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Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1in tumors of epithelial, NK- and B-cell origins
© Li et al; licensee BioMed Central Ltd. 2012
Received: 18 October 2011
Accepted: 29 March 2012
Published: 29 March 2012
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.
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.
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.
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.
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 . Two immediate-early (IE) proteins, BZLF1 (Zta) and BRLF1 (Rta), are essential to the switch from latent to lytic infection .
Epigenetic regulation of EBV genome is a fundamental regulatory mechanism determining different types of EBV infections in its associated tumors [5–8]. Several latent or lytic genes, including EBV nuclear antigens (EBNA-2, 3A, 3B, 3 C), latent membrane protein 1 (LMP1), IE antigens (Zta, Rta) and lytic cycle viral kinases, have been identified tightly controlled by the CpG methylation of various EBV promoters [1, 5, 9, 10], such as W promoter (Wp), C promoter (Cp), Q promoter (Qp), F promoter (Fp), LMP1 promoters (ED-L1 and ED-L1E promoters), Z promoter (Zp) and R promoter (Rp). The precise epigenetic regulation ensures the production of viral progeny without releasing viral antigens detectable by host immune system. Meanwhile, reactivation of viral genes from latency by demethylation agents could serve as a therapeutic strategy for EBV-associated tumors [1, 10–12].
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 [15–17], 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.
Cell lines and tumor samples
B95-8 is an EBV-immortalized lymphoblastoid cell line. Rael, Akata, Wanyonyi (Wan), Raji, Namalwa, and AG876 are EBV-positive BL cell lines [13, 14]. SNK6 and NK-YS are EBV-positive NK-cell lymphoma cell lines [13, 14]. C666-1 is EBV-positive NPC cell line . SNU719 and YCCEL1 (Rha, Tao, et al, unpublished) are EBV-positive gastric carcinoma cell lines [19, 20]. All cell lines were cultivated at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1 mmol/L glutamine, 100 U/ml penicillin and streptomycin (Invitrogen, CA). Cell lines were maintained at 37°C in cRPMI 1640. 5-aza-dC (Sigma-Aldrich, St Louis, MO) was used at different concentrations and time to treat Rael, NK-YS and C666-1 cell lines. Other cell lines were treated with 10 μmol/L 5-aza-dC (Aza, Sigma-Aldrich, St. Louis, MO) for 3 days or further treated with 100 nmol/L trichostatin A (TSA, Cayman Chemical Co., Ann Arbor, MI) for additional ~16 h as described previously [21, 22]. Archival tumor DNA samples have been previously described [21, 23–25]. The study was approved by Johns Hopkins Medicine Institutional Review Board.
Semiquantitative reverse transcription (RT)-PCR
Sequences of primers used in this study
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 bisulfite-converted 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
Methylation status of Zp and Rp in EBV-positive cell lines of epithelial, NK- or B-cell origins
Methylation status of Zp and Rp in EBV-associated cell lines and tumors
EBV-associated cell lines and tumors
Zp methylation status
(Methylated CpG sites, %)
Rp methylation status
(methylated CpG sites, %)
Nude mice-passaged tumors
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
Restoration of BZLF1 and BRLF1expression by demethylation in EBV-positive cell lines
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 . 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 EBV-positive tumors with rare cell undergoing spontaneous lytic infection, methylation status represents the latent genome . 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 . 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 .
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 epigenetics-related EBV pathogenesis and further develop target therapy for EBV-associated tumors.
We thank Prof Gopesh Srivastava for the NK-YS and SNK6 cell lines. This study was supported by Joint Research Fund for Hong Kong and Macao Young Scholars (#30928012) and National Natural Science Foundation of China (#81071634, #81172582).
- Tao Q, Young LS, Woodman CB, Murray PG: Epstein-Barr virus (EBV) and its associated human cancers-genetics, epigenetics, pathobiology and novel therapeutics. Front Biosci. 2006, 11: 2672-2713. 10.2741/2000.View ArticlePubMedGoogle Scholar
- Young LS, Rickinson AB: Epstein-Barr virus: 40 years on. Nat Rev Cancer. 2004, 4 (10): 757-768. 10.1038/nrc1452.View ArticlePubMedGoogle Scholar
- Young LS, Murray PG: Epstein-Barr virus and oncogenesis: from latent genes to tumours. Oncogene. 2003, 22 (33): 5108-5121. 10.1038/sj.onc.1206556.View ArticlePubMedGoogle Scholar
- Amon W, Farrell PJ: Reactivation of Epstein-Barr virus from latency. Rev Med Virol. 2005, 15 (3): 149-156. 10.1002/rmv.456.View ArticlePubMedGoogle Scholar
- Niller HH, Wolf H, Ay E, Minarovits J: Epigenetic dysregulation of epstein-barr virus latency and development of autoimmune disease. Adv Exp Med Biol. 2011, 711: 82-102. 10.1007/978-1-4419-8216-2_7.View ArticlePubMedGoogle Scholar
- Niller HH, Wolf H, Minarovits J: Epigenetic dysregulation of the host cell genome in Epstein-Barr virus-associated neoplasia. Semin Cancer Biol. 2009, 19 (3): 158-164. 10.1016/j.semcancer.2009.02.012.View ArticlePubMedGoogle Scholar
- Ambinder RF, Robertson KD, Tao Q: DNA methylation and the Epstein-Barr virus. Semin Cancer Biol. 1999, 9 (5): 369-375. 10.1006/scbi.1999.0137.View ArticlePubMedGoogle Scholar
- Flower K, Hellen E, Newport MJ, Jones S, Sinclair AJ: Evaluation of a prediction protocol to identify potential targets of epigenetic reprogramming by the cancer associated Epstein Barr virus. PLoS One. 2010, 5 (2): e9443-10.1371/journal.pone.0009443.View ArticlePubMedPubMed CentralGoogle Scholar
- Takacs M, Banati F, Koroknai A, Segesdi J, Salamon D, Wolf H, Niller HH, Minarovits J: Epigenetic regulation of latent Epstein-Barr virus promoters. Biochim Biophys Acta. 2010, 1799 (3-4): 228-235.View ArticlePubMedGoogle Scholar
- Tao Q, Robertson KD: Stealth technology: how Epstein-Barr virus utilizes DNA methylation to cloak itself from immune detection. Clin Immunol. 2003, 109 (1): 53-63. 10.1016/S1521-6616(03)00198-0.View ArticlePubMedGoogle Scholar
- Tao Q, Chan AT: Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments. Expert Rev Mol Med. 2007, 9 (12): 1-24.View ArticlePubMedGoogle Scholar
- Chan AT, Tao Q, Robertson KD, Flinn IW, Mann RB, Klencke B, Kwan WH, Leung TW, Johnson PJ, Ambinder RF: Azacitidine induces demethylation of the Epstein-Barr virus genome in tumors. J Clin Oncol. 2004, 22 (8): 1373-1381. 10.1200/JCO.2004.04.185.View ArticlePubMedGoogle Scholar
- Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF: The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive. J Virol. 1998, 72 (9): 7075-7083.PubMedPubMed CentralGoogle Scholar
- Tao Q, Swinnen LJ, Yang J, Srivastava G, Robertson KD, Ambinder RF: Methylation status of the Epstein-Barr virus major latent promoter C in iatrogenic B cell lymphoproliferative disease. Application of PCR-based analysis. Am J Pathol. 1999, 155 (2): 619-625. 10.1016/S0002-9440(10)65157-7.View ArticlePubMedPubMed CentralGoogle Scholar
- Dickerson SJ, Xing Y, Robinson AR, Seaman WT, Gruffat H, Kenney SC: Methylation-dependent binding of the epstein-barr virus BZLF1 protein to viral promoters. PLoS Pathog. 2009, 5 (3): e1000356-10.1371/journal.ppat.1000356.View ArticlePubMedPubMed CentralGoogle Scholar
- Bhende PM, Seaman WT, Delecluse HJ, Kenney SC: The EBV lytic switch protein, Z, preferentially binds to and activates the methylated viral genome. Nat Genet. 2004, 36 (10): 1099-1104. 10.1038/ng1424.View ArticlePubMedGoogle Scholar
- Karlsson QH, Schelcher C, Verrall E, Petosa C, Sinclair AJ: Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein. PLoS Pathog. 2008, 4 (3): e1000005-10.1371/journal.ppat.1000005.View ArticlePubMedPubMed CentralGoogle Scholar
- Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS, Wong N, Whitney BM, Lee JC: Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer. 1999, 83 (1): 121-126. 10.1002/(SICI)1097-0215(19990924)83:1<121::AID-IJC21>3.0.CO;2-F.View ArticlePubMedGoogle Scholar
- Oh ST, Seo JS, Moon UY, Kang KH, Shin DJ, Yoon SK, Kim WH, Park JG, Lee SK: A naturally derived gastric cancer cell line shows latency I Epstein-Barr virus infection closely resembling EBV-associated gastric cancer. Virology. 2004, 320 (2): 330-336. 10.1016/j.virol.2003.12.005.View ArticlePubMedGoogle Scholar
- Oh ST, Cha JH, Shin DJ, Yoon SK, Lee SK: Establishment and characterization of an in vivo model for Epstein-Barr virus positive gastric carcinoma. J Med Virol. 2007, 79 (9): 1343-1348. 10.1002/jmv.20876.View ArticlePubMedGoogle Scholar
- Qiu GH, Tan LK, Loh KS, Lim CY, Srivastava G, Tsai ST, Tsao SW, Tao Q: The candidate tumor suppressor gene BLU, located at the commonly deleted region 3p21.3, is an E2F-regulated, stress-responsive gene and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma. Oncogene. 2004, 23 (27): 4793-4806. 10.1038/sj.onc.1207632.View ArticlePubMedGoogle Scholar
- Ying J, Li H, Seng TJ, Langford C, Srivastava G, Tsao SW, Putti T, Murray P, Chan AT, Tao Q: Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene. 2006, 25 (7): 1070-1080. 10.1038/sj.onc.1209154.View ArticlePubMedGoogle Scholar
- Seng TJ, Low JS, Li H, Cui Y, Goh HK, Wong ML, Srivastava G, Sidransky D, Califano J, Steenbergen RD, et al: The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation. Oncogene. 2007, 26 (6): 934-944. 10.1038/sj.onc.1209839.View ArticlePubMedGoogle Scholar
- Wang Y, Li J, Cui Y, Li T, Ng KM, Geng H, Li H, Shu XS, Liu W, Luo B, et al: CMTM3, located at the critical tumor suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis. Cancer Res. 2009, 69 (12): 5194-5201. 10.1158/0008-5472.CAN-08-3694.View ArticlePubMedGoogle Scholar
- Cheng Y, Geng H, Cheng SH, Liang P, Bai Y, Li J, Srivastava G, Ng MH, Fukagawa T, Wu X, et al: KRAB zinc finger protein ZNF382 is a proapoptotic tumor suppressor that represses multiple oncogenes and is commonly silenced in multiple carcinomas. Cancer Res. 2010, 70 (16): 6516-6526. 10.1158/0008-5472.CAN-09-4566.View ArticlePubMedGoogle Scholar
- Tao Q, Huang H, Geiman TM, Lim CY, Fu L, Qiu GH, Robertson KD: Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells. Hum Mol Genet. 2002, 11 (18): 2091-2102. 10.1093/hmg/11.18.2091.View ArticlePubMedGoogle Scholar
- Pritchett RF, Hayward SD, Kieff ED: DNA of Epstein-Barr virus. I. Comparative studies of the DNA of Epstein-Barr virus from HR-1 and B95-8 cells: size, structure, and relatedness. J Virol. 1975, 15 (3): 556-559.PubMedPubMed CentralGoogle Scholar
- Henderson S, Huen D, Rowe M, Dawson C, Johnson G, Rickinson A: Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc Natl Acad Sci USA. 1993, 90 (18): 8479-8483. 10.1073/pnas.90.18.8479.View ArticlePubMedPubMed CentralGoogle Scholar
- Woodcock DM, Adams JK, Cooper IA: Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin, and the effect of DNA replication inhibition. Biochim Biophys Acta. 1982, 696 (1): 15-22.View ArticlePubMedGoogle Scholar
- Chang PJ, Chang YS, Liu ST: Role of Rta in the translation of bicistronic BZLF1 of Epstein-Barr virus. J Virol. 1998, 72 (6): 5128-5136.PubMedPubMed CentralGoogle Scholar
- Chen C, Li D, Guo N: Regulation of cellular and viral protein expression by the Epstein-Barr virus transcriptional regulator Zta: implications for therapy of EBV associated tumors. Cancer Biol Ther. 2009, 8 (11): 987-995.View ArticlePubMedGoogle Scholar
- Heather J, Flower K, Isaac S, Sinclair AJ: The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1. J Gen Virol. 2009, 90 (Pt 6): 1450-1454.View ArticlePubMedPubMed CentralGoogle Scholar
- Bhende PM, Seaman WT, Delecluse HJ, Kenney SC: BZLF1 activation of the methylated form of the BRLF1 immediate-early promoter is regulated by BZLF1 residue 186. J Virol. 2005, 79 (12): 7338-7348. 10.1128/JVI.79.12.7338-7348.2005.View ArticlePubMedPubMed CentralGoogle Scholar
- Bergbauer M, Kalla M, Schmeinck A, Gobel C, Rothbauer U, Eck S, Benet-Pages A, Strom TM, Hammerschmidt W: CpG-methylation regulates a class of Epstein-Barr virus promoters. PLoS Pathog. 2010, 6 (9): e1001114-10.1371/journal.ppat.1001114.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoo CB, Jones PA: Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov. 2006, 5 (1): 37-50. 10.1038/nrd1930.View ArticlePubMedGoogle Scholar
- Taberlay PC, Jones PA: DNA methylation and cancer. Prog Drug Res. 2011, 67: 1-23.PubMedGoogle Scholar
- Egger G, Liang G, Aparicio A, Jones PA: Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004, 429 (6990): 457-463. 10.1038/nature02625.View ArticlePubMedGoogle Scholar
- Lubbert M, Suciu S, Baila L, Ruter BH, Platzbecker U, Giagounidis A, Selleslag D, Labar B, Germing U, Salih HR, et al: Low-dose decitabine versus best supportive care in elderly patients with intermediate- or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. J Clin Oncol. 2011, 29 (15): 1987-1996. 10.1200/JCO.2010.30.9245.View ArticlePubMedGoogle Scholar
- Prebet T, Gore SD, Esterni B, Gardin C, Itzykson R, Thepot S, Dreyfus F, Rauzy OB, Recher C, Ades L, et al: Outcome of High-Risk Myelodysplastic Syndrome After Azacitidine Treatment Failure. J Clin Oncol. 2011, 29 (24): 3322-3327. 10.1200/JCO.2011.35.8135.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang X, Lay F, Han H, Jones PA: Targeting DNA methylation for epigenetic therapy. Trends Pharmacol Sci. 2010, 31 (11): 536-546. 10.1016/j.tips.2010.08.001.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2407/12/125/prepub
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