Methylation ofDiscrete Sites within theEnhancer Region Regulates theActivity oftheEpstein-Barr Virus BamHIW Promoter in Burkitt LymphomaLines

Eight ofthenineviral antigens knowntobeexpressed ininvitro Epstein-Barr virus(EBV)-transformed B-lymphoblastoid cell lines aredownregulated inEBV-carrying Burkitt lymphomas (BL). OnlyEBNA1canbe detected inBLbiopsies andBL-derived cell lines thatmaintain therepresentative phenotype during culture in vitro (group IBLlines). Thisrestricted pattern ofviral geneexpression isaccompanied byextensive EBVDNA methylation andcanbereversed bytreatment withthedemethylating agent 5-azacytidine. Transcription ofthe genesencoding thesixtransformation-associated EBNAscanbeinitiated fromoneoftwopromoters located in theBamHICandW regions, respectively, ofthevirus genome.We showthatdiscrete sites within theBamHI W enhancer region are methylated inthegroupIBL lines Rael, Cheptage, andElijah andbecome unmethylated after 5-azacytidine treatment thatinduces theexpression ofEBNA2.Demethylation correlates withactivation oftranscription fromtheBamHIW promoter as determined bySiprotection analysis. Reporter plasmids inwhichtheW enhancer sequenceswerelinked tothechloramphenicol acetyltransferase genewere active inuntreated Rael, Cheptage, andEliahcells, demonstrating thatalloftherequired transcription factors are present ingroup IBL cells.Conversely, invitro methylation oftheenhancer sequencesabolished their activity. Theresults suggest that methylation ofcontrol regions intheEBVgenome may play a critical role fortheregulation ofviral geneexpression intumorcells. Epstein-Barr virus (EBV)isa potenttransforming agent forhumanB lymphocytes andhasbeenimplicated inthe pathogenesis ofhumanmalignancies like Burkitt lymphoma (BL),nasopharyngeal carcinoma, andlymphoproliferative disorders inimmunocompromised individuals (for areview,