Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors

EBV has previously been shown to induce RNA polymerase III activity via TFIIIC expression in both lymphoid and epithelial cells [29], although the viral gene product responsible for this induction remained unidentified. Affymetrix array analysis revealed a 2.6-fold induction of RNA encoding the 102kd subunit of TFIIIC (TFIIIC102) in response to EBNA1 [35], indicating that this EBV latent gene product may be responsible for inducing TFIIIC expression. To confirm this observation, immunohistochemical staining for TFIIIC102 was conducted and revealed a clear induction of TFIIIC102 in cells expressing EBNA1 (Figure 1). However, the Ad/AH-EBNA1-cl3 cells that were used in these experiments overexpressed EBNA1 [35]. In order to ensure that the observed effects of EBNA were not due to non-physiological levels of expression, another Ad/AH cell line, Ad/AH-EBNA1-cl8, that stably expresses EBNA1 at levels comparable to those seen in EBV latently infected cells (Figure 2) was used in all the following experiments and is referred to as Ad/AH-EBNA1. The EBNA1 derivatives of AGS and Hone-1 cells were previously shown to express physiological levels of EBNA1 [35].

To ascertain EBNA1's capability to induce multiple TFIIIC subunits, Q-PCR was carried out for all 5 TFIIIC subunits that have been shown to respond to EBV [29]. In order to compare directly the effect of EBNA1 and EBV on TFIII induction, Ad/AH-EBV cells were included in this analysis. Transcripts encoding TFIIIC subunits were induced in EBNA1-expressing derivatives of 3 distinct carcinoma cell models, Ad/AH, AGS and HONE-1 (Figure 3). As observed previously [29], the pattern of subunit mRNAs induced varied between cell types, but in each case EBNA1 induced a statistically significant enhancement of the expression of at least two TFIIIC subunits. The effect was most pronounced in Ad/AH and HONE-1 cells. In contrast, there was no significant increase in mRNAs encoding the TFIIIB subunits Bdp1, Brf1 and TBP (Figure 3). In all cases the effect of EBNA1 in Ad/AH cells essentially mirrored that of EBV. We conclude that EBNA1 can have a selective effect on expression of TFIIIC.

To verify that the transcriptional enhancement resulted in increased protein expression the levels of TFIIIC subunits expressed in Ad/AH, Ad/AH-EBNA1 and Ad/AH-EBV cells were examined by immunoblotting (Figure 4). The levels of 4 of the 5 subunits were found to be induced by EBNA1 to levels equivalent to those seen following EBV infection. For technical reasons we were unable to confirm the expression of TFIIIC-102. However, in view of the fact that translation of the other 4 subunits reflects the induction of the mRNA and that immunohistochemistry (Figure 1) shows induction of TFIIIC-102 in response to EBNA1, we conclude that TFIIIC-102 protein is upregulated and, like EBV, EBNA1 induces the expression of all 5 TFIIIC subunits in Ad/AH cells.

EBV has previously been shown to increase the expression of several pol III targets [29, 36, 37]. To determine whether this could be due to the expression of EBNA1, RT-PCR for a number of cellular pol III-transcribed genes was carried out across the panel of EBNA1-expressing epithelial cells. Expression of EBNA1 in all cell lines resulted in increases in expression of the cellular pol III-transcribed genes tRNA^Tyr, 7SL RNA and 5S rRNA (Figure 5), similar to those seen in EBV-infected epithelial cells. Therefore, EBNA1 may be responsible, at least in part, for the increase in expression of endogenous pol III products that is induced by EBV. Although the induction is quantitatively modest, a comparable level of tRNA overexpression has been shown to be capable of proliferative and oncogenic effects in immortalised mouse embryonic fibroblasts [38].

In addition to the influence of pol III-specific transcription factors, EBER expression is reliant upon factors more typically associated with pol II transcription, such as ATF-2 which has been shown to be increased in EBV-infected cells [29]. ATF-2 was also found to bind and activate the promoter of the gene encoding TFIIIC220 [29]. Consistent with our previous study [31], a robust increase in levels of ATF-2 mRNA was observed in EBNA1-expressing Ad/AH cells (Figure 6a), along with an increase in the total, mono- and dual-phosphorylated forms of the protein (Figure 6b, 6c, 6d).

c-Myc has a binding site upstream of the EBER genes [21] and has been shown to be transcriptionally induced in EBNA1-expressing Ad/AH cells [35]. This upregulation was confirmed by RT-PCR analysis (Figure 6e). To assess whether the enhanced expression was associated with an increase in the transcriptional activation capacity of c-Myc, Ad/AH-Neo and -EBNA1 cells stably expressing a functionally inducible c-Myc fusion protein (c-MycER) were assessed for c-Myc-mediated activation of a reporter construct following functional induction of the c-Myc fusion protein with 4-hydroxytamoxifen (4-OHT) (Figure 6f). At all tamoxifen concentrations, enhancement of c-Myc function was observed in both Neo- and EBNA1-expressing cells, with activity being around 4- to 5-fold higher in EBNA1-expressing cells compared with their Neo counterparts. This is consistent with there being an increased transcription-enhancing activity of c-Myc in the presence of EBNA1. In both Ad/AH-Neo and -EBNA1 cells, the increase of c-Myc activity in response to 4-OHT treatment was titratable, with increased 4-OHT concentrations resulting in increased levels of c-Myc-induced transcriptional activation.

Previously, we have used ChIP assays to show that EBNA1 is present at the promoters of several AP-1 subunits, including ATF-2, that are transcriptionally induced by EBNA1 in epithelial cells [31]. Here, we use a variant (HaloChIP) of conventional ChIP to demonstrate that EBNA1 is present at the promoters of genes encoding EBER-associated transcription factors that are induced in EBNA1-expressing cells. Using a transiently-expressed fusion protein construct (HaloEBNA1), we were able specifically to pull-down EBNA1 and associated DNA in an antibody-independent manner. The tagged version of EBNA1 is bound covalently by a resin; a specific interaction that can be prevented by a blocking ligand. We first validated the HaloChIP method by performing a comparison of HaloChIP with conventional ChIP in EBV-infected Ad/AH cells using an anti-EBNA1 antibody that, in such assays [39] had already been demonstrated to be competent to enrich the known EBNA1-binding DS region of EBV DNA and the cellular c-jun promoter region [31]. The enrichment obtained using the two methods was very similar (compare the c-jun data in Figure 7A). Using HaloChIP in Ad/AH cells, we were able to confirm that the EBNA1-fusion protein is present at the ATF-2 and c-Myc promoters through observation of a substantial enrichment of promoter DNA in experimental samples, compared with control samples (Figure 7A). Furthermore, we demonstrate that EBNA1 is present at the promoters of all TFIIIC subunit genes except TFIIIC63. This is consistent with the fact that TFIIIC63 was unique amongst the TFIIIC subunit genes in not responding to EBNA1 in Ad/AH cells. We also did not observe any enrichment of promoter DNA for TFIIIB genes or indeed cellular pol III-transcribed genes encoding tRNA^Tyr, 5S RNA and 7SL RNA. The GAPDH promoter, included as a negative control, was not enriched in experimental samples compared with controls.

Although EBNA1 was not associated with the promoters of cellular pol III-transcribed genes it was of interest to determine whether the enhancement of EBER expression in EBNA1-expressing cells could be attributed to EBNA1 binding to the viral EBER promoters. Since, in the EBV genome, the EBER genes are located in close proximity to the EBNA1-binding OriP region [40], this study had to be performed in the Ad/AH-EBERs cell line, (see Methods) in which the EBERs are stably expressed from their native promoter elements in the absence of OriP which would have hampered the interpretation of ChIP assay data. HaloChIP assays performed in this cell line revealed a level of enrichment of the ATF2 promoter similar to that observed in Ad/AH-EBV cells. In contrast, as with the cellular pol III-transcribed genes, there was no enrichment of promoter DNA for the EBERs (Figure 7A, 7B).

The data presented above confirm that EBNA1 is able to induce several factors involved in the regulation of EBER expression (TFIIIC, ATF-2 and c-Myc). However, these experiments fail to illustrate definitively that EBNA1 increases EBER expression. To address this, an EBNA1-expressing plasmid (pSG5 EBNA1) was titrated into the Ad/AH-EBERs cell line and the effect on EBER expression was assessed by RT-PCR (Figure 8a). Small amounts (down to 1ng) of EBNA1-encoding plasmid DNA were sufficient to increase levels of expression of both EBER1 and EBER2. This increase can be attributed to changes in shared components of the pol III machinery, since levels of tRNA transcripts are also increased. It is noteworthy that even the lowest level of EBNA1 is sufficient to elicit the responses and that overexpression is not required. Levels of EBER expression are shown to be comparable to those seen in the Burkitt's Lymphoma (BL) cell line Namalwa (Figure 8b).

As a converse of the experiments described above, ablation of EBNA1 function through introduction and expression of dnEBNA1 (EBNA1 M1) in EBV-infected Ad/AH cells resulted in a robust decrease in levels of both EBER 1 and EBER 2 expression (Figure 8c).

To confirm the influence of EBNA1-induced transcription factors ATF-2 and c-Myc on EBER expression, we used shRNA-mediated knock-down of these factors in EBV-infected Ad/AH cells and examined, using RT-PCR, the effect on EBER expression. shRNA targeting ATF-2 robustly reduced ATF-2 expression and, as a result, expression of both EBER1 and EBER2 was significantly diminished (Figure 9a).

shRNA targeting c-Myc also successfully reduced levels of c-Myc transcription in EBV-infected Ad/AH cells, and was associated with a diminished level of EBER1 but no apparent reduction in that of EBER2 (Figure 9b). This is consistent with the observation that only the EBER1 gene contains an E-Box (c-Myc binding site) upstream of its transcriptional start site [21]. Previously, c-Myc has been shown to increase levels of pol III transcription directly by interacting with TFIIIB subunits [41]. Consistent with this, RT-PCR conducted for endogenous pol III targets (Figure 9b) showed that shRNA-mediated depletion of c-Myc reduced expression of cellular pol III products (data shown for tRNA^Tyr).

To assess further the contribution of EBNA1's ability to enhance the transcriptional activation potential of c-Myc leading to increased EBER expression, an Ad/AH cell line was generated which stably expressed both c-MycER and the EBERs (including their upstream transcriptional regulatory regions). Following functional induction of c-Myc, levels of EBER expression were assessed (Figure 9c). After 24 hours of induction, a clear increase in the level of EBER1 was observed, but only a very modest increase in that of EBER2. This result is consistent with the shRNA data and supports the idea of the E-boxes upstream of EBER1 being important in terms of transcriptional regulation of the EBERs. To confirm this, an Ad/AH cell line was generated which stably carried both c-MycER and the EBERs, including their upstream transcriptional regulatory regions (TATA, SP1 and ATF sites), but with a ~230 bp region containing the E-boxes excised (see Materials and Methods). Again, levels of EBER expression were assessed by RT-PCR following induction (Figure 9d). Levels of EBER1 or EBER2 expression were not significantly increased upon functional induction of c-Myc in this cell line, again indicating that c-Myc influences EBER transcription via the upstream E-boxes.

The potential relevance in vivo of our observations of EBNA1-induced induction of TFIIIC102 was addressed by immunohistochemical staining of EBV-positive NPC tumour biopsies (Figure 10a - d). In 18 out of 19 NPC biopsies, nuclear staining for TFIIIC102 appeared significantly stronger in tumour cells than in infiltrating non-tumour cells. Additionally, microarray analysis of RNA extracted from 15 samples of microdissected EBV-positive NPC tumour cells compared with 4 samples from normal epithelial cells, revealed consistent induction of TFIIIC102 mRNA in the tumours (Figure 10e). Expression of other TFIIIC subunits was variable and so low in many samples as to be called "absent" by the analysis software.

The EBNA1 expression plasmid pSG5 EBNA1 [33], c-Myc reporter pX-CMVp-Luc [21] and dominant-negative EBNA1 expression vector EBNA1-M1 [56] were as described. A plasmid containing a functionally-inducible c-Myc gene (a fusion between the hormone binding domain of the oestrogen receptor and c-Myc) [57] was obtained from C. Tselepis. An EcoRI fragment containing the fusion (c-MycER) was ligated into pcDNA3.1/zeo (Invitrogen). The pUC19 EBERs plasmid was generated by inserting the 1 kb SacI - EcoRI subfragment from the EcoRI J fragment of the EBV genome into the corresponding sites of pUC19, with a puromycin resistance cassette [58] being inserted at the SalI site. For generation of pUC19 Puro EBERs ΔX, the X-box c-Myc binding site was excised from pUC19 EBERs plasmid using SacI and PmlI, excising bases -352 to -125 relative to the EBER1 transcriptional start site. A control Renilla luciferase plasmid (pRL-TK) was from Promega.

Ad/AH (a human adenocarcinoma cell line derived from the nasopharynx), HONE-1 (an EBV-negative NPC cell line) and AGS (human gastric carcinoma-derived) cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and 1% penicillin-streptomycin solution (Sigma-Aldrich). HONE-1 and AGS cells stably expressing EBNA1 and the Ad/AH line stably infected with a recombinant EBV were described previously [35]. An Ad/AH cell line that stably expresses physiological levels of EBNA1 (Figure 2) was derived as described previously [35].

Ad/AH-Neo and -EBNA1 cells stably expressing the c-MycER fusion protein were generated by transfection of cells with c-MycER plasmid followed by drug selection with Zeocin (Invitrogen). Ad/AH-EBERs cell lines (and derivatives) were generated by stable transfection of pUC19 EBERs plasmid, followed by puromycin drug selection of polyclonal EBER-positive cell populations.

Dual luciferase reporter assays were performed according to the manufacturer's instructions (Promega) with cells cultured in RPMI 1640 supplemented with 0.5% FCS, 2 mM L-glutamine, and 1% penicillin-streptomycin solution. Cells were transfected with plasmids using Lipofectamine (Invitrogen) according to the manufacturer's instructions. All assays were carried out in biological and technical triplicate and are represented as the mean of three independent experiments.

Cells were subjected to shRNA directed against c-Myc (pGIPZ 152051, OpenBiosystems) and ATF-2 (pLKO.1 13713, OpenBiosystems) using transfection techniques previously outlined. RNA was extracted from cells 72 h post-transfection.

ChIP assays were performed as described previously [31] using an EBNA1 antibody (chEBNA1) and conditions described by Chau & Lieberman (2004) [39]. A rabbit isotype control antibody was obtained from Santa Cruz. Precipitated target sequences were identified by PCR and quantitated by densitometry as described below.

HaloCHIP assays were performed according to the manufacturer's instructions (Promega, UK). HaloEBNA1 fusion protein-encoding plasmid was generated using PCR based cloning techniques, with pseudo-wild type EBNA1 (with deleted Gly/Ala repeat region) fused to HaloTag protein (N-terminal) in pFC14K-CMV backbone plasmid (Promega UK). 0.5 μg HaloEBNA1 plasmid was transfected per 10 cm² dish. Dishes were formaldehyde cross-linked 24 h post transfection before manufacturer's protocols were followed precisely.

PCR was performed using GoTaq Green Polymerase Mastermix (Promega UK). Primer sets for promoter regions were designed with the aid of literature searches for known promoter regions. All primer sets were based upon regions within 1 Kb upstream of transcriptional start sites. Exceptionally, primer sets for cellular pol III-transcribed genes tRNA^Tyr and 5S RNA were based, due to their intragenic promoters, on regions internal to the gene. 7SL RNA's promoter regions are both intragenic and upstream of the transcriptional start site therefore primer sets for both regions were used. Primer sets are listed in Table 1. The linear range of each PCR reaction was determined empirically through semi-quantitative PCR to determine experimental cycle numbers.

RNA for RT-PCR was reverse transcribed with Superscript III, (Invitrogen) following the manufacturer's protocol, using random primers (Promega UK). Resultant cDNA was then used for PCR reactions as above. Primers are listed in Table 2. Standard immunoblotting procedures [35] were used to detect total ATF-2, (mouse MAB1536, 1 mg/ml, Santa Cruz), mono- (Thr69) and dual-phosphorylated (Thr69/71) ATF-2 (mouse MAB1536 1 mg/ml, Cell Signaling Technology), EBNA1 (human serum, 1:500), actin (mouse AC-38, 1:20 000, Sigma). Immunoblotting for TFIIIC subunits was performed as described previously [29]. Immunohistochemical staining and EBER in situ hybridisation were performed on biopsies used in previous studies [59] and were conducted as described therein. For immunohistochemistry, antibody 3238 to TFIIIC-102 [29] was used at a dilution of 1:100 following standard techniques. Immunofluorescence was carried out on Ad/AH-Neo and -EBNA1 cells using standard procedures [35] with total ATF-2 antibody (mouse MAB1536, 0.5 mg/ml, Santa Cruz).

Densitometry was conducted on UV transilluminator images using ImageJ 1.37 V (http://​rsbweb.​nih.​gov/​ij/​). Bands were analysed in technical triplicate, normalising against background image levels and, if appropriate, internal controls. In all instances, densitometry was conducted in biological triplicate.

RT-qPCR for cellular genes was performed using ready synthesised Taqman primer and probe (FAM labelled) mixes purchased from Applied Biosystems (see Table 3). Each multiplexed RT-qPCR reaction consisted of 5 μl (50 ng) of cDNA, 1 μl of ABI test primers and probe mix, 0.5 μl of huGAPDH primer and probe (VIC labelled) mix, 10 μl of 2× sensimix (Quantace) and DEPC water to 20 μl. Reactions were performed in biological and technical triplicate and analysed on an ABI 7500 Fast Real-time PCR machine. Data were analysed using the 2^-ddCt method [60].

Full details of the protocol will be presented elsewhere (C. Hu et al., in preparation). Briefly, normal epithelial cells and EBV-positive NPC tumour cells were isolated by laser-capture microdissection from frozen biopsies. Total cellular RNA was extracted using a Qiagen RNEasy kit followed by amplification, labelling and hybridisation to Affymetrix U133 Plus2 arrays essentially as previously described [61]. After scanning, intensity values were analysed using GCOS software (Affymetrix).