Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.
The Epstein-Barr Virus (EBV) latency C promoter (Cp) is the origin of transcripts for six viral proteins. The promoter is active in lymphoblastoid B-cell lines but silent in many EBV-associated tumors and tumor cell lines. In these latter cell lines, the viral episome is hypermethylated in the vicinity of this promoter. We show that in such a cell line (Rael, a Burkitt's lymphoma line), 5-azacytidine inhibits DNA methyltransferase, brings about demethylation of EBV genomes, activates Cp transcription, and induces the expression of EBNA-2. Investigation of the phenomenon demonstrates the importance of the methylation status of a particular CpG site for the regulation of the Cp: (i) genomic sequencing shows that this site is methylated when the Cp is inactive and is not methylated when the promoter is active; (ii) methylation or transition mutation at this site abolishes complex formation with a cellular binding activity (CBF2) as determined by electrophoretic mobility shift analyses, competition binding analyses, and DNase I footprinting; and (iii) a single C --> T transition mutation at this site is associated with a marked reduction (> 50-fold) of transcriptional activity in a reporter plasmid. Thus, the CBF2 binding activity is shown to be methylation sensitive and crucial to EBNA-2-mediated activation of the Cp.
Robertson
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KD
Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
Hayward
S D
SD
Ling
P D
PD
Samid
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D
Ambinder
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RF
D000956:Antigens, Viral / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology*; D001483:Base Sequence; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D018983:DNA Footprinting; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019309:Epstein-Barr Virus Nuclear Antigens; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D008745:Methylation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects; D017735:Virus Latency
D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D019309:Epstein-Barr Virus Nuclear Antigens; D012333:RNA, Messenger; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine