Methylation of discrete sites within the enhancer region regulates the activity of the Epstein-Barr virus BamHI W promoter in Burkitt lymphoma lines.
Eight of the nine viral antigens known to be expressed in in vitro Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines are downregulated in EBV-carrying Burkitt lymphomas (BL). Only EBNA1 can be detected in BL biopsies and BL-derived cell lines that maintain the representative phenotype during culture in vitro (group I BL lines). This restricted pattern of viral gene expression is accompanied by extensive EBV DNA methylation and can be reversed by treatment with the demethylating agent 5-azacytidine. Transcription of the genes encoding the six transformation-associated EBNAs can be initiated from one of two promoters located in the BamHI C and W regions, respectively, of the virus genome. We show that discrete sites within the BamHI W enhancer region are methylated in the group I BL lines Rael, Cheptage, and Elijah and become unmethylated after 5-azacytidine treatment that induces the expression of EBNA2. Demethylation correlates with activation of transcription from the BamHI W promoter as determined by S1 protection analysis. Reporter plasmids in which the W enhancer sequences were linked to the chloramphenicol acetyltransferase gene were active in untreated Rael, Cheptage, and Elijah cells, demonstrating that all of the required transcription factors are present in group I BL cells. Conversely, in vitro methylation of the enhancer sequences abolished their activity. The results suggest that methylation of control regions in the EBV genome may play a critical role for the regulation of viral gene expression in tumor cells.
Department of Clinical Chemistry, Gothenburg University, Sahlgren's Hospital, Sweden.
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Jansson
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Masucci
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Rymo
D000956:Antigens, Viral / Q000235:genetics* / Q000378:metabolism; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D002051:Burkitt Lymphoma; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D019309:Epstein-Barr Virus Nuclear Antigens; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D008745:Methylation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D000956:Antigens, Viral; D004274:DNA, Recombinant; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D015245:Deoxyribonuclease BamHI; D001374:Azacitidine