Functional Interaction of Nuclear Factor Y and ... - Journal of Virology

JOURNAL OF VIROLOGY, Jan. 2003, p. 821–829 0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.2.821–829.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 2

Functional Interaction of Nuclear Factor Y and Sp1 Is Required for Activation of the Epstein-Barr Virus C Promoter Cecilia Borestro ¨m, Henrik Zetterberg, Kristian Liff, and Lars Rymo* Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Go ¨teborg University, S-413 45 Gothenburg, Sweden Received 24 June 2002/Accepted 14 October 2002

Two Epstein-Barr virus (EBV) latent cycle promoters, Wp and Cp, are activated sequentially during virus-induced transformation of primary B lymphocytes. Immediately postinfection, viral transcription initiates from Wp, leading to expression of EBV nuclear antigen 2 (EBNA2) and EBNA5. Within 36 h, there is a switch in promoter usage from Wp to the upstream Cp, which leads to expression of EBNA1 to EBNA6. EBNA2 appears to be required for the Wp-to-Cp switch, but the switching mechanism is not fully understood at the molecular level. In a previous investigation we showed that there is an EBNA2-independent activity of reporter constructs containing deletion fragments of Cp in B-lymphoid cell lines, and we demonstrated that Cp activity is highly dependent on several cellular transcription factors, including nuclear factor Y (NF-Y) and Sp1. In the present work, we analyzed the effect of NF-Y on Cp activity in greater detail. We demonstrate that (i) a dominant negative analogue of NF-Y abolishes Cp activity, (ii) NF-Y and Sp1 costimulate Cp, and (iii) the oriPI-EBNA1-induced transactivation of Cp requires concomitant expression of NF-Y and Sp1, although additional factors seem necessary for optimal activation. Furthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2 function is regulated by estrogen, we demonstrate that inactivation of EBNA2 results in decreased expression of NF-Y and down-regulation of Cp. On reconstitution of the EBNA2 function, the cells enter the cell cycle, NF-Y levels increase, and a concomitant Wp-to-Cp switch occurs. Taken together, our results suggest that NF-Y is essential for Cp activation and that up-regulation of NF-Y may contribute to a successful Wp-to-Cp switch during B-cell transformation. lines (LCLs), where all six nuclear antigens (EBNA1 through EBNA6) (6) as well as all three LMPs are expressed (latency III) (18). After in vitro infection of B lymphocytes, transcription is initiated from the W promoter (Wp) in the BamHI W repeat region of the EBV genome, leading to the expression of EBNA2 and EBNA5 (1, 2, 49, 56). Within 36 h there is a switch in promoter usage from Wp to the upstream Cp in the BamHI C region, leading to the expression of EBNA1 through EBNA6 (57). The mechanism behind this switch is not fully understood at the molecular level, but EBNA2 appears to play an important role (61). The regulation of Cp has been the subject of several investigations, and a number of positive cis-acting transcription regulatory elements for Cp activity have been identified in the regions up- and downstream of the promoter (Fig. 1) (19, 28, 40, 43–45, 53, 60). EBNA1 homodimers activate Cp by binding to a subelement of the latency origin of replication (oriPI), which functions as an EBNA1-dependent enhancer of Cp (45, 52). In a previous investigation, we showed that Cp activation is highly dependent on several promoter-proximal regulatory elements (41). A central finding was the identification of a GC-rich sequence in the ⫺99/⫺91 Cp region, which contains overlapping binding sites for Sp1 and Egr-1. This region is essential for Cp activity, and our results suggest that Sp1 is a positive regulator and Egr-1 is a negative regulator. Moreover, we demonstrated that the NF-Y transcription factor interacts with the previously identified CCAAT box in the ⫺71/⫺63 Cp region (44), which appears to be essential for promoter activity (41). NF-Y, also referred to as CP1 or CBF, is a ubiquitous mul-

Epstein-Barr virus (EBV) is an exclusively human lymphotropic herpesvirus that infects more than 90% of the population worldwide (46). The virus is the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disorder (39), and is additionally associated with various malignancies including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and lymphoproliferative syndromes in immunocompromised individuals (22). However, most immunocompetent individuals harbor the virus for life within latently infected resting memory B-cells; this latent infection causes no symptoms (25, 54). In vitro, EBV efficiently transforms resting B cells into activated lymphoblasts. These perpetually dividing cells express a repertoire of viral antigens (EBNA1-6 and LMP1), all of which have been directly implicated in the immortalization process (22). EBV can adopt four distinct programs of latency (latency 0, I, II, and III), in which different combinations of the EBV gene products (EBV nuclear antigen 1 [EBNA1] through EBNA6 and latent membrane protein 1 [LMP1], LMP2A, and LMP2B) are detected. The various combinations range from latent EBV infection in healthy individuals, where only LMP2A is consistently detectable (latency 0) (37, 38, 54), to the cells of biopsy specimens from Burkitt’s lymphoma patients where only EBNA1 is expressed (latency I) (46, 54), and further to acute infectious mononucleosis, lymphoproliferative syndromes in immunocompromised individuals, and lymphoblastoid cell * Corresponding author. Mailing address: Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden. Phone: 46 31 342 40 80. Fax: 46 31 82 84 58. E-mail: [email protected]. 821

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FIG. 1. Sequences involved in the regulation of the EBV Cp. (A) Schematic illustration of the region upstream of Cp. The sequence coordinates are from the DNA sequence of the B95-8 EBV genome (3). The bent arrow indicates the Cp transcription start site at position 11336. Boxes indicate the presence of previously identified cis-acting elements, including oriP, which is composed of oriPI and oriPII, a glucocorticoid-responsive element (GRE), and an EBNA2-responsive enhancer (E2RE). (B) Fragments of the region upstream of Cp present in different CAT reporter plasmids. (C) Detailed map of previously identified promoter-proximal transcriptional elements (41) and their relationships to fragments present in CAT reporter plasmids used in this study. Open boxes indicate transcriptional elements. Numbers in panel B are positions in relation to the Cp transcription initiation site (⫹1).

timeric protein that is composed of three highly conserved subunits, NF-YA, NF-YB, and NF-YC (4, 15, 51), all required for DNA binding (51). This complex is thought to play a general role in transcriptional regulation by binding to the CCAAT motif found in many eukaryotic genes, leading to the recruitment of upstream DNA-binding transcription factors to the proximal promoter complex (36, 58). Recently, a physical interaction between NF-YA and Sp1 was demonstrated (30, 48). The present report describes a functional analysis of the importance of the NF-Y transcription factor for Cp activity in B-lymphoid cell lines of different phenotypes. We also investigate the putative cooperation of NF-Y and Sp1 in Cp activation, as well as the role of NF-Y in the switch from Wp to Cp usage. MATERIALS AND METHODS Plasmids. The expression vector for dominant negative NF-YA, dnNF-YA, was kindly provided by R. Mantovani (Universita` di Milano, Milan, Italy). Its empty vector was engineered by excision of the dnNF-YA exon with endonucleases EcoRI and BglII. The pCI-EBNA1 expression plasmid was constructed by PCR amplification of a cloned BamHI K fragment of the EBV genome (nucleotides 107565 to 110429), which was inserted into the SmaI site of the pCI vector (Promega, Madison, Wis.) by a blunt-end ligation. The pPac expression vectors for the NF-Y subunits A, B, and C were generously provided by T. Osborne (University of California, Irvine, Calif.). The pPacSp1 expression vector and its parent pPac were kindly provided by G. Suske (Klinikum der Philipps-Universita¨t Marburg, Marburg, Germany). The pPac expression vector for EBNA1, pPacEBNA1, and the plasmid pgSV40CAT, which carries the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the simian virus 40 (SV40) early promoter, have been described previously (41). The series of CAT reporter plasmids with different 5⬘ deletions of Cp with or without oriPI, pgCp(⫺170)CAT, pgCp(⫺1024)CAT, pgCp(oriPI/⫺170)CAT pgCp(oriPI/ ⫺1024)CAT, and pgCp(⫺3889)CAT, has been described previously (41). oriPI is essential for significant Cp activity in group III phenotype cells (40, 44). Furthermore, in combination with presynthesized EBNA1, this sequence enhances the nuclear importation of plasmids on transfection, partly due to the strong nuclear localization signal of EBNA1 (29). To overcome this difference in nuclear importation, when transfecting reporter plasmids containing oriPI into

J. VIROL. EBNA1-expressing cells, this sequence was also inserted in the expression vector for dnNF-YA and its empty vector. This was achieved by excision of the oriPI fragment from the previously described pgCp(oriPI/⫺111)CAT (41), with endonuclease PstI and insertion of the fragment into the NdeI-linearized expression vector for dnNF-YA and its empty vector by a blunt-end ligation. Cell culture, transient transfections, and CAT assay. DG75 is an EBV-negative BL cell line (5). Rael is an EBV-positive BL cell line with a latency I expression pattern (26). The cbc-Rael line was obtained by in vitro infection of cord blood cells with the Rael virus strain and has a latency III expression pattern (11). The IB4 line was derived by transforming human placental lymphocytes with the B95-8 strain and has a latency III expression pattern (24). EREB2–5 is a transformed LCL expressing a conditional mutant of EBNA2 (ER-EBNA2) that requires 1 ␮M estrogen in the medium for the activation of the EBNA2 function (21). It was established by infection of primary B cells with the EBNA2deficient P3HR-1 virus strain complemented with an EBNA2-estrogen receptor fusion construct. Estrogen withdrawal leads to inactivation of EBNA2 and entrance of the cells into the resting phase of the cell cycle. All lymphoid cell lines were maintained as suspension cultures in RPMI 1640 (Gibco, Life Technologies Inc., Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco, Life Technologies Inc.), streptomycin, and penicillin. For estrogen withdrawal, EREB2-5 cells were washed three times with and resuspended in complete medium without estrogen. Schneider’s Drosophila line 2 (SL2) is a Drosophila cell line that expresses neither the Sp factors (8) nor the NF-Y transcription factors (31) endogenously. The SL2 cells were grown in room temperature in Schneider’s Drosophila medium (Gibco, Life Technologies Inc.) supplemented with 10% fetal calf serum, streptomycin, and penicillin. All cell lines except SL2 were transfected by electroporation with a Gene Pulser system (Bio-Rad Laboratories, Hercules, Calif.) (42). Transfections were performed with 5 ⫻ 106 DG75, Rael, and IB4 cells and 7 ⫻ 106 cbc-Rael cells, with constant amounts of reporter plasmid (1 to 10 ␮g) and increasing amounts of the expression vector for dnNF-YA. The empty vector for dnNF-YA was added to keep the total amount of DNA constant in all transfections. At 3 days posttransfection, the cells were harvested. Cell extracts were prepared by three rounds of freezing and thawing and analyzed for CAT activity as described by Ricksten et al. (47). Storage phosphor screens were exposed and scanned in a PhosphorImager or Typhoon 9200 (Molecular Dynamics, Sunnyvale, Calif.), and the signals were quantified using ImageQuant software (Molecular Dynamics). SL2 cells were transfected using the calcium phosphate-DNA precipitation method (9). A total of 4 ⫻ 106 SL2 cells were plated in 60-mm dishes 24 h prior to transfection. The cells were cotransfected with 10 ␮g of different CAT reporter constructs and different amounts and combinations of pPacSp1, pPacEBNA1, pPacNF-Y-A/B/C, and the parental vector pPac. The total amount of expression vector was adjusted with the parental empty vector, pPac. Cells were harvested 2 days posttransfection. Cell extracts were prepared and CAT activity was analyzed as described above. Isolation of B lymphocytes. EBV-negative B lymphocytes were purified from buffy coat from an EBV-negative donor, using the B-cell negative isolation kit (Dynal Biotech ASA, Oslo, Norway). The final B-cell preparation was 70% pure, as determined by flow cytometric analysis in a FACScan apparatus (BD Biosciences, San Jose, Calif.) using phycoerythrin-conjugated anti-CD19, antiCD14, and anti-CD45 monoclonal antibodies (BD Biosciences). Immunoblot analysis. EREB2-5 cells, both resting and proliferating, were collected by centrifugation and washed with phosphate-buffered saline (PBS) (180 mM NaCl, 3.6 mM KCl, 11 mM Na2HPO4, 2.0 mM KH2PO4). Cell pellets were frozen in liquid nitrogen and stored at ⫺80°C. The cells were lysed by sonication in lysis buffer (1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl [pH 8.0], protease inhibitor cocktail [Complete EDTA-free; Roche, Bromma, Sweden]) and cleared by centrifugation. The protein concentration of the lysates was determined (Bradford protein assay; Bio-Rad), and the total amount of protein was standardized by dilution with lysis buffer. The protein extracts (50 ␮g/sample) were separated on 10% Bis-Tris sodium dodecyl sulfatepolyacrylamide gels (Invitrogen, Paisley, United Kingdom) and blotted to nitrocellulose membranes (Hybond C-extra; Amersham Biosciences, Uppsala, Sweden). The membranes were blocked with 5% nonfat dry milk in PBS and incubated overnight at 4°C with mouse anti-NF-YA antibody diluted 1:1,000 (Pharmingen, San Diego, Calif.) or goat anti-actin antibody diluted 1:1,000 (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) in PBS–0.5% nonfat dry milk. After repeated washings in PBS containing 0.1% Tween 20, the membranes were incubated for 1.5 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Bio-Rad) diluted 1:10,000 or HRP-conjugated rabbit anti-goat antibody (Bio-Rad) diluted 1:10,000. The membranes were washed in PBS containing 0.1% Tween 20, and the proteins were visualized by enhanced chemiluminescence procedures (Phototope-HRP Western blot detection system), as described by the manufacturer of the reagents

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NF-Y AND Sp1 COREGULATE THE EBV C PROMOTER

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FIG. 2. oriPI-EBNA1-independent Cp activity is inhibited by a dominant negative analogue of NF-YA. The reporter plasmid pgCp(⫺1024)CAT was cotransfected with increasing amounts of expression vector for dnNF-YA into EBV-negative DG75 cells (A) and group I phenotype Rael cells (B). An identical control series was performed using the reporter plasmid pgSV40CAT. CAT activities are percentages of the activity obtained with pgCp(⫺1024)CAT and pgSV40CAT, respectively, cotransfected with the empty vector for dnNF-YA alone. The 100% values in panel A correspond to 200% conversion of substrate for pgCp(⫺1024)CAT and 200% for pgSV40CAT (measured values times dilution factor), and the background level was 2.6%. The 100% values in panel B correspond to 35% conversion of substrate for pgCp(⫺1024)CAT and 33% for pgSV40CAT, and the background level was 0.5%. The values in panels A and B are means of three and five independent transfection series, respectively. Error bars indicate standard errors of the means.

(Cell Signalling Technology, Beverly, Mass.). To verify a dose-dependent increase in EBNA1 expression in SL2 cells transfected with pPacEBNA1, immunoblot analysis of whole-cell extracts was performed as described above. Polyclonal human serum CW with high titers of antibody to EBNA1, diluted 1:50, and alkaline phosphatase-conjugated secondary rabbit anti-human antibody (Dako, Glostrup, Denmark), diluted 1:1,000, were used. RNA analysis. Total cellular RNA was isolated from EREB2-5 cells by using TRI REAGENT (Sigma, Steinheim, Germany), treated with RQ1 DNase (Promega), and stored at ⫺80°C. S1 endonuclease protection assays of the Cp and Wp transcripts were carried out as described previously (40). Briefly, transcription initiated at Wp or Cp was determined by using single-stranded oligonucleotides corresponding to nucleotides 11312 to 11370 (oligo E-125) and 14366 to 14410 (oligo E-126), respectively, of the L strand of B95-8 EBV DNA. A nonhomologous tail of 6 nucleotides was added at the 3⬘ end of the oligonucleotides, in order to distinguish incomplete digestion from protection of the probe by transcripts initiated further upstream. RNA samples and 50 to 100 fmol 32P-endlabeled oligonucleotides were heated to 85°C for 10 min and incubated at 37°C for 18 h for hybridization. Subsequently, the hybridization mixture was treated with S1 endonuclease (750 U/ml) at 22°C for 1 h, and this was followed by inactivation and ethanol precipitation. Protected fragments were fractionated by electrophoresis in a denaturing 12% polyacrylamide gel. Gels were dried, and signals were detected by exposure of storage phosphor screens, which were scanned in a Typhoon 9200. The expected sizes of fragments protected by RNA initiated at Cp and Wp are 35 and 30 nucleotides, respectively.

RESULTS A dominant negative analogue of NF-YA abolishes the activity of Cp. To evaluate the functional significance of the heteromeric NF-Y protein for Cp activity, we cotransfected a reporter plasmid, carrying the CAT reporter gene under the control of the Cp, pgCp(⫺1024)CAT, with increasing amounts of a plasmid that expresses a mutant form of the NF-YA subunit (dnNF-YA) in EBV-negative DG75 cells and in group I phenotype Rael cells. It is known that the NF-YA mutant inhibits NF-Y-dependent transcription by sequestering the NF-YB and NF-YC subunits in defective complexes, which are unable to bind DNA. The mutant thus functions as a dominant negative transcription factor (34). In all experiments, the basal Cp activity (100%) was assessed by cotransfecting the empty

vector for dnNF-YA with pgCp(⫺1024)CAT. Analysis of CAT activity demonstrated that overexpression of dnNF-YA completely abrogated Cp activity and that this effect was dose dependent (Fig. 2). Control experiments were performed to verify the specificity of the dominant negative effect of dnNFYA. Hence, a reporter gene driven by the NF-Y-independent SV40 early promoter was also cotransfected with increasing amounts of expression plasmid for dnNF-YA (Fig. 2). The overexpression of dnNF-YA did not decrease SV40 early promoter activity, confirming that dnNF-YA specifically downregulates NF-Y-dependent promoters. To investigate the role of NF-Y in oriPI-EBNA1-dependent activation of Cp, a similar series of experiments was performed. Plasmids containing the oriPI sequence benefit from increased nuclear import in EBNA1-expressing cells, partly due to the strong nuclear localization signal of EBNA1 (29). Thus, to ensure equal nuclear import, all plasmids used in the transfection series presented in Fig. 3 contained the oriPI sequence of the EBV genome. Transfections were executed in DG75 cells simultaneously cotransfected with pCI-EBNA1 (Fig. 3A), group I phenotype Rael cells (Fig. 3B), group III phenotype cbc-Rael (Fig. 3C), and IB4 cells (Fig. 3D), using pgCp(oriPI/⫺1024)CAT and pgCp(⫺3889)CAT reporter plasmids, together with increasing amounts of the oriPI-containing expression vector for dnNF-YA. Identical control series were performed using the reporter plasmid pgSV40CAT. These experiments confirm our results that Cp activity is dependent on NF-Y and extend them by showing that dnNF-YA also abolished oriPI-EBNA1-dependent Cp activity without having a significant effect on the NF-Y-independent SV40 early promoter. Functional interaction of NF-Y and Sp1 is required for activation of Cp. Cooperative interactions among transcription factors are important for the regulation of a number of gene promoters (30, 31, 48). We have previously shown that Cp

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FIG. 3. oriPI-EBNA1-dependent Cp activity is inhibited by a dominant negative analogue of NF-YA. The reporter plasmids pgCp(oriPI/ ⫺1024)CAT and pgCp(⫺3889)CAT were cotransfected with increasing amounts of expression vector for dnNF-YA into EBV-negative DG75 cells simultaneously cotransfected with 1 ␮g of pCI-EBNA1 (A), group I phenotype Rael cells (B), group III phenotype cbc-Rael cells (C), and group III phenotype IB4 cells (D). Identical control series were performed using the reporter plasmid pgSV40CAT. CAT activities are percentages of the activity obtained with pgCp(oriPI/⫺1024)CAT, pgCp(⫺3889)CAT, and pgSV40CAT, cotransfected with the empty vector for dnNF-YA alone. (A) The 100% values correspond to 210% conversion of substrate for pgCp(⫺3889)CAT, 130% for pgCp(oriPI/⫺1024)CAT, and 240% for pgSV40CAT (measured values multiplied by dilution factor), and the background level was 7%. (B) The 100% values correspond to 38% for pgCp(⫺3889)CAT, 53% for pgCp(oriPI/⫺1024)CAT, and 49% for pgSV40CAT, and the background level was 0.7%. (C) The 100% values correspond to 5% for pgCp(⫺3889)CAT, 76% for pgCp(oriPI/⫺1024)CAT (measured value multiplied by dilution factor), and 4% for pgSV40CAT, and the background level was 0.5%. (D) The 100% values correspond to 4% for pgCp(⫺3889)CAT, 92% for pgCp(oriPI/ ⫺1024)CAT (measured values multiplied by dilution factor), and 39% for pgSV40CAT and the background level was 1%. The values are means of three independent transfection series. Error bars indicate standard errors of the means. The differences in 100% values between different cell lines are to a large extent due to different transfection efficiencies, as described previously (41). The group III phenotype cbc-Rael and IB4 cell lines display the lowest transfection efficiency, and the group I phenotype Rael and the EBV-negative DG75 cell lines display the highest. It should also be noted that the 100% values in Fig. 2A and 3A cannot be used for estimation of EBNA1-induced transactivation of Cp, since different amounts of reporter plasmid were used in the two experiments.

activation is dependent on promoter-proximal Sp sites and the NF-Y-binding CCAAT box (41). To determine whether Sp1 cooperates with NF-Y at Cp, pgCp(oriPI/⫺170)CAT, pgCp(oriPI/⫺1024)CAT, pgCp(⫺3889)CAT, and pgCp(⫺1024)CAT were cotransfected with Drosophila expression vectors encoding Sp1 and the three NF-Y subunits into SL2 cells (Fig. 4). These cells lack endogenous expression of Sp1 and NF-Y. Whereas expression of either Sp1 or NF-Y activated Cp to a low degree, simultaneous expression of both factors resulted in a dramatic increase in promoter activity, independently of EBNA1. These results implied that Sp1 and NF-Y act synergistically to drive transcription from Cp. To study the requirement of Sp1 and NF-Y for the EBNA1-mediated

up-regulation of Cp, a Drosophila expression vector for EBNA1 was included in the experiments shown in Fig. 4. EBNA1 alone or together with only one of the factors failed to activate an oriPI-containing Cp reporter construct. However, EBNA1 in conjunction with both Sp1 and NF-Y resulted in a significant increase in activation when the pgCp(oriPI/⫺170)CAT and pgCp(oriPI/⫺1024)CAT reporter plasmids were used, in contrast to the effect of simultaneous expression of only Sp1 and NF-Y. As expected pgCp(⫺1024)CAT, which lacks oriPI, was not further activated by EBNA1; unexpectedly, neither was pgCp(⫺3889)CAT. The latter result could be due to the relatively high NF-Y- and Sp1-induced activation of this

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FIG. 4. Functional interaction of NF-Y and Sp1 is required for activation of Cp. To determine whether NF-Y cooperates with Sp1 and EBNA1 at Cp, the reporter plasmids pgCp(oriPI/⫺170)CAT, pgCp(oriPI/⫺1024)CAT, pgCp(⫺3889)CAT, and pgCp(⫺1024)CAT were cotransfected into SL2 cells with Drosophila expression vectors encoding the three NF-Y subunits, Sp1, and EBNA1 (pPacNF-YA, pPacNFYB, pPacNF-YC, pPacSp1, and pPacEBNA1). Column 1 represents relative CAT activities of reporter plasmids cotransfected with the empty pPac vector alone. The 100% values correspond to 50% conversion of substrate for pgCp(oriPI/⫺170)CAT, 100% for pgCp(oriPI/ ⫺1024)CAT, 260% for pgCp(⫺3889)CAT, and 110% for pgCp(⫺1024)CAT (measured values multiplied by dilution factor). The values are means of three independent transfection series. Error bars indicate standard errors of the means.

construct (260%, compared to 50, 100, and 110% of the other constructs [Fig. 4]). The low transactivation effect of EBNA1 in SL2 cells could reflect a low level of expression of EBNA1 from the pPac plasmid. To address this question, a titration of EBNA1 levels was performed on the basis of a constant amount of Drosophila expression plasmids for Sp1 and the three NF-Y subunits (Fig. 5). A dose-dependent increase in EBNA1 expression was confirmed by immunoblot analysis (data not shown). A two- to fourfold increase in activity was measured in all reporter constructs, pgCp(⫺3889)CAT included, when the amount of EBNA1 expression plasmid was increased in several steps. In conclusion, a functional interaction of NF-Y and Sp1 seems to be necessary for the Cp transactivation function of oriPI-EBNA1. However, the transactivation efficiency of EBNA1 in SL2 cells was low compared to the results of earlier investigations in EBVnegative cells (29, 52), indicating that additional factors, not expressed in SL2 cells, may be important for efficient oriPIEBNA1-induced Cp activation. The expression of NF-YA is low in resting and high in proliferating B-lymphoid cells. NF-YA levels vary in certain cell types during the cell cycle (33). To investigate if NF-Y contributes to the Wp-to-Cp switch in the initial stages of transformation of resting B cells, we used the EREB2-5 cell line (21). These cells carry the EBNA2-deleted virus strain P3HR-1 and a plasmid that expresses an EBNA2-estrogen receptor fusion protein. This protein renders EBNA2 function dependent on the presence of estrogen in the cell cul-


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FIG. 5. EBNA1 expression up-regulates the activity of Cp reporter plasmids in SL2 cells. The reporter plasmids pgCp(oriPI/⫺170)CAT, pgCp(oriPI/⫺1024)CAT, and pgCp(⫺3889)CAT were cotransfected with increasing amounts of pPacEBNA1 and constant amounts (25 ng) of pPacSp1, pPacNF-YA, pPacNF-YB, and pPacNF-YC. Column 1 represents relative CAT activities of reporter plasmids cotransfected with the empty pPac vector alone. The 100% values correspond to 31% conversion of substrate for pgCp(oriPI/⫺170)CAT, 58% for pgCp(oriPI/⫺1024)CAT, and 210% for pgCp(⫺3889)CAT (measured values multiplied by dilution factor). The values are means of three independent transfection series. Error bars indicate standard errors of the means.

ture medium. Estrogen withdrawal leads to the inactivation of EBNA2, which is followed by entrance of the cells into a quiescent, nonproliferating state reminiscent of normal, resting B lymphocytes (21). We studied NF-YA expression in resting as well as proliferating EREB2-5 cells by using immunoblot assays with ␤-actin as an internal standard (Fig. 6A). The results showed that the expression of NF-YA was abrogated as the cells entered the resting phase of the cell cycle. As estrogen was added to the growth medium and the cells reentered the proliferating phase, the expression of NF-YA was restored. A previous study has shown that proliferating EREB2-5 cells use Cp while resting cells use Wp (61). This effect was attributed to the lack of EBNA2 in estrogen-depleted cells. In the present investigation, we determined the activity of the endogenous Cp in EREB2-5 cells at different times after withdrawal and addition of estrogen and related the promoter usage to NF-YA levels in the cells (Fig. 6A and B). The results showed a rapid decline of Cp usage after withdrawal of estrogen. The decline came as early as 24 h after withdrawal, i.e., before the decrease in NF-YA levels. Thus, the rapid down-regulation of Cp usage probably depended to a large extent on the loss of intranuclear EBNA2 function. On the other hand, addition of estrogen did not result in a prompt up-regulation of Cp, in spite of an immediate translocation of ER-EBNA2 from the cytoplasm to the nucleus, where it should exert its Cpactivating function. It took 48 h after addition of estrogen before the Cp activity was fully increased. This increase was strongly correlated to up-regulation of NF-YA in three independent experiments. S1 nuclease analysis of Wp-initiated transcripts (data not shown) verified the previously described inverse correlation between Cp and Wp usage

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FIG. 6. The expression of NF-YA is low in resting and high in proliferating B-lymphoid cells. EREB2-5 cells were analyzed at different times after withdrawal and addition of estrogen. (A) Immunoblot analysis of NF-YA and ␤-actin at the indicated times. (B) S1 nuclease protection assay of RNA harvested at the indicated times. Arrowheads indicate the positions of size markers. Protected fragments corresponding to transcripts initiated at Cp are indicated by a solid arrow. (C) Immunoblot analysis of NF-YA and ␤-actin in resting and proliferating EREB2-5 cells and primary B cells from an EBV-negative donor.

(61). To verify the biological significance of the low NF-YA levels in resting EREB2-5 cells, primary B cells from an EBV-negative donor were isolated and analyzed for NF-YA expression. The NF-YA levels in the primary B cells were as low as in the resting EREB2-5 cells (Fig. 6C). Taken together, the results corroborate the conclusion that NF-Y is important for Cp activity and also indicate that NF-Y may

play a role in the Wp-to-Cp switch during B-cell transformation. DISCUSSION We have previously shown that the transcriptional regulatory activity of Cp is highly dependent on binding of NF-Y to

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a promoter-proximal CCAAT box (41). In the present investigation, we corroborate the dependence of Cp activation on NF-Y by showing that a dominant negative mutant of NF-YA abolishes both oriPI-EBNA1-independent and -dependent upregulation of Cp. Moreover, we show that a functional interaction of NF-Y and Sp1 is required for Cp activity in the Drosophila SL2 cell line. The concomitant expression of NF-Y and Sp1 is also necessary for the transactivating function of the oriPI-EBNA1 complex on Cp activity. The biological significance of NF-Y is emphasized by the finding that up-regulation of NF-Y expression seems to be required for the Wp-to-Cp switch in growth-arrested EREB2-5 cells, as they are triggered to enter the cell cycle. NF-Y is a ubiquitous multimeric transcription factor, also referred to as CP1 or CBF, that consists of NF-YA, NF-YB, and NF-YC subunits with molecular masses of 42, 36, and 40 kDa, respectively (23, 32, 51, 59). NF-YB and NF-YC contain conserved histone fold motifs and form a dimer that interacts with NF-YA. All three NF-Y subunits are required for binding to the CCAAT motif (23, 51). To further elucidate the role of NF-Y in Cp regulation, we used a mutated NF-YA analogue, which forms a functionally impotent trimeric complex with the NF-YB and NF-YC subunits that is unable to bind the cognate DNA motif. This dominant negative mutant has been widely used to characterize NF-Y-dependent promoters (7, 10, 12, 35). We showed that the overexpression of dnNF-YA almost completely abolished oriPI-EBNA1-independent and -dependent Cp activity (Fig. 2 and 3), demonstrating that (i) NF-Y is indeed important for Cp activity and (ii) other CCAAT boxbinding transcription factors cannot compensate for the loss of NF-Y function. Our earlier studies on promoter-proximal regulatory elements in Cp documented the importance of Sp1 for EBNA gene transcription initiated at Cp. Mutation of either the CCAAT box or the Sp sites completely eliminated Cp activity (41). In the present study, we showed that NF-Y does not work in isolation but, rather, operates in conjunction with Sp1 to regulate Cp. Expression of both Sp1 and NF-Y in Drosophila cells led to amplification of Cp activity far beyond that seen with either transcription factor alone. Additionally, the activation was also beyond what was seen in our previous investigation, where Sp1 was expressed alone, and a very large excess of the pPacSp1 plasmid had to be used to obtain a measurable Cp activation (41). In conclusion, the results show that Sp1 and NF-Y activate Cp synergistically, implying a direct or indirect functional interaction. Interestingly, recent investigations have demonstrated a physical interaction between these two transcription factors both in vitro and in vivo (30, 48). It is a well-established fact that oriPI in conjunction with EBNA1 functions as a transcriptional enhancer of Cp (45, 52). The oriPI-EBNA1 complex is also necessary to induce Cp activity in reporter plasmids transfected into group III phenotype cells (40, 44). The mechanism for the interaction between the oriPI-EBNA1 complex and Cp is, however, not understood at the molecular level. In a previous study, we showed that the minimal oriPI-EBNA1-responsive Cp region comprises sequences between positions ⫺111 and ⫹76 relative to the Cp transcription start site (41). This region contains one Sp site and the CCAAT box. To investigate the requirement of NF-Y and Sp1 for the oriPI-EBNA1-enhancer function, we cotrans-


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fected SL2 cells with Drosophila expression factors for Sp1, NF-Y, and EBNA1. The results revealed an absolute requirement of both Sp1 and NF-Y for the oriPI-EBNA1-mediated up-regulation of Cp. However, the EBNA1-mediated transactivation of Cp in SL2 cells was relatively low (two- to fourfold) compared to what has been reported from cotransfection studies with EBV-negative lymphoid and nonlymphoid cells (generally ⬎10-fold) (29, 40, 52). One explanation for the low transactivation efficiency would be a low level of expression from the pPacEBNA1 expression vector in SL2 cells or a defective importation of EBNA1 to the nuclei of this cell type. These explanations are, however, not valid, as shown in our previous study, in which we detected high levels of EBNA1 expression in the nuclei of transfected SL2 cells (41). Moreover, a dose-dependent increase in EBNA1 expression was detected by immunoblot analysis when the amount of pPacEBNA1 was increased in the transfection experiments represented in Fig. 5. Conceivably, expression of additional factors in SL2 cells does not occur at levels necessary for optimal oriPI-EBNA1 transactivation efficiency. It is, however, also possible that the relative amounts of Sp1, NF-Y, and EBNA1 in transiently transfected SL2 cells are suboptimal for the formation of a fully active Cp-enhancer complex. Interestingly, NF-Y recognizes CCAAT boxes in several genes involved in regulation of cell growth, including murine ribonucleotide reductase R2 (13), murine E2F-1 (16), cyclin B1 (20), cdc2, cyclin A, cdc25C (62), and human thymidine kinase (14). Recent studies also demonstrated that stable expression of a dominant negative mutant of NF-Y in mouse fibroblasts resulted in retardation of cell growth and inhibition of the transcription of various cellular genes (17), implying that NF-Y may be crucial for cell cycle progression. These observations led us to hypothesize that there may be a difference in NF-Y expression in resting and proliferating B lymphocytes. EREB2-5 cells provide a useful system to study this hypothesis, since estrogen withdrawal from the cell culture medium arrests these cells in the cell cycle and induces a phenotype which resembles that of normal, resting B lymphocytes. Activation of EBNA2 with estrogen induces cell proliferation and an LCLlike phenotype (21). Previously, Yoo et al. investigated Cp and Wp activity in EREB2-5 cells grown in the presence of various concentrations of estrogen (61). Estrogen-depleted resting cells used only Wp, whereas Cp was quiescent. This finding was attributed to the lack of intranuclear EBNA2 function. In this study, we measured the levels of the NF-YA subunit at different times after withdrawal or addition of estrogen to the cell medium and related NF-YA expression to Cp and Wp activity, measured as steady-state levels of Cp- and Wp-initiated transcripts by S1 nuclease mapping. The experiments revealed that resting EREB2-5 cells expressed low levels of NF-YA whereas proliferating EREB2-5 cells expressed high levels. After withdrawal of estrogen, Cp usage declined rapidly before the level of NF-YA had decreased, probably reflecting loss of EBNA2 function. These data corroborate the importance of EBNA2 for regulating Cp in the viral context. However, on addition of estrogen and reconstitution of EBNA2 function, the Cp usage was not fully up-regulated until the level of NF-YA was restored. These data indicate that NF-Y may be an important factor for the Wp-to-Cp switch. In agreement with this hypothesis, primary B lymphocytes isolated from an EBV-negative

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donor expressed low levels of NF-Y, which may explain why the first EBV promoter to be activated on infection is Wp, not Cp, and why the Wp-to-Cp switch coincides with triggering of the cellular growth program. Generally, there appear to be at least two levels of transcriptional regulation: one involving chromatin unfolding and another involving the transcriptional machinery at RNA polymerase II promoters. The first level may be a prerequisite for the second, which then can proceed independently (27). Most of the present investigation was focused on this second level of regulation. Through transient transfections, we showed that Cp fragment-containing reporter plasmids, which are unmethylated and packaged in nucleosomes and have an open chromatin conformation, require coexpression of Sp1 and NF-Y for activation. Using similar reporter plasmids, we have previously shown that Cp in this context was active in BL group I cells and that activation depended on neither the presence of the EBNA2-responsive enhancer (E2RE) sequence nor the expression of EBNA2 (41). Cp in the endogenous viral genome was, however, inactive. This apparent contradiction is possibly explained by the observation by Salamon et al. that the activity of Cp in the context of the endogenous virus correlated inversely with the methylation state of the proximal part of the promoter (50). Thus, the mechanism for the silencing of the endogenous Cp in the cells could be methylation of CpG motifs. In the present investigation, the endogenous Cp was silent in the estrogen-depleted EREB2-5 cells and was subsequently activated by the release of biologically active EBNA2, due to addition of estrogen to the medium. EBNA2 is not known to be able to override the effect of DNA methylation. We suggest that Cp is not hypermethylated in the EREB2-5 cells and that activation is coupled to the ability of EBNA2 to interact with coactivators possessing intrinsic histone acetyltransferase activity, including p300, CBP, and PCAF (55). Assuming that the viral promoter sequence is stabilized in a condensed chromatin conformation in the silent state, one conceivable role of EBNA2 would be to recruit these coactivators to Cp. This might lead to destabilization of chromatin structure, allowing the recruitment of Sp1, NF-Y, and presumably additional factors to the promoter and the subsequent activation of transcription. In conclusion, the results presented herein emphasize the biological significance of NF-Y in the activation of Cp. NF-Y is undoubtedly essential for constitutive Cp activity and may, together with EBNA2 and epigenetic events such as methylation of transcription factor-binding sites and histone modifications, contribute to a successful Wp-to-Cp switch. We are, however, very well aware that the temporal coincidence of increased NF-Y expression and the Wp-to-Cp switch, on triggering of the cell growth program in the EREB2-5 cell line, is merely consistent with and cannot be interpreted as proof of a causal relationship. It certainly does not exclude EBNA2 as an important factor for the switch, and it is also possible that EBNA2, directly or indirectly, contributes to the increased expression of NF-Y. ACKNOWLEDGMENTS We thank Roberto Mantovani for the dominant-negative NF-YA expression plasmid; Timothy Osborne for the pPacNF-YA, pPacNFYB, and pPacNF-YC expression plasmids; Guntram Suske for the

J. VIROL. pPacSp1 expression plasmid; and Bettina Kempkes for the EREB2-5 cell line. Additionally, we thank the Section for Flow Cytometry at the Department of Clinical Chemistry, Sahlgrenska University Hospital, for help with the FACS analyses. This study was supported by grants from the Swedish Medical Research Council (project 5667), the Swedish Cancer Society, the Sahlgrenska University Hospital, the Go ¨teborg Medical Society, the Assar Gabrielsson’s Foundation for Clinical Research, and the King Gustav V Jubilee Clinic Cancer Research Foundation. REFERENCES 1. Alfieri, C., M. Birkenbach, and E. Kieff. 1991. Early events in Epstein-Barr virus infection of human B lymphocytes. Virology 181:595–608. 2. Allday, M. J., D. H. Crawford, and B. E. Griffin. 1989. Epstein-Barr virus latent gene expression during the initiation of B cell immortalization. J. Gen. Virol. 70:1755–1764. 3. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. 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