1 Microbiology and Tumorbiology Center (MTC), Karolinska Institute, Nobels väg 16, Box 280, S-171 77 Stockholm, Sweden
2 Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden
3 Acibadem Genetic Diagnostic Center, Libadiye Cad, Bogazici Sitesi, Goztepe, 34724 Istanbul, Turkey
Correspondence
I. Ernberg
Ingemar.Ernberg{at}mtc.ki.se
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ABSTRACT |
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INTRODUCTION |
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EBNA1 binds as a dimer to the palindromic core consensus 16 bp 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 affinity for the FR and the lowest for the binding sites in Qp (Ambinder et al., 1990
; Rawlins et al., 1985
). In the prototype B95-8 virus, the FR comprises 20 copies of a 30 bp repeat element containing the EBNA1 core-binding site and functions as an EBNA1-dependent enhancer for Cp (Längle-Rouault et al., 1998
; Nilsson et al., 1993
; Sugden & Warren, 1989
). EBNA1 is expressed from Cp and is also essential for transactivation of Cp (Puglielli et al., 1996
; Wysokenski & Yates, 1989
). Full FREBNA1-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 identified in the regions upstream and downstream 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-1 and members of the C/EBP transcription-factor family (Boreström 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.
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METHODS |
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The luciferase reporter vector containing the FR upstream of a thymidine kinase promoter (pT81luc-FR) was constructed by PCR amplification of FR using B95-8 DNA as template (nt 74018044; 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/170Cp)Luc was constructed by using the oriPI (FR) and 170Cp fragments (nt 73158190 and 1116611412, respectively) from the previously described pg(oriPI/170Cp)CAT (Nilsson et al., 2001
) (Fig. 1b
). The fragments were inserted into the pGL3Basic vector (Promega). All constructs were verified by using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems).
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Nuclear extracts were prepared for electrophoretic mobility-shift assays (EMSAs) as follows. Cells were suspended in a hypotonic buffer (10 mM HEPES, 10 mM KCl, 0·1 mM EDTA, 0·1 mM EGTA). After 15 min on ice, NP-40 was added to a concentration of 0·25 %, samples were centrifuged and nuclear proteins were extracted from the pellets by using 100 µl 20 mM HEPES, 25 % glycerol, 0·4 M NaCl and 1 mM EDTA at 4 °C for 15 min. Supernatants were collected and kept at 80 °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 for 1 h at room temperature with 5 % dried milk made up in PBS/0·1 % Tween 20, the membranes were probed overnight at 4 °C with the following antibodies: anti--actin (diluted 1 : 5000; Sigma); anti-Oct1 (Upstates), anti-Oct-2 and anti-Bob.1 (Santa Cruz Biotechnologies) (all diluted 1 : 2000) and, for EBNA1, serum from an EBV-positive donor (diluted 1 : 4000). The secondary antibody used was conjugated to horseradish peroxidase, and bound immunocomplexes were detected by enhanced chemiluminescence (Amersham Biosciences).
EMSA.
EMSA was performed as follows. An oligonucleotide FR probe was end-labelled with [-32P]dCTP and purified by native PAGE (10 % gel). Nuclear extracts were prepared as described above and 3 µg was used in each band-shift assay, in the presence of 1 µg poly(dI-dC), 1 mM Tris/HCl (pH 7·5), 100 mM NaCl, 5 mM MgCl2, 0·5 mM dithiothreitol and 2000 c.p.m. probe and mixed at room temperature. Samples were loaded on to a native polyacrylamide gel (4 %) and electrophoresed at 250 V for 1 h. After drying the gel, autoradiography was performed overnight. Unlabelled oligonucleotides were used for competition at 50x molar excess. Anti-Oct-1 (Upstates), anti-Oct-2, anti-Bob.1 (Santa Cruz Biotechnology) and anti-Otx-1, a mAb specific for EBNA1 (a kind gift from Dr Jaap Middeldorp, Free University of Amsterdam, the Netherlands), were used for supershifting. The antibodies were added 15 min before the probe and poly(dI-dC).
Transient transfections and luciferase assays.
Co-transfections were performed at least three times in triplicate in 293A and DG75 cells, using a constant amount of reporter plasmid [0·5 µg pT81luc-FR and/or 1 µg p(oriPI/170Cp)Luc] and varying amounts of expression vector for EBNA1, Oct-1, Oct-2 or Bob.1. pcDNA-3 was added to equalize the amount of DNA in each transfection. -Galactosidase expression from the co-transfected pCMV-
-gal vector was used for normalization of variation in transfection efficiency. Another luciferase vector, RSV-luc, was used as a positive control.
293A cells were seeded in 6 cm plates and transfected by using FuGENE 6 transfection reagent (Roche) when 75 % confluent. Transfection of DG75 cells was performed with 8x106 cells by using electroporation (960 µF, 280 mV). Luciferase activity was measured in one-fifth of the whole-cell extract at 48 h post-transfection by using a luciferase assay system (Promega). Control transfections using an expression vector for green fluorescent protein showed that approximately 10 % of the DG75 cells were transfected by using this method.
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RESULTS AND DISCUSSION |
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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-specific (Fig. 2
). An excess of unlabelled FR fragment competed efficiently with these complexes (not shown). Complex I (cI), cIII and part of cIV were Oct-specific (Fig. 2
, OCTA), which was further confirmed by antibody supershifting. cI was shown to contain Oct-1, whilst cII contained EBNA1 and low amounts of Oct-1 and Oct-2, and cIII and cIV contained Oct-2. Antibodies against the B cell-specific Oct cofactor Bob.1 (also known as OBF-1 and 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, EBNA1 and 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 EBNA1 and the Oct proteins bound to different parts of the FR probe (Fig. 3a
).
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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-2 can repress the tyrosine hydroxylase gene promoter (Dawson et al., 1994) and Oct-2 represses the herpes simplex 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 configurations 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.1 in the complex (Tomilin et al., 2000
). In the FR, there are three interspersed ATATAAAT motifs that best match the consensus octamer.
Oct proteins and EBNA1 showed 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 EBNA1 was 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 steric hindrance in binding both EBNA1 and Oct protein to a single FR repeat. Despite this, there is still the possibility that EBNA1 binds to one FR repeat and the Oct protein to another. As shown by Ambinder et al. (1990)
, the affinity of the EBNA1DNA interaction, and presumably also the OctDNA interaction, varies detectably with the small sequence variations detected in the FR motifs. It seems that EBNA1 and 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 EBNA1 motifs with reduced binding affinity for EBNA1. The relative concentrations of the proteins can also play a role in vivo. EBNA1 expression is relatively low in latency I cell lines and the EBNA1 signal may be drowned by higher levels of Oct proteins. In vivo, the configuration of the entire DNA region from the FR to Cp and the multimerization of EBNA1 following 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 significance of our findings relates to the B cell-specific regulation of viral latency depending on Cp.
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ACKNOWLEDGEMENTS |
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Received 18 September 2004;
accepted 26 January 2005.
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