Transcriptional activation of the Epstein-Barr Virus latency C promoter after 5-azacytidine treatment: Evidence that demethylation at a single CpG site is crucial

Keith D. Robertson, S. Diane Hayward, Paul D. Ling, Dvorit Samid, Richard F. Ambinder

Research output: Contribution to journalArticlepeer-review

104 Scopus citations

Abstract

The Epstein-Barr Virus (EBV) latency C promoter (C(P)) 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 C(P) 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 C(P): (i) genomic sequencing shows that this site is methylated when the C(P) 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 C(P).

Original languageEnglish (US)
Pages (from-to)6150-6159
Number of pages10
JournalMolecular and cellular biology
Volume15
Issue number11
DOIs
StatePublished - Nov 1995

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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