Epstein - Barr Virus Transforming Protein LMP-1 Alters B Cells Gene Expression by Promoting Accumulation of the Oncoprotein ΔNp73α

Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.


Introduction
Epstein-Barr virus, also known as human herpesvirus 4 (HHV4), belongs to the gammaherpesvirus family and is largely spread as it can be detected in 90% of the worldwide population. EBV infects B cells and, in most cases, does not lead to any clinical manifestations. However, when EBV infection occurs during adolescence or young adulthood, it may cause infectious mononucleosis, a benign lymphoproliferative disease. A minority of EBV infections result in the development of several types of human B cell malignancies, including Burkitt's lymphoma (BL), Hodgkin and non-Hodgkin lymphomas [1]. In addition, EBV has been clearly associated with epithelial cancers, i.e. nasopharyngeal carcinoma (NPC) and a sub-set of gastric carcinoma [1]. The risk of developing EBV-induced malignancies is significantly increased in immuno-compromised individuals, such as AIDS patients and organ-transplant recipients. In vitro EBV efficiently infects human resting B cells and transforms them into proliferating lymphoblastoid cell lines (LCLs) [2]. Similar to other herpesviruses, the EBV life cycle includes a latent and non-productive phase, as well as a lytic phase leading to the production of the virus progeny. After primary infection, EBV persists lifelong in a latent state in a sub-population of resting memory B cells [3]. Recent studies led to a model of EBV persistence whereby different viral transcription programs were used within the context of the normal biology of B lymphocytes in order to carry out its life cycle [4], [5]. Eleven genes can be expressed in the latency phases, namely the EBV nuclear antigens (EBNA) 1, 2, 3A, 3B, 3C, LP, the latent membrane proteins (LMP) 1, 2A, 2B, the untranslated EBER-1 and EBER-2 RNAs, as well as multiple microRNAs [2]. Based on the expression pattern of the different latency genes, four latency phases have been identified so far. Type I latency is normally present in Burkitt's lymphoma and is associated with the expression of EBNA-1 as well as EBERs and miRNAs. Type II latency is frequently detected in Hodgkin's lymphoma and nasopharyngeal carcinoma, and is linked to the expression of EBNA-1, LMP-1, LMP-2A, LMP-2B, EBERs and miRNAs. Type III latency is characterized by the expression of all 11 latency genes and is mainly found in lymphoproliferative diseases in immunocompromised individuals and in in vitro EBV-transformed LCLs. Finally, type IV latency is associated with the infectious mononucleosis and is less well defined, since the expression pattern of the latency genes may differ in different patients [6].
LMP-1 is the major EBV oncoprotein and displays transforming activities in in vitro and in vivo models [2]. It is an integral membrane protein composed of a short cytoplasmic aminoterminal domain, six hydrophobic transmembrane domains, and a cytoplasmic carboxy-terminal domain [2]. LMP-1 exerts its transforming properties by functioning as a member of the tumour necrosis factor receptor (TNFR) superfamily leading to the a constitutive activation of several cellular signaling pathways [7][8][9]. In particular, LMP-1 activates the nuclear factor-kappa B (NF-kB) signaling pathway, thus promoting cell growth and inhibition of apoptosis. In addition to LMP-1, other EBV latent proteins, i.e. EBNA-2, EBNA-3A and EBNA-3C, are involved in the immortalization of primary B cells. The role of EBNA-2 is mainly mediated by its ability to modulate the transcription of host and viral genes, while EBNA-3C plays a direct role in cellular transformation by inactivating the products of tumor suppressor genes, such as retinoblastoma (pRb) and p53 [10][11][12][13][14]. Interestingly, as for other oncogenic viruses, e.g. human papillomavirus type 16 (HPV16) [15], EBV developed multiple and redundant mechanisms to inactivate p53-regulated pathways. Indeed, EBNA-3C is able to alter the p53 transcription activity via direct binding as well as by inducing stabilization of p53 inhibitors, such as mdm2 and Gemin3 [10,12,14].
p73 is a closely p53-related transcription factor that shows functional similarity to p53 [16,17]. The impact of EBV on p73 has been poorly investigated so far, although initial findings indicate that, similarly to p53, p73 is targeted by EBV. Indeed, p73 expression was found down-regulated in EBVpositive gastric carcinoma by heavy methylation of CpG islands within its promoter [18]. In addition, it has been recently shown that EBNA-3C attenuates p73 expression in LCLs by targeting the transcription factor E2F-1 [19]. However, it is likely that, in line with the previous findings on p53, EBV has developed multiple mechanisms to alter p73 function. In addition, due to the variability in the expression pattern of the latent genes in EBV-positive cancer cells, it is possible that more than one viral protein has the ability to target the p53/p73 pathway.
We have previously shown that cutaneous HPV38 which belongs to an HPV subgroup potentially associated with the development of non-melanoma skin cancer (NMSC), is able to induce the accumulation of DNp73a which in turn alters the transcriptional functions of p53 and p73 [20,21]. DNp73a is a p73 isoform that lacks transactivation (TA) domain and is accumulated in several tumors [22]. Most importantly, increase in DNp73a protein levels correlates with poor outcome of the disease and bad response to therapy [23][24][25].
Here we show that EBV LMP-1 activates DNp73a expression in B cells by favoring the recruitment of p73 to DNp73 promoter. This phenomenon appeared to be mediated by c-Jun NH2terminal kinase 1 (JNK-1), and resulted in the inhibition of p53regulated genes encoding key anti-proliferative regulators.

EBV induces DNp73 transcriptional activation in B cells
Several isoforms of DNp73 have been identified, that can be generated by alternative splicing at the 59 region of the p73 mRNA or by transcriptional initiation from a promoter (p2) within the p73 gene [26,27]. We have previously shown that HPV38 induces the accumulation of DNp73a mRNA generated by the p2 promoter [20]. We therefore evaluated whether the levels of this specific DNp73 transcript were induced by EBV. DNp73a mRNA levels were determined in EBV-positive and EBV-negative B-cell lines by RT-PCR using specific primers. Six LCLs expressed high levels of DNp73a transcript, while no signal was detected in the EBV-negative B lymphoma cell line BJAB ( Figure 1A). To assess that the over-expression of DNp73a was indeed linked to EBV infection, we infected primary B cells with recombinant EBV and analyzed DNp73a mRNA levels by quantitative RT-PCR. As previously shown, DNp73a is not expressed in primary B cells ( Figure 1B) [28]. In contrast, an increase in DNp73 mRNA levels was observed between 12-36 hours post-EBV infection which correlated with LMP1 transcript levels ( Figure 1B). DNp73 mRNA accumulation was also observed in cancer B-cell lines, RPMI, upon EBV infection cells ( Figure 1C). Several C-terminus DNp73 isoforms have been characterized, that are generated by alternative splicing (a, b, c, e). The isoform alpha plays a key role in altering the p53/p73 functions and is over-expressed in several human cancers [29]. RT-PCR experiments with specific DNp73 isoform primers confirmed that alpha, and not beta, DNp73 is expressed in EBV-infected B-cells ( Figure 1D). Accordingly, immunoblotting with a p73 antibody revealed a 65-70 kD protein band in LCLs that comigrated with the DNp73a ectopically expressed in HEK 293 cells ( Figure 1E).
Taken together, these data showed that EBV specifically activates DNp73a transcription in primary and cancer B-cells.

LMP-1 plays a central role in EBV-mediated up-regulation of DNp73a
Studies on other oncogenic viruses demonstrated that alterations of p53-regulated pathways are normally induced by the viral oncoproteins [15,30]. Figure 1B showed a correlation between DNp73a and LMP-1 expression levels, supporting the possible involvement of the viral oncoprotein in DNp73a upregulation. Therefore, we determined later whether the major EBV transforming protein, LMP-1, was responsible for DNp73a accumulation. RPMI cells were transduced with empty (pLXSN)

Author Summary
Approximately 20% of worldwide human cancers have been associated with viral infections. Many oncogenic viruses exert their transforming properties by inactivating the products of tumour suppressor genes. One of the best characterized events induced by ongocenic viruses is the inactivation of the transcriptional factors p53. The mucosal high-risk HPV types, EBV, HTLV-1 and KSHV, via their viral proteins, are able to target p53 by distinct mechanisms. We have recently described a novel p53 inactivation mechanism of some cutaneous beta HPV types which have been suggested to be associated with skin carcinogenesis. Beta HPV38 induces accumulation of the p53 antagonist, DNp73a which in turn silences the expression of the p53regulated genes. Here we report that also EBV, via the oncoprotein LMP-1, induces the expression of DNp73a which is dependent on the recruitment of p73 on DNp73 promoter and the activation of JNK-1. The recruitment of p73 to the DNp73 promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of a transcriptional repressive polycomb 2 complex. We also show that DNp73a plays an important role in transformation of primary human B cells and regulates the expression of a large number of cellular genes that encode proteins linked to cancer development, including lymphomagenesis.
or LMP-1 expressing retrovirus (pLXSN-LMP-1) and DNp73a transcript and protein levels were determined by RT-PCR and immunoblotting, respectively. Both DNp73a mRNA and protein levels were strongly increased in RPMI/LMP-1 cells in comparison to cells infected with the empty retroviral vector (Figures 2A  and B). To further demonstrate the role of LMP-1 in DNp73a upregulation, we infected RPMI cells with a wild-type or mutated EBV, in which the LMP-1 gene was deleted (EBVDLMP-1). Two RPMI/EBVDLMP-1 cell lines were generated by two independent infections and DNp73a expression levels were compared with the ones of mock infected cells as well as RPMI/EBV. Deletion of LMP-1 gene from the EBV genome abolished DNp73a upregulation, as shown by RT-PCR and immunoblot analyses (Figures 3 A and B). Transduction of RPMI/EBVDLMP-1 cells with a recombinant retrovirus expressing LMP-1 (pLXSN LMP-1) restored the ability of EBV to promote DNp73a mRNA and protein accumulation ( Figures 3C and D).
In summary, these data highlight the central role of LMP-1 in EBV-mediated DNp73a up-regulation.

p73 activates DNp73a expression in LMP-1-expressing cells
The p73 p2 internal promoter contains a p53 responsive element (RE) which can be activated by both p53 and p73 [31][32][33]. Therefore, we evaluated whether p53 and/or p73 are involved in the regulation of p2 promoter in the presence or absence of LMP-1. Chromatin immune precipitation (ChIP) experiments using the p53 null SaOS-2 cells as experimental model showed that LMP-1 over-expression increased p73 binding affinity for the RE within the p2 promoter, while it did not influence p53 recruitment to the same site ( Figure 4A). ChIP experiments with anti p73 antibody in RPMI cells transduced with empty (pLXSN) or pLXSN-LMP-1 retrovirus also showed an increased binding of p73 to the p2 promoter in presence of LMP1 ( Figure 4B). In addition, DNA pull-down experiments, in which a biotinylated DNA probe containing a region of the p2 promoter encompassing the p53RE was incubated with cellular extracts of RPMI or RPMI LMP-1 cells, showed that LMP-1 increased p73 efficiency in binding DNA ( Figure 4C). Silencing of p73 expression in LCL by shRNA correlated with down-regulation of DNp73a mRNA levels ( Figure 4D). In contrast, targeting p53 with a siRNA in the same cells did not alter DNp73a levels ( Figure 4D), indicating that p53 is not involved in the transcriptional regulation of DNp73a in LCLs.
We conclude from this set of data that LMP-1-mediated DNp73a transcriptional activation is partly due to the recruitment of p73 to the p2 promoter.

JNK-1 is involved in LMP-1-mediated activation of DNp73a expression
It is known that LMP-1 stimulates JNK-1, which in turn leads to p73 phosphorylation and increase in its transcriptional activity [34][35][36]. We therefore determined whether JNK-1 is involved in DNp73a accumulation mediated by LMP-1. Ectopic levels of JNK-1 in BJAB cells induced DNp73a accumulation ( Figure 5A). In addition, treating LCLs with a specific inhibitor of JNK (SP600125) led to a time-dependent decrease in DNp73a mRNA levels ( Figure 5B). JNK-1 down-regulation in LCL by siRNA also led to a decrease in DNp73a mRNA and protein levels ( Figures 5C and D).
LMP-1 transforming activities lie mostly on two distinct domains in its cytoplasmic C-terminus, namely C-terminal activation region 1 (CTAR1) (amino acids 187-231) and CTAR2 (amino acids 351-386). As CTAR2 LMP-1 mutant (LMP-1/378 stop) is unable to activate JNK-1 [37], we determined whether deletion of CTAR2 affected LMP-1 ability to promote p73 and DNp73a accumulation. We first transfected SaOS-2 cells with HA tagged p73 in the presence of wild-type or 378 stop-mutant LMP-1. ChIP experiments performed with an HA antibody showed that only the wild-type LMP-1 increased p73 recruitment to p2 promoter ( Figure 5E). According to previous data [35], immunoblotting showed that wild-type LMP-1, but not the LMP-1 378 stop-mutant that is unable to activate JNK-1, induced p73 accumulation ( Figure 5F). Finally, DNp73a expression was only detected in RPMI/EBVDLMP-1 cells containing the wild-type LMP-1 and not the LMP-1 378 stop-mutant ( Figure 5G). LMP-1 is also able, via the CTAR1 and CTAR2, to activate the cellular kinase p38 which in turn activates p73 [38][39][40]. However, p38 inhibition in LCLs by a chemical inhibitor did not result in a decrease of mRNA levels of DNp73a (data not shown), indicating that a different mechanism is involved in the event.
Together, these data highlight a crucial role of JNK-1 in LMP-1-mediated accumulation of p73 and DNp73a.

LMP-1 induces epigenetic changes on the p2 promoter
Emerging lines of evidence show that epigenetic changes play an important role in carcinogenesis [41]. Accordingly, several oncogenic viruses, including EBV, are able to hijack the epigenetic machinery in order to de-regulate cellular gene expression, to persist in the host cell and complete the viral cycle [42]. EZH2 is a component of the Polycomb 2 complex and is able to methylate the Histone H3 on Lysine 27, leading to chromatin condensation and gene silencing [43]. Interestingly, it has been recently shown that, upon interferon alpha treatment, EZH2 inhibits DNp73a expression in hepato-cellular carcinoma cells (HCC) by direct binding to the p2 promoter [44]. Therefore, we next determined whether in primary B cells and LCL the observed alterations in DNp73a expression may be ascribed to changes in the EZH2 recruitment to p2 promoter. ChIP experiments showed that EZH2 binds the p53 RE within the p2 promoter only in primary B cells, but not in LCL ( Figure 6A). A similar pattern was observed in ChIP experiments performed with an antibody that specifically recognized H3K27 methylated form ( Figure 6A). In contrast, acetylation on lysine 9 of the Histone H3, a marker of transcriptionally active chromatin, was increased at the p53/p73 RE in LCLs in comparison to primary B cells ( Figure 6A). In addition, p73 was more efficiently recruited to p53/p73 RE in LCLs than primary B Cells ( Figure 6B). Loss of EZH2 at DNp73a promoter appeared to be dependent on LMP-1 expression in LCLs and RPMI cells ( Figure 6C and D).
According to these results, histone H4 hyperacetylation, another event associated with active transcription, is strongly enhanced at DNp73a promoter in LCLs in comparison to primary human B cells ( Figure 6E). JNK can induce H4 hyperacetylation to regulate gene expression [45], accordingly inhibition of JNK-1 by siRNA or chemical inhibitor in LCLs resulted in the decrease of histone H4 hyperacetylation at DNp73a promoter ( Figure 6E and F). In addition, the recruitment of p73 to the DNp73a promoter in LCL is strongly reduced upon JNK-1 inhibition ( Figure 6F).
Immunoblotting showed that EZH2 is weakly detected in primary B cells, while its protein levels are considerably elevated in LCLs ( Figure 6G and data not shown). Similarly, expression of LMP-1 in RPMI cells infected with EBV-DLMP-1 led to a substantial increase in EZH2 protein levels ( Figure 6H). To determine whether the increase of EZH2 levels and decrease of its recruitment to DNp73 promoter in LMP-1 cells was due to changes in its localization, we performed cellular fractionation experiments followed by immunoblotting. LMP-1 expression did not alter EZH2 cellular localization, which appeared to be exclusively nuclear in primary B cells and LCLs ( Figure 6I). However, immuno-fluorescence experiments with an anti-EZH2 antibody highlighted a different pattern of staining in primary B cells and LCLs, indicating that LMP-1 induced a redistribution of EZH2 in the nucleus ( Figure 6J). Indeed, EZH2 staining appears to be punctuated in primary B cells, while it is more diffuse in LCLs ( Figure 6J).
Together the data show that DNp73a expression mediated by EBV LMP-1 correlates with the release of EZH2 from the DNp73 promoter, which results in the opening of the chromatin and in an increased access to transcription factors.

DNp73a expression is required for EBV infected cells survival
To investigate the role of DNp73a in EBV infected cells, we down-regulated its protein levels by expressing an anti DNp73a anti sense oligo-nucleotide (AS). The sense oligo-nucleotide (S) was used as negative control. Figure 7A shows that DNp73a was efficiently down-regulated in a dose-dependent manner by AS in LCLs. Most importantly, DNp73a down-regulation led to PARP cleavage, indicating that DNp73a is involved in the inhibition of apoptosis in EBV infected cells ( Figure 7A). FACS analysis confirmed that transfection with AS promoted cellular death ( Figure 7B). To gain more insight into the biological significance of DNp73a expression in EBV infected cells, we compared the transcriptome profiling by RNA-seq of LCLs transfected by S or AS at two different concentrations. The expression levels of approximately 253 genes were found differentially expressed (214 up-regulated, and 39 down-regulated, p value,0.01) in cells transfected with S and AS (Tables S2 and S3, supplementary material). Functional analysis of the data was conducted as explained in Materials and Methods. It has been previously shown that DNp73a, but not DNp63a, plays a role in nervous system development and in the prevention of nerve growth factor-induced The real time PCR and the quantification of the % of p73 binding to the p73-RE within the DNp73 promoter, was performed as explained in the legend of Figure 4A. The data are the mean of three independent experiments. The difference in the p73 levels recruited to DNp73a promoter in presence and absence of LMP-1 is statistically significant ((p = 0.000254) (C) After the DNA pull-down assay the levels of p53 and p73 proteins binding to biotinylated DNA probe were analysed by immunoblotting. (D) LCLs were transfected with scrambled RNA and siRNA for p53 (sip53) or transduced with lentivirus carrying two different OmicsLink p73 shRNAs (shp73-1, shp73-2) and scramble shRNA. Thirty-six hours after transfection or one-week post lentivirus transduction cells were collected and processed for RNA extraction and cDNA synthesis. The levels of p53, p73 and DNp73a transcript were determined by quantitative RT-PCR. The data are the mean of three independent experiments. The difference in mRNA DNp73a levels in scramble or sip53 transfected cells was not significant, while it was statistically significant between scramble and shp73-1 (p value = 0.0022) and scramble and shp73-2 (p value = 0.005). doi:10.1371/journal.ppat.1003186.g004 p53-mediated apoptosis [46]. Accordingly, we observed that the decrease in DNp73a levels by AS in LCL also led to the deregulation of several genes encoding products that are involved in development, e.g. in neurogenesis and in the regulation of apoptosis in neurons (Tables S2 and S3, supplementary material), indicating the specificity of our approach and validating our RNA seq analysis. Most importantly, AS DNp73a up-regulated genes encoding proteins, which play crucial roles in cellular transformation processes, such as apoptosis, cell cycle, DNA-repair and signaling pathways, i.e. NF-kB, Notch, RAS, and toll-like receptors (TLRs) (Table S2 and S3, supplementary material). In agreement with the functions of DNp73a as an antagonist of p53, several p53-regulated pro-apoptotic genes were found up-regulated in LCLs transfected by AS ( Figure 7C and Tables S2 and S3, supplementary material). Interestingly, the inhibition of DNp73a by AS in LCLs resulted in a strong increase of PLK2 mRNA levels. PLK2 is a p53-regulated gene and encodes a serine threonine kinase which is involved in cell cycle regulation and cellular response to stresses. Its expression has been often found silenced by promoter methylation in Burkitt's Lymphomas [47]. ChIP experiments in SaOS-2 cells expressing HA-tagged-DNp73a demonstrated its binding to the promoter of PLK2 to the p53 binding site 1, which was further increased in the presence of LMP-1, while p73 recruitment to the same promoter was not influenced by the viral oncoprotein ( Figure 7D). To corroborate the RNA-seq data, we have down-regulated DNp73a and determined PLK2 mRNA levels by quantitative PCR. Figure 7E shows that PLK2 and Pig3 are down-regulated in LCL in comparison to primary B cells. In addition down-regulation of DNp73a resulted in a rescue of their expression confirming the RNA seq data. Another gene that was found up-regulated in DNp73a AS-transfected LCLs is KLHDC8B which also appears to be associated with lymphomagenesis and is often found mutated in familiar and sporadic Hodgkin lymphomas [48]. KLHDC8B mRNA levels were increased by 3.66 and 18.44 folds in LCLs transfected with low and high doses of DNp73a AS, respectively (p value = 0.001693). Taken together, these data show that in LCL, DNp73a plays a key role in regulating cellular genes, the products of which exert important functions in cellular transformation, including two genes, PLK2 and KLHDC8B, that have been previously reported to be associated with lymphomagenesis.

Discussion
Oncogenic viruses share the ability to target key pathways involved in preventing cellular transformation, considerably increasing the probability of an infected cell to evolve towards malignancy. One of the best characterized mechanisms of oncogenic viruses is the ability to inhibit the function of the tumor suppressor p53, a transcription factor that can trigger cell cycle arrest or apoptosis in response to stress or DNA damage [49]. Many oncogenic viruses, such as HR mucosal HPV types [15], EBV [10,12,14], Human T-cell Lymphotropic Virus (HTLV-1) [50,51], Kaposi's sarcoma-associated herpesvirus (KSHV) [52][53][54] have developed strategies to inactivate p53. We have recently described a novel mechanism of deregulation of p53 transcriptional functions by the beta cutaneous HPV38 which appears, together with other beta HPV types, to be linked to skin carcinogenesis. HPV38 E7 oncoprotein promotes accumulation of DNp73a increasing its transcription and protein half-life ( [20,21] and our unpublished data). In turn, DNp73a competes with p53 for binding to p53 RE elements, preventing the activation of p53regulated genes. Although the involvement of HPV38 in NMSC is still under debate, the demonstration that also other wellestablished oncogenic viruses promote DNp73a accumulation will further highlight the importance of the event and corroborate the potential role of HPV38 in human carcinogenesis. In this study, we show that the oncoprotein LMP-1 from EBV activates the transcription of DNp73, favoring the recruitment of p73 to cis p53 element of DNp73 p2 promoter. We also demonstrated that LMP-1-mediated up-regulation of DNp73a transcription is dependent on JNK-1, a kinase strongly activated by LMP-1. JNK-1 inhibition by different means strongly decreased DNp73a expression in EBVinfected cells. In addition, expression of ectopic levels of JNK-1 in the EBV-negative B-lymphoma cell line, BJAB, resulted in the activation of DNp73a transcription. Accordingly, LMP-1 mutants lacking the JNK-1 activating domain (CTAR2) did not influence the DNp73a expression levels. It is well established that several amino acid residues of p73 are phosphorylated by JNK-1 [34]. Therefore, it is likely that p73 recruitment to the DNp73 promoter is mediated by its JNK-1-dependent phosphorylation. Additional experiments are required to confirm this hypothesis and establish whether the p73 affinity for DNp73 promoter is determined by phosphorylation of one or more specific amino acids. Previous studies have shown that inhibition of JNK-1 in LMP-1 expressing cells led to decrease of cdc2 levels and cell cycle arrest [55]. In our experimental model the inhibition of cdc2 in LCLs by the chemical inhibitor roscovitine slightly affected DNp73a levels (data not shown).
JNK-1 could also induce DNp73 transcription by an alternative mechanism via activation of the proto-oncogene c-Jun. It has been shown that p73 acts in synergistic manner with c-Jun in promoting cellular survival [56]. This event is well explained by their cooperative ability to activate the transcription of specific subsets of cellular genes. ChIP-seq experiments have revealed the presence of AP1-binding motifs in close proximity to the p73 cis elements in promoters of genes encoding proteins with antiapoptotic functions [57]. Brigati et al. have shown that TPA treatment of Germinal Center B cells, able to induce DNp73a expression, also leads to binding of c-Jun to an AP1 site which was located on the promoter of DNp73a, just upstream the p53/p73 RE [28]. According to these findings, we observed that in LCLs c-Jun is recruited to an AP1 cis element closely located to the p53/ p73RE of DNp73 promoter (our unpublished data).
ChIP experiments in primary and EBV-immortalized B cells showed that activation of DNp73 promoter by the recruitment of p73 correlated with the displacement of the polycomb 2 complex component EZH2 and epigenetic changes. The apparently paradoxical finding that EBV infected B cells, despite the increased intracellular levels of EZH2, show reduced amount of EZH2 and lower levels in H3K27 methylation on the promoter of DNp73, recalls the scenario observed in HPV16 E6/E7 expressing cells [58]. Hyland et al. observed increased levels of EZH2 in the presence of HPV16 E6 and E7 proteins, which correlated with a decrease of H3K27 methylation. The authors explained that this phenomenon was due to an increase in KDM6A and KDM6B levels, two demethylase enzymes, and a decrease in BMI1, a Polycomb1 protein which stabilizes Polycomb 2-mediated methylation. According to this model, EBV is able to trigger accumulation of KDM6B via LMP-1 ( [59] and our unpublished data) as well as a reduction of BMI1 levels (our unpublished data). Accumulation of EZH2 in cells expressing LMP-1 could be a consequence of post-translational modifications that negatively regulate its enzymatic activity. Accordingly, it has been previously shown that phosphorylation of EZH2 by AKT on serine 21 suppresses methylation of lysine 27 in Histone 3 [60]. It has been reported that EBV LMP-1 triggers the AKT pathway [61] which is often found activated in NPC and Hodgkin's lymphomas [62,63]. Based on these findings, we could speculate that the loss of EZH2 recruitment to DNp73 promoter is due to serine 21 phosphorylation. We are currently assessing this hypothesis.
High levels of p73 and DNp73 have been observed in B cell chronic lymphocytic Leukemia [64]. Although resting B cells do not express DNp73, epigenetic changes leading to DNp73 upregulation were observed in the activated B cells compartment of the germinative center of the tonsil. Thus, it is likely that the mechanisms characterized in EBV-infected cells in this study may also occur in different scenarios independently of the presence of the viral oncoprotein.
To evaluate the biological significance of EBV-mediated DNp73a over-expression in the transformation of B cell, we down-regulated DNp73a expression by AS in LCL, and compared the cellular expression profiling with one of the S transfected LCL. Decrease in DNp73a levels led to the alteration of the expression of cellular genes linked to neurogenesis as well as to the regulation of apoptosis in neurons. These results are consistent with the known in vivo functions of DNp73 in brain development and in the prevention of nerve growth factor induced p53-mediated apoptosis [46]. An additional cluster of genes that appeared downregulated by DNp73a in LCL is a group of Homeobox genes. To our knowledge, this is the first time that DNp73a has been shown to be implicated in the regulation of HOX genes. Since both HOX genes and DNp73a are aberrantly expressed in cancer cells, these findings, if confirmed, could further contribute to the understanding of the events associated with carcinogenesis [22,65].
Loss of PLK2 expression by promoter CpG island methylation is one of the most common epigenetic events in B-cell lymphomas [47]. It is worth noting that we found PLK2 gene strongly upregulated upon inhibition of DNp73a in LCL. It is well known that PLK2 promoter is positively regulated by p53. Thus, it is highly likely that DNp73a induces PLK2 down-regulation by altering the p53 transcriptional function. For the first time, our data provide evidence for a link between PLK2 expression silencing and DNp73a in EBV-infected cells. Our findings also suggest that Figure 6. EBV infection of B cells determines epigenetic changes on the p2 promoter. (A and B). Primary B cells were infected and immortalized with EBV (LCL). At the day of the extraction and 2-weeks post infection, primary and immortalized B cell respectively, were fixed and processed for quantitative ChIP analysis. (A) ChIP was carried out using EZH2, Histone 3 Trimethylated Lysine 27 (H3K27), Histone 3 acetylated Lysine 9 (H3K9Ac) antibodies and IgG antibody as negative control. After ChIP the eluted DNA was used as template for quantitative PCR with primers spanning the p73RE within the DNp73 promoter. Part of the total chromatin fraction (1/10) was processed at the same time and used as input. After subtracting the background of the unspecific binding (ChIP for IgG), the amount of promoter specifically bound by each protein was expressed as a percentage of the total amount of DNp73 promoter (% of input). (B) Quantitative-ChIP for p73 was carried out and analyzed as described in the legend of Figure 6A. The data shown in (A) and (B) are the mean of three independent experiments with primary B cells of three different donors. The differences between the % of binding to the promoter in primary B cells and in LCL are significant (p value = 0.01 for EZH2, p value = 0.01 for H3K27Met, p value = 0.002 for H3K9Ac). (C) Primary B cells or primary B cells infected with wild-type EBV (EBV) or EBV lacking the entire LMP-1 gene (EBVDLMP-1) were fixed and processed for EZH2 ChIP by using the LowCell ChIP Kit (Diogenode). Quantitative ChIP analysis was performed according to the manufacturers' protocol. The data are the mean of two independent experiments. The difference between the % of EZH2 binding to p2 in primary and LCL is significant (p value = 0.01). (D) RPMI-pLXSN (pLXSN) and RPMI-pLXSN-LMP-1 (LMP-1), RPMI EBV and RPMI EBVDLMP-1 were fixed and processed for ChIP for EZH2 as described in figure 6C. The difference in the levels of EZH2 recruited to DNp73a promoter in the different conditions is significant (pLXSN vesus LMP-1 p value = 0.038, EBVDLMP-1 versus EBV p value = 0.008). (E) Primary and LCLs non-transfected or transfected with scramble (Scr) or JNK-1 siRNA were fixed and processed for quantitative ChIP analysis using an antibody against the acetylated form of histone H4 following the procedure described in the legend of Figure 6A. The data are the mean of three independent experiments. The difference in the levels of H4Ac binding to DNp73a promoter in the different conditions is significant (primary B cells versus LCL p value = 0.002, scramble siRNA versus JNK-1 siRNA p value = 0.04). (F) LCLs were cultured in absence (DMSO) or presence of 20 mM of JNK-1 inhibitor for 5 hours, fixed and processed for quantitative ChIP analysis using an antibody against the acetylated form of histone H4 or p73 following the procedure described in the legend of Figure 6A. LMP-1 increases the affinity of DNp73a for PLK2 promoter without altering its protein levels (HA-DNp73a: Figure 7D and our unpublished data). It is possible that the viral oncoprotein, independently of its ability to positively regulate the DNp73a transcription, may increase DNp73a affinity for PLK2 promoter by promoting post-translational modifications.
Similarly to PLK2, KLHDC8B has been linked to lymphomagenesis. Multiple cases of Hodgkin's lymphoma with translocations or polymorphisms affecting KLHDC8B have been reported in the same family [66], indicating that inhibition of its expression plays a role in B cell transformation. Our data show that the expression of KLHDC8B was restored upon DNp73a down-regulation. According to the well characterized DNp73a ability to act as an inhibitor of p53, the expression levels of different p53 responsive genes (MDM2, APAF1, GADD45, BAX, CCND1, etc.) increased signifi-cantly after DNp73 down-regulation, leading to apoptosis. Other subgroups of genes that resulted to be regulated by DNp73a are the ones involved in lymphocyte migration and proliferation, cytokine production and innate immune response. As a whole, the RNA seq data indicate that in EBV infected cells DNp73a may contribute to the development of EBV-associated disease in several ways, e.g. by inhibiting apoptosis, promoting B cell growth, as well as by modulating host defence machinery allowing EBV persistence.
In summary, our data underline the important function of DNp73a in EBV-induced cellular transformation, unveiling novel links between its accumulation and deregulation of the expression of many cellular genes. However, the degree to which DNp73a oncogenenic effects exceed tumor suppressor effects of p73 activation remains to be better determined. LCLs treated with S-high (2 mg), AS-low (0.5 mg) and AS-high (2 mg) were used to perform RNAseq. The p53 target genes which were significantly deregulated (p value,0,01 EdgeR software) in S vs. AS were represented in the histogram and expressed as relative RPMK values. (D) SaOS-2 cells were transfected with different pcDNA3 constructs in the indicated combinations. After 36 hours, ChIP was performed using an anti HA-tag antibody and followed by real-time PCR, using primers flanking the p53-RE BS1 within the PLK2 promoter. The percentage of binding of p73 and DNp73 to PLK2 promoter was determined as described in the legend of Figure 4A. (E) LCLs were transfected with 2 mg of DNp73a S (S-high) and 3 increasing concentration of DNp73a AS (0.5 mg, AS-low; 1 mg, AS-medium; 2 mg, AS-high). Thirty-six hours after transfection, cells were collected and processed for RNA extraction. Pig3 and PLK2 mRNA levels were determined by quantitative RT-PCR. The data are the mean of two independent experiments. The difference of Pig3 or PLK2 mRNA levels in LCLs transfected with S and AS is statistically significant (p values = 0.02 and 0.01 for Pig3 and PLK2 respectively). doi:10.1371/journal.ppat.1003186.g007
Immuno-fluorescence in primary and immortalized B cells was performed as described in Fathallah et al. [67].
For FACS staining, cells were collected and washed twice in PBS, then stained with Propidium iodide (PI) at the final concentration of 5 mg/ml. Subsequently, cells were analyzed for the % of dead cells by FACS CANTO (Becton Dickinson).

Gene expression silencing
Gene silencing of SAPK/JNK was performed using SignalSilence SAPK/JNK siRNAI (6269 cell signalling). Cells (8610 5 ) were transfected with siRNA to the final concentration of 100 nM by oligofectamine (invitrogene) according to the manufacture protocol. p53 gene silencing was performed as in Accardi et al. [20]. DNp73a levels were down-regulated by electroporating 1.5610 6 cells with either 0.5 or 2 mg of AS or S oligos (for AS and S oligos sequences please see [20]). Cells electroporation was performed by Neon Transfection System, using a pulse voltage of 1350 v and a pulse width of 30 ms. To specifically silence p73 isoform we used the target sequence: 59-CAGACAGCACC-TACTTCGA-39 spanning from +71 to +90 bp downstream of the transcription start codon was cloned in OmicsLink shRNA Expression system containing a puromycin selection marker (HSH018180-6-HIVmH1, OS395979, GeneCopoeia). As negative control, a scrambled shRNA (sH1) was used. Lentivirus production was performed as previously described [70]. Lentiviral suspension was added to 1.5610 6 LCLs and selection with puromycin (0.8 mg/ml) was initiated 24 hours later. One week post infection cells were collected and processed for the different experiments.

RT-PCR and Quantitative PCR
Total cellular RNA was extracted from cells using the Absolutely RNA Miniprep kit (Stratagene). RNA Reverse transcription to cDNA was carried out by RevertAid H Minus M-MuLV Reverse Transcriptase (MBI Fermentas) according to manufacturer's protocol. Quantitative PCR (Q-PCR) was performed in duplicate in each experiment as previously described [67]. The primer sequences used for RT and Q-PCR are indicated in Table S1A (supplementary material).

DNA pull-down
A biotinylated fragment of DNp73 promoter was generated by PCR using the genomic DNA as template and a biotinylated primer Fw 59-Btndt CTGGTGGGTTTAATTA-39 and a nonbiotinylated primer Rev 59-AGGAGCCGAGGATGCTGG-39 (Sigma). Cells were re-suspended in lysis buffer HKMG (10 mM HEPES, pH7.9, 100 mM KCl, 5 mM MgCl2, 10% Glycerol, 1 mM DTT and 0.5% NP-40) incubated in ice for 10 min, then lysed by sonication (25% Amp, 1 min). One mg of total cellular extracts was pre-cleared with 40 ml of streptavidine-agarose beads (Amersham Bioscience) for 1 hour at 4uC, and then incubated with 2 mg of purified DNA biotinylated probe for 16 hours at 4uC. Poly dI-dC (40 mg) was added to the reaction to avoid unspecific binding. DNA bound proteins were recovered by incubating with 60 ml of streptavidine-agarose beads for 1 hour at 4uC and washed several times with HKMG buffer [71]. Beads were resuspended in 16 SDS-PAGE loading buffer and analyzed by immunoblotting.

RNA sequencing
RNA integrity and quantification of the total cellular RNA from LCLs tranfected with DNp73a AS and S oligo-nucleotide were characterized by measuring the 28s/18s rRNA ratio and RIN using the Agilent 2100 bioanalyzer instrument, and the Agilent RNA 6000 Nano kit. 5 mg of total cellular RNAs was depleted from rRNA using the Invitrogen RiboMinus Eukaryote kit according to manufacturer's standard protocol. The absence of 28s/18s rRNA was checked on the Agilent 2100 bioanalyzer instrument. Five hundred ng of each sample were enzymatically fragmented using 1 unit of RNase III provided in the SOLiD Total RNA-seq Kit, incubated at 37uC for 10 minutes and cleaned up using the Invitrogen RiboMinus Concentration Module according to manufacturer's standard protocol. RNA yield and size distribution were assessed with the Agilent 2100 Bioanalyzer instrument and the Agilent RNA 6000 Pico kit. The amplified whole transcriptome library for each sample was constructed according to Lifetechnologies's SOLiD Total RNAseq Kit protocol (PN 4452437 Rev.B). To summarize, adaptors were hybridised and ligated to 100 ng of fragmented rRNAdepleted total RNAs followed by the construction and subsequent purifications of cDNAs using successively the SOLiD Total RNAseq Kit and the Agencourt AMPure beads. cDNAs were then barcoded, amplified with 15 cycles of PCR and purified using the Invitrogen PureLink PCR Micro Kit. Yield and size distribution of the amplified DNA libraries were assessed with the Agilent 2100 Bioanalyzer instrument and the Agilent DNA 1000 kit. After minimizing the DNA in the 25-200 bp range, 0.4 pM of each barcoded libraries were pooled at equimolar concentrations prior to template bead preparation, in which the pooled library is clonally amplified by emulsion PCR following the Lifetechnologies's SOLiD EZ bead E80 protocols (PN 4441486 Rev. D, 4443494 Rev. D, 4443496 Rev. D). Two hundred and forty ml of emPCR beads were 39 modified and deposited on 4 lanes FlowChip before being incubated for 60 minutes at 37uC. The forward 50 bp reads sequencing chemistry was applied.

RNA seq data analysis
The secondary and tertiary analyses was done with LifeScope software v. 2.5.1 from Life Technologies (Build ID:LifeScope-v2.5.1-r0_102906_20120406100430) The raw data (xsq files) from each lane were grouped per sample (based on the barcodes) before launching the standard RNA seq workflow on the 3 samples (EBV_sense, EBV_antisens1, EBV_antisens2). We kept all the standard parameters as advised by Life Technologies. This workflow includes 3 modules: the ''mapping analysis'' for which we used hg19 as reference genome, the ''coverage analysis'' and the ''count known genes and exons analysis''.
After reads mapping, the R/Bioconductor package edgeR (empirical analysis of digital gene expression data in R) was used to study differential gene expression [72]. After fitting a negative binomial model, data obtained from antisense samples were grouped before applying the ''common dispersion'' function in edgeR. Next, differential gene expression was determined using the exact test. Heatmaps and gene set expression comparisons were performed with BRB-ArrayTools software Version 4.2.1. To this end, reads were RPKM (Reads Per Kilobase of exon model per Million mapped reads) normalized and corresponding gene lists were filtered for selected pathways (Table S3, supplementary material).

Statistical analyses
Statistical significance was determined by Student T test. The p value of each experiment is indicated in the corresponding Figure  legend. Error bars in the graphs represent the standard deviation.