream enhancer of the Epstein–Barr virus(EBV)latent C promoter(Cp)that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1.Here,it was shown that Cp could also be activated by octamer-binding factor(Oct)proteins.Physical binding to the FR by the cellular transcription factors Oct-1and Oct-2was demonstrated by using an electrophoretic mobility-shift assay.Furthermore,Oct-1in combination with co-regulator Bob.1,or Oct-2alone,could
drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells.Cp controlled by the FR was also activated by binding of Oct-2to the FR. This may have direct implications for B cell-speci?c regulation of Cp.
INTRODUCTION
The172kb Epstein–Barr virus(EBV)genome is maintained in latently infected B cells as a circular episome and rep-lication of this episome is activated from oriP once every cell cycle in a process that involves direct binding of the EBV nuclear protein1(EBNA1)to oriP.EBNA1is essential for maintenance of the EBV genome in latently infected cells and is also expressed during latency in B cells(Chen et al., 1995).EBNA1also regulates viral transcription in latency via direct interaction with three key promoters:the latent membrane protein1(LMP1),C and Q promoters(LMP1p, Cp and Qp,respectively;Gahn&Sugden,1995;Sugden& Warren,1989;Sung et al.,1994).
EBNA1binds as a dimer to the palindromic core con-sensus16bp sequence G(A/T)TAGCATATGCTA(C/T)C, which can be found in several copies at three different sites in the EBV genome:in the dyad-symmetry element and the family of repeats(FR)in oriP upstream of Cp and downstream of Qp(Ambinder et al.,1990;Reisman& Sugden,1986;Wysokenski&Yates,1989;Yates et al.,1984). Due to sequence variation in the binding motif,EBNA1 has the strongest af?nity for the FR and the lowest for the binding sites in Qp(Ambinder et al.,1990;Rawlins et al., 1985).In the prototype B95-8virus,the FR comprises20 copies of a30bp repeat element containing the EBNA1 core-binding site and functions as an EBNA1-dependent enhancer for Cp(La¨ngle-Rouault et al.,1998;Nilsson et al., 1993;Sugden&Warren,1989).EBNA1is expressed from Cp and is also essential for transactivation of Cp(Puglielli et al.,1996;Wysokenski&Yates,1989).Full FR–EBNA1-mediated transactivation of Cp requires at least eight EBNA1-binding sites within the FR(Zetterberg et al.,2004). Several other cis-acting transcription-regulatory elements have been identi?ed in the regions upstream and down-stream of the promoter,e.g.a glucocorticoid-responsive element(Kupfer&Summers,1990),an EBNA2-responsive enhancer(Jin&Speck,1992;Sung et al.,1991)and binding sites for NF-Y,Sp1,Egr-1and members of the C/EBP transcription-factor family(Borestro¨m et al.,2003;Nilsson et al.,2001).
Here,we demonstrated that members of the octamer-binding factor(Oct)family of transcription factors could bind to and activate transcription via the FR,which is of interest in relation to viral promoter regulation in B cells and epithelial cells.
METHODS
Plasmids and PCR.The expression vectors Rc/CMV-EBNA1 (Levitskaya et al.,1995),pEV3S1.Oct-1(from W.Schaffner,Institute for Molecular Biology,University of Zurich,Switzerland),WS2 (Oct-2.6)(from T.Wirth,Department of Physiological Chemistry, University of Ulm,Germany)and pEV-OBF1(Bob.1)(from P. Matthias,Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation,Basel,Switzerland)are all regulated through cytomegalovirus(CMV)promoters.
0008-0620G2005SGM Printed in Great Britain1261 Journal of General Virology(2005),86,1261–1267DOI10.1099/vir.0.80620-0
The luciferase reporter vector containing the FR upstream of a thymidine kinase promoter(pT81luc-FR)was constructed by PCR ampli?cation of FR using B95-8DNA as template(nt7401–8044;see Fig.3b).This fragment was inserted in the pT81luc plasmid(Nordeen, 1988)by using the pGEM-T Easy Vector system(Promega)according to the manufacturer’s manual.The luciferase reporter vector p(oriPI/ 2170Cp)Luc was constructed by using the oriPI(FR)and2170Cp fragments(nt7315–8190and11166–11412,respectively)from the previously described pg(oriPI/2170Cp)CAT(Nilsson et al.,2001) (Fig.1b).The fragments were inserted into the pGL3Basic vector (Promega).All constructs were veri?ed by using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit(Applied Biosystems). Cell lines,nuclear extracts and Western blotting.DG75is an EBV-negative Burkitt’s lymphoma cell line(Ben-Bassat et al.,1977). Rael is an EBV-positive Burkitt’s lymphoma cell line with a latency I expression pattern(Klein et al.,1972).The CBMI-Ral-STO cell line was obtained by in vitro infection of cord-blood cells with virus rescued from Rael cells and had a latency III expression pattern (Ernberg et al.,1989).All lymphoid cell lines were maintained as suspension cultures in RPMI1640medium(Sigma)supplemented with10%fetal bovine serum(FBS),streptomycin and penicillin (Sigma).QBI-HEK293A cells(293A),a human embryonic kidney monolayer epithelial-cell line,were cultured in minimal essential medium(Gibco-BRL,Life Technologies)supplemented with8% FBS,penicillin and streptomycin.Nuclear extracts were prepared for electrophoretic mobility-shift assays(EMSAs)as follows.Cells were suspended in a hypotonic buffer(10mM HEPES,10mM KCl,0?1mM EDTA,0?1mM EGTA). After15min on ice,NP-40was added to a concentration of0?25%, samples were centrifuged and nuclear proteins were extracted from the pellets by using100m l20mM HEPES,25%glycerol,0?4M NaCl and1mM EDTA at4u C for15min.Supernatants were collected and kept at280u C.The protein concentration of the nuclear extracts was determined by using the Dc protein assay(Bio-Rad).Equal amounts of nuclear extract were loaded in each lane.Proteins were fractionated by SDS-PAGE(9%gel)and transferred to nitrocellulose membranes. After blocking for1h at room temperature with5%dried milk made up in PBS/0?1%Tween20,the membranes were probed overnight at4u C with the following antibodies:anti-b-actin(diluted1:5000; Sigma);anti-Oct1(Upstates),anti-Oct-2and anti-Bob.1(Santa Cruz Biotechnologies)(all diluted1:2000)and,for EBNA1,serum from an EBV-positive donor(diluted1:4000).The secondary antibody used was conjugated to horseradish peroxidase,and bound immuno-complexes were detected by enhanced chemiluminescence(Amersham Biosciences).
EMSA.EMSA was performed as follows.An oligonucleotide FR probe was end-labelled with[c-32P]dCTP and puri?ed by native PAGE(10%gel).Nuclear extracts were prepared as described above and3m g was used in each band-shift assay,in the presence of1m g poly(dI-dC),1mM Tris/HCl(pH7?5),100mM NaCl,5mM MgCl2,0?5mM dithiothreitol and2000c.p.m.probe and mixed at room temperature.Samples were loaded on to a native polyacryl-amide gel(4%)and electrophoresed at250V for1h.After drying the gel,autoradiography was performed overnight.Unlabelled oligo-nucleotides were used for competition at506molar excess.Anti-Oct-1(Upstates),anti-Oct-2,anti-Bob.1(Santa Cruz Biotechnology) and anti-Otx-1,a mAb speci?c for EBNA1(a kind gift from Dr Jaap Middeldorp,Free University of Amsterdam,the Netherlands),were used for supershifting.The antibodies were added15min before the probe and poly(dI-dC).
Transient transfections and luciferase assays.Co-transfections were performed at least three times in triplicate in293A and DG75 cells,using a constant amount of reporter plasmid[0?5m g pT81luc-FR and/or1m g p(oriPI/2170Cp)Luc]and varying amounts of expression vector for EBNA1,Oct-1,Oct-2or Bob.1.pcDNA-3was added to equalize the amount of DNA in each transfection.b-Galactosidase expression from the co-transfected pCMV-b-gal vector was used for normalization of variation in transfection ef?ciency. Another luciferase vector,RSV-luc,was used as a positive control. 293A cells were seeded in6cm plates and transfected by using FuGENE 6transfection reagent(Roche)when75%con?uent.Transfection of DG75cells was performed with~86106cells by using electropora-tion(960m F,280mV).Luciferase activity was measured in one-?fth of the whole-cell extract at48h post-transfection by using a luciferase assay system(Promega).Control transfections using an expression vector for green?uorescent protein showed that approximately10% of the DG75cells were transfected by using this method. RESULTS AND DISCUSSION
In the FR sequence,slightly overlapping the25bp fragment that is protected in DNase-protection assays in EBV-positive cells(Rawlins et al.,1985),we found a putative Oct-binding site(Fig.1b).The Oct family of transcription factors was originally identi?ed as transcriptional regulators when bound to the octamer motif ATGCAAAT.Oct-1is expressed ubiquitously in all types of tissues(Mu¨ller et al.,1988; Sturm et al.,1988).It interacts with a variety of other
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Fig.1.Schematic illustration of the oriP region upstream of
Cp.(a)Schematic illustration of the region upstream of Cp.
Sequence coordinates are from the DNA sequence of the
B95-8EBV genome.The arrow indicates the Cp transcription
start site at nt11336.(b)About20repeated motifs,of30bases
each,constitute the FR.The sequence of several of these repeats
is shown,illustrating some of the sequence variation in the FR.
Each repeat contains a core EBNA1-binding element(shaded).
The motif resembling octamer-binding sites is indicated by an
open box or is underlined on the complementary strand.
(c)Detailed map of promoter-proximal transcriptional elements
present in p(oriPI/”170Cp)Luc.Numbers are positions in relation
to the Cp transcription-initiation site(+1).
1262Journal of General Virology86 J.Almqvist and others
transcriptional regulators and activates transcription of small nuclear RNA,histone H2B and immunoglobulin genes (Das &Herr,1993;Scheidereit et al .,1987;Tanaka et al .,1988).Oct-2is tissue-speci?c for neuronal cells and B cells.It is involved in activating transcription from promoters of the immunoglobulin genes (Scheidereit et al .,1987).The primary Oct-2RNA transcript is subject to alternative splicing,resulting in at least eight different isoforms of the protein (Lillycrop &Latchman,1992;Liu et al .,1995;Wirth et al .,1991).The splicing pattern differs between B cells and neurons.Oct-2.1and Oct-2.2are most prevalent in B cells,whereas Oct-2.4and Oct-2.5are the predominant isoforms in neuronal cells.For overexpression of Oct-2,we used a cloned cDNA of Oct-2.6,which is expressed in B cells and has a splicing pattern similar to those of Oct-2.1and Oct-2.2(Wirth et al .,1991).Two methods were used to demonstrate the interaction of Oct proteins with the FR:EMSA and a luciferase reporter assay.We performed an EMSA to see whether an FR-derived probe (Fig.3b)could form complexes with proteins extracted from the EBV-positive Burkitt’s lymphoma cell line Rael.The FR probe formed several different protein complexes that were FR-speci?c (Fig.2).An excess of unlabelled FR fragment competed ef?ciently with these complexes (not shown).Complex I (cI),cIII and part of cIV were Oct-speci?c (Fig.2,OCTA),which was further con?rmed by antibody supershifting.cI was shown to con-tain Oct-1,whilst cII contained EBNA1and low amounts of Oct-1and Oct-2,and cIII and cIV contained Oct-2.Antibodies against the B cell-speci?c Oct cofactor Bob.1(also known as OBF-1and OCA-B)only resulted in a weak reduction in intensity of cI and cII.Several complexes contained more than one protein and,taken together,this suggested a possible interaction between Oct proteins,EBNA1and the FR in cis .In an attempt to further map the Oct-binding site in the FR,we divided the probe into two parts (Fig.3b)so that the putative Oct-binding site and the EBNA1-binding site were separated.As suspected,the result showed that EBNA1and the Oct proteins bound to different parts of the FR probe (Fig.3a).
To further explore this novel interaction between Oct pro-teins and the FR,we set up a model system to evaluate its effect on transcription.We constructed a luciferase reporter vector (pT81luc-FR)containing the FR upstream of a thymidine kinase promoter (Fig.4c).Our results con?rmed
previous observations (La
¨ngle-Rouault et al .,1998)that EBNA1enhanced luciferase gene expression in 293A cells and showed that our reporter-vector system worked as
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Fig.2.Oct-1,Oct-2and EBNA1form com-plexes with a probe representing a single repeat of the FR.Nuclear extracts (NE)of latency I Rael cells were used in an EMSA and complexes were supershifted by using speci?c https://www.wendangku.net/doc/c83261177.html,petition with a cold FR probe (FR)and octamer sequence (OCTA)are shown on the right.Roman numerals to the left indicate the different complexes.
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Fig.3.EMSA on nuclear extract (NE)from Rael cells.(a)EMSA of a single,full-length repeat of the FR as a probe,or probes consisting of a single repeat of the FR divided into two (Part I and Part II).Note that the Part I and Part II probes are slightly smaller than the control probe in the left-hand lane.(b)Probe of the single repeat of the FR used in the EMSA.The probe was divided into two,Part I and Part II,indicated by an arrow,for the re?ned binding analysis shown in lanes 2and 3in (a).Part I contained the EBNA1-binding site (shaded)and Part II represented a probe containing the putative Oct-binding site (open box).Asterisks indicate bases protected in the DNase-protection assay of Jones et al .(1989).
https://www.wendangku.net/doc/c83261177.html,
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expected (Fig.4a).In the same reporter system,we found that overexpression of Oct-2alone,but not Oct-1,could upregulate the FR-dependent heterologous promoter in 293A cells in the same way as EBNA1(Fig.4a).However,Oct-1could activate transcription together with the B cell-speci?c co-activator Bob.1(Fig.4a).Bob.1on its own did not induce transcription (data not shown).Thus,EBNA1enhancement of transcription through the FR could be replaced by Oct proteins.We also detected a small additive effect of co-transfection of EBNA1with Oct-2or with Oct-1plus Bob.1(Fig.4b).In the physiological setting,EBNA1and Oct proteins may cooperate in the regulation of Cp.293A cells offer a readily transfectable model cell line,although this cellular environment lacks the natural background of EBV infection in B cells.We therefore also carried out transfection of the EBV-negative human B-cell line DG75.EBNA1activated transcription more ef?ciently in DG75than in 293A cells and reached saturation with 0?5m g EBNA1vector (data not shown).Oct-1together with
Bob.1,and Oct-2alone,both activated transcription through the FR–luciferase reporter system (Fig.5).Oct-1also showed a low activation on its own without Bob.1(data not shown),probably due to the presence of endogenous Bob.1in B cells.When transfecting DG75cells with Oct-2,or Bob.1plus Oct-1,together with EBNA1,we could not detect the additive effect that we saw in the 293A system.This difference in 293A cells can most probably be attributed to the different background of endogenous regulatory factors in B cells,including Oct-2,compared with epithelial cells.Unfortunately,this reporter system could not be used easily in other EBV-carrying B-cell lines,as they showed much lower transfection ef?ciencies,and endogenous EBV genomes and EBNA1seemed to interfere with the results in a complex manner.Fig.5(b)shows the endogenous-expression levels of Oct-1,Oct-2and Bob.1in 293A and DG75cells.Control Western blots of lysates from cells transfected for the luciferase assay showed that the cooperating effect of EBNA1and the Oct proteins was indeed a result of interaction with the FR sequence (Fig.5c)
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Fig.4.Oct proteins activate transcription from an FR reporter vector.(a)Luciferase activity obtained in 293A cells co-transfected with pT81luc-FR and increasing amounts (m g)of EBNA1,Oct-2,Oct-1and/or Bob.1expression vectors as indicated.Activity is shown as fold activation of the negative-control reporter vector alone (0?5m g pT81luc-FR;?rst black bar).Transfections were done in triplicate.Error bars indicate SEM .(b)Luciferase activity obtained in 293A cells transfected with pT81luc-FR and varying amounts (m g)of expression vectors for EBNA1,Oct-2.6,Oct-1and Bob.1as indicated.(c)Luciferase reporter vector pT81luc-FR used in the transfection experiments.The FR corresponds to the region between nt 7401and 8044in the EBV genome and was inserted upstream of a thymidine kinase promoter in front of the luciferase (luc )gene.Grey bars indicate co-transfection of EBNA1and an Oct protein.
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and not due to any direct effects on the vector-expression system.It clearly showed that there was no effect of co-transfection,either on Oct-2or on EBNA1expression levels.To investigate the effect of Oct proteins on the natural EBV promoter,we used another FR–luciferase construct,p(oriPI/2170Cp)Luc,containing parts of the natural Cp of EBV.Vector (1m g)was transfected into 293A cells together with different amounts of Oct-2.6.Both EBNA1and Oct-2.6enhanced promoter activity on their own (Fig.6).
Thus,we demonstrated that Oct transcription factors can functionally interact with the major enhancer in EBV con-trol of transcription in latency,the FR.This is extremely interesting in view of the overwhelming documentation of B cell-speci?c regulation of EBV latency,involving Cp and
cellular and EBV-speci?c transcription factors (Nilsson et al .,1993;Sung et al .,1991).
Oct proteins may be involved in both activation and repression.In certain binding conformations,Oct factors cooperate with activating co-regulators,such as Bob.1,but there is also considerable evidence that Oct can recruit inhibitory molecules;for example,the neuronal forms of Oct-2can repress the tyrosine hydroxylase gene promoter (Dawson et al .,1994)and Oct-2represses the herpes sim-plex virus (HSV)immediate-early promoter in neurons (Lillycrop et al .,1991).This potentially enables Oct-dependent regulation to be involved in the switches between different forms of latency.
The sequence of the FR contains imperfect consensus octamer-binding sites.However,it is already well-established that Oct transcription factors can interact with DNA in several different con?gurations and with binding motifs that diverge extensively from the so-called octamer consensus motif (ATGCAAAT)that is found in all promoter enhancers within the IgH locus.One example is the TAATGARAT motif in HSV (Lillycrop &Latchman,1992)and another is the more recently discovered MORE and PORE sequences that can either exclude or include Bob.1in the complex (Tomilin et al .,2000).In the FR,there are three interspersed ATATAAAT motifs that best match the consensus octamer.
Oct proteins and EBNA1showed a preference for binding by themselves to the FR probe,although we also found some indication of combined binding in one of the EMSA complexes (cII).Binding of Oct and EBNA1was mapped to different ends of the FR repeat.The results of a DNase-protection assay (Jones et al .,1989),together with our results from the EMSA,suggest that there may be some
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2 m g Fig.5.(a)Luciferase activity obtained in DG75cells transfected with pT81luc-FR and varying amounts (m g)of the expression vectors for EBNA1,Oct-1,Bob.1and Oct-2.6as indicated.Error bars indicate SEM .(b)Western blot analysis comparing protein levels of Oct-2,Bob.1and Oct-1in 293A and DG75cells.The membrane was probed with protein-speci?c antibodies as indicated on the left.(c)Western blot analysis of DG75cells transfected with varying amounts (m g)of expression vectors for EBNA1and Oct-2as indicated.An increase in the amount of Oct-2vector added did not affect the amount of EBNA1expressed and EBNA1did not affect Oct-2expression.Proteins were identi?ed by using speci?c antibodies as indicated on the left.Grey bars indicate co-transfection of EBNA1and an Oct
protein.
Fig.6.Oct proteins activate transcription through a Cp-containing reporter vector.Luciferase activity obtained in 293A cells co-transfected with p(oriPI/”170Cp)Luc and increasing amounts (m g)of EBNA1and Oct-2.6expression vector,as indicated,is shown as fold activation of the negative-control reporter vector alone [1m g p(oriPI/”170Cp)Luc;?rst black bar ].Transfections were done in triplicate.Error bars indicate SEM .https://www.wendangku.net/doc/c83261177.html,
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steric hindrance in binding both EBNA1and Oct protein to a single FR repeat.Despite this,there is still the possibility that EBNA1binds to one FR repeat and the Oct protein to another.As shown by Ambinder et al.(1990),the af?nity of the EBNA1–DNA interaction,and presumably also the Oct–DNA interaction,varies detectably with the small sequence variations detected in the FR motifs.It seems that EBNA1and Oct proteins can both bind together to longer probes with two repeats,but the number of complexes then also increases and complicates the analysis(data not shown).It is interesting to note that the Oct-binding motifs in the FR that mostly resemble the consensus Oct site are in fact adjacent to variant EBNA1motifs with reduced binding af?nity for EBNA1.The relative concentrations of the proteins can also play a role in vivo.EBNA1expression is relatively low in latency I cell lines and the EBNA1signal may be drowned by higher levels of Oct proteins.In vivo, the con?guration of the entire DNA region from the FR to Cp and the multimerization of EBNA1following DNA binding is likely to confer additional conditions for recruitment of transcription factors and their co-factors.
Although the in vivo situation is more complex than our model,we propose that the biological signi?cance of our ?ndings relates to the B cell-speci?c regulation of viral latency depending on Cp. ACKNOWLEDGEMENTS
J.A.was a recipient of a fellowship from the Swedish Foundation for Strategic Research(SSF),Infection and Vaccinology PhD programme. This work was also supported by the Swedish Cancer Society,the Swedish Research Council,the Swedish Children Cancer Society and the Board for Internationalization of Research(STINT).S.P.is supported by the SSF and the Swedish Cancer Society.We are grateful for the technical assistance of Anita Westman and to Stephen Malin (MTC,now at The University of Vienna,Austria)for valuable comments.The EBNA1expression vector was kindly provided by Professor Maria Masucci,MTC,Karolinska Institutet,Sweden. REFERENCES
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Oct regulation through EBV oriP