Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins

Background Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples. Methods We evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins. Results We found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1. Conclusions Hypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.


Background
Epstein Barr virus (EBV) is a tumor virus associated with multiple human malignancies of lymphoid or epithelial origin, including Burkitt lymphoma (BL), Hodgkin disease (HD), nasopharyngeal carcinoma (NPC), gastric carcinoma (GsCa), nasal NK-lymphoma and posttransplant lymphoproliferative disease (PTLD), with more than 90% of adults infected in the world [1,2]. EBV has two types of infection in cells: latent or lytic. It persists in the human host as lifelong latent infection, which requires periodically reactivation of lytic genes and viral replication for maintaining its latency [3]. Two immediate-early (IE) proteins, BZLF1 (Zta) and BRLF1 (Rta), are essential to the switch from latent to lytic infection [4].
Our previous work characterized the CpG methylation of EBV major latent promoters Qp, Fp and Cp by genomic sequencing [13,14]. Recent studies of IE genes have been focused on the methylation-dependent binding and activation of Zta to Rp and other viral promoters [15][16][17], while the overall methylation status of Zp and Rp in tumor cells still remains unclear. Here, we studied the methylation profiles of Zp and Rp in a series of EBV-positive cell lines and primary tumors of epithelial, NK-or B-cell origin. We also evaluated the effect of demethylation agent on the reactivation of BZLF1 and BRLF1 in EBV-positive cell lines.

Semiquantitative reverse transcription (RT)-PCR
Reverse transcription using random hexamer and RT-PCR using Go-Taq (Promega, Madison, WI) were performed as previously, with GAPDH as a control [26]. Sequences of primers are listed in Table 1.

DNA bisulfite treatment and methylation analysis
Bisulfite modification of DNA, methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS) were carried out as described [14,26]. Bisulfite-treated DNA was PCR amplified with strand-specific primers (for bisulfiteconverted top strand of Zp and Rp) for BGS. MSP and BGS primers of Zp and Rp are listed in Table 1.

Analysis of CpG sites in Z and R promoters
The two IE transcripts derived from EBV genome are shown in Figure 1A, BZLF1 and BRLF1 are located adjacent to each other on the EBV genome. BZLF1 mRNA (~1.0 kb) can be transcribed from both Zp and Rp, but its protein product Zta mainly derived from Zp. BRLF1 mRNA (~4.0 kb) is transcribed from Rp and encodes Rta. We evaluated the distribution of CpG sites and transcriptional regulatory motifs in Zp and Rp. In Zp, only few CpG sites exist in the "ZI" and "ZII" motifs, which are the critical elements for activation of Zp by binding of several transcript factors including CREB, ATF1/2, MEF2D and Smad3/Smad4. The other negative regulatory motifs in Zp, like the proximal ZV element for binding of zinc-finger E-box binding factor (ZEB), and distal elements for binding of transcription factors YY1 and E-box binding protein (E2-2), show scattered distribution of CpG sites ( Figure 1B). In Rp, two Zta-responsive elements (RpZRE2 and RpZRE3) contain several CpG sites, mainly responsible for BRLF1 transcription, while other regulatory elements, such as Sp1, EGR-1, contain only 1-2 CpG sites ( Figure 1C). Thus, promoter CpG methylation of Zp and Rp may regulate BZLF1 and BRLF1 activation in EBV life cycle.

Methylation status of Zp and Rp in EBV-positive cell lines of epithelial, NK-or B-cell origins
We then examined the expression of BZLF1 and BRLF1 in EBV-positive tumor cell lines. Results showed that BZLF1 and BRLF1 were readily expressed in EBV-positive cell lines (C666-1, YCCEL1, B95-8, and AG876), with expression of early lytic gene BHRF1 and late lytic gene BLLF1 also detected in these cell lines. In addition, weak expression of BRLF1 was detected in Raji cells but without BZLF1 (Figure 2A). SUN719 showed only trace expression of BZLF1, BRLF1 and lytic BHRF1 but weak expression of BLLF1.
Next, we used methylation-specific PCR (MSP) to evaluate the promoter methylation of Zp and Rp. Primers were designed to specifically amplify a region containing the dense CpG sites in Zp and Rp. Zp and Rp methylation was detected in all EBV-positive cell lines, while unmethylated alleles were mainly seen in cell lines expressing BZLF1 and/or BRLF1 ( Figure 2B). To further confirm the MSP results and characterize the methylation status of Zp and Rp in more detail, we performed high-resolution bisulfite genome sequencing (BGS) analysis of 18 CpG sites and 20 CpG sites spanning Zp and Rp, respectively. In EBV-positive BL cell lines Real, Akata, Namalwa and Raji, dense methylation was observed at both Zp and Rp, while relatively more unmethylated alleles in Zp and Rp were detected in B95-8 and Wan cell lines which are with high level of spontaneous EBV lytic replication [27,28]. Notably, three CpG sites (#12-14) in Zp were always unmethylated ( Figure 2C, D, Table 2). These results suggest that promoter methylation is closely related to the transcriptional repression of BZLF1 and BRLF1 in EBV-positive cell lines.

Zp and Rp methylation in EBV-positive tumors
EBV-positive tumors of epithelial or lymphoid origin including NPC, BL and PTLD samples, as well as two nude mice-passaged undifferentiated NPC tumors (C15 and C18) were studied. MSP analysis showed that Zp and Rp methylation was detected in virtually all 38 NPC tumors, with unmethylated Zp and Rp only detected in rare cases ( Figure 3A, B), well correlated with the general silencing of BZLF1 and BRLF1 in NPC ( Figure 3C).
We further studied the detailed methylation profiles of Zp and Rp by BGS in EBV-positive tumors. Results revealed that both Zp and Rp were heavily methylated in all studied samples, but relatively more unmethylated alleles in Zp were observed in PTLD patients ( Figure 4A, B). Again, we observed that the three CpG sites (#12- 14) in Zp were unmethylated in virtually all studied tumors ( Figure 4A). These results are summarized in Table 2.

Restoration of BZLF1 and BRLF1 expression by demethylation in EBV-positive cell lines
To determine whether methylation directly mediates the transcriptional repression of BZLF1 and BRLF1, Rael, NK-YS and C666-1 were treated with 5-aza-2'deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor. Rael treated with 5-aza-dC at different concentrations (0.005, 0.015, 0.03, 0.06, 0.5 and 1 μM) for 72 h. MSP analysis showed that unmethylated Rp and Zp alleles were increased in a dose-dependent manner, with significant demethylation of Rp and Zp observed by treatment with 1 μM 5-aza-dC. Subsequently, 1 μM of 5-aza-dC was chosen to evaluate the demethylation of Zp and Rp for different indicated times (5 h, 24 h, 30 h, 48 h) in Rael cell line. It was found that both Zp and Rp were demethylated in a time-dependent manner ( Figure 5A, B). Similarly, NK-YS and C666-1 cells treated with 5-aza-dC at different concentrations showed obvious demethylation of Zp and Rp, compared with untreated cells (Figure 5C, D). Concomitantly, Zp and Rp alleles were partially demethylated after 5-aza-dC treatment by high-resolution BGS analysis ( Figure 5E, F, Table 2), We further found that after 5-aza-dC demethylation or combined with the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) treatment, the expression of BZLF1 and BRLF1 was dramatically increased in EBV-positive epithelial cell lines, as measured by RT-PCR, along with obviously induced expression of early lytic gene BHRF1 and late lytic gene BLLF1 ( Figure 6A, B, C), suggesting the initiation of EBV-lytic cascade by      Figure 6D), indicating epigenetic-mediated silencing of BZLF1 and BRLF1 in EBV-associated epithelial tumors.

Discussion
This study characterized the CpG methylation profiles of EBV immediate-early lytic promoters Zp and Rp in cell lines and tumors of epithelial or lymphoid origin, and further evaluated the reactivation of BZLF1 and BRLF1 by demethylation treatment. We found that Zp and Rp were frequently methylated in all EBV-positive cell lines and tumors, whereas unmethylated Zp and Rp were mainly present in EBV-positive cell lines with lytic activities, along with the expression of BZLF1 and BRFL1. We did not observe major difference in Zp and Rp methylation in cell lines/or tumors of epithelial, NK-or B-cell origin.
We also demonstrated that demethylation of Zp and Rp by treatment with 5-aza-dC alone or combined with TSA resulted in the re-expression of BZLF1 and BRLF1 and activation of EBV lytic cycle. It has been identified that DNA synthesis inhibitors have no effect on DNA methylation by using four different inhibitors of DNA replication [29]. Although DNA synthesis inhibitors will delay some of the cytosine methylation, all delayed DNA methylation will be finally completed prior to the subsequent S phase. Thus, in our study, DNA methylation inhibitors are mainly responsible for Zp and Rp demethylation and initiation of lytic cascade, while other events indirectly leading to EBV reactivation also cannot be ruled out.
In EBV-positive cell lines except for B95-8, Wan and AG876, only very few % of cells or no cell undergoing spontaneous lytic infection without much lytic virion DNA, thus the methylation status of Zp and Rp detected represents the latent viral genome but not the virion genome, while in B95-8, Wan and AG876 cell lines with significant % of cells undergoing spontaneous lytic infection, the methylation status detected probably represents both latent and viral genomes ( Figure 2C, D). Similarly, in EBVpositive tumors with rare cell undergoing spontaneous lytic infection, methylation status represents the latent genome [1]. In our study, lower level of methylation was only observed in B95-8 and Wan cell lines, but not in other EBV-positive cell lines and primary tumors, consistent with the high level of spontaneous EBV lytic replication only in B95-8 and Wan.
The existence of a small proportion of cells expressing viral lytic genes is essential for the success maintenance of EBV latency in host cells with a highly methylated viral genome [1,3]. As viral transactivator proteins, Zta is unique to initiate the entire EBV lytic cascade by transactivating a series of lytic gene promoters, but Rta appears to be more effective in epithelial cells [30,31]. Increased evidences have shown that Zta initiates EBV lytic infection mainly from a methylated viral genome, whereas Rta initiates lytic infection mainly from an unmethylated genome [15,16,32,33]. Rp methylation inhibits Rta expression, however it enhances the ability of Zta to activate Rp [16]. In line with reported studies, we found that either the Rp-ZRE2 (CpG site #13- 14) or/and the ZRE3 (CpG site #15) were heavily methylated in virtually all EBV-positive BL, LCL and NPC cell lines and tumors, but less methylated in Wan and B95-8 cells with basal lytic activity. A CpG methylation-free zone (three CpG sites #12-14) in Zp, located in regulatory elements YY1 and E2-2, is possibly responsible for the initial activation of BZLF1. Thus, Zp and Rp are regulated by both CpG methylation and cellular transcription factors, indicating the complexity of the regulation of BZLF1 and BRLF1 in EBV-associated tumors [34].
DNA methylation plays a crucial role in allowing EBV to escape from the detection of host immune system. Conversely, pharmacologic reversal of viral gene methylation and activation of gene expression would resensitize host's immune surveillance or enhance its response to immunogenic viral antigens [10]. The efficacy of DNA methyltransferase inhibitors in hematologic diseases, including myelodysplastic syndromes (MDS) and acute myeloid leukemia, has been successfully evaluated by multiple clinical trials [35][36][37]. 5-azacytidine has been now approved as the first-line treatment of high-risk MDS [38,39]. Our group has firstly reported the achievement of successful demethylation of EBV viral antigen promoters including Cp, Wp, LMP1p, Zp and Rp, in NPC patients with azacitidine treatment [1,[10][11][12]. When combined with other chemotherapy drugs and histone

Conclusions
Collectively, our study found that frequent silencing of BZLF1 and BRLF1 by hypermethylation of Zp and Rp could be reactivated by demethylation agent, resulting in the initiation of the EBV lytic cascade in EBV-associated tumors. Our study helps to understand epigeneticsrelated EBV pathogenesis and further develop target therapy for EBV-associated tumors.