title: Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins.
abstract: 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.
authors.affiliation: Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer, Hong Kong, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Li
authors.forename: Xianwei
authors.initials: X
authors.lastname: Su
authors.forename: Gigi Ching Gee
authors.initials: GC
authors.lastname: Choi
authors.forename: Ya
authors.initials: Y
authors.lastname: Cao
authors.forename: Richard F
authors.initials: RF
authors.lastname: Ambinder
authors.forename: Qian
authors.initials: Q
authors.lastname: Tao
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D017781:Genes, Immediate-Early / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: C068382:BRLF1 protein, Human herpesvirus 4; C063676:BZLF1 protein, Herpesvirus 4, Human; D017874:Immediate-Early Proteins; D015534:Trans-Activators