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 (C-p) is the origin of transcripts for six viral proteins, The promoter is active in lymphob lastoid B-cell lines but silent in many EBV-associated tumors and tumo r cell lines, In these latter cell lines, the viral episome is hyperme thylated in the vicinity of this promoter, We show that in such a cell line (Rael, a Burkitt's lymphoma line), 5-azacytidine inhibits DNA me thyltransferase, 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 stat us 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 inactiv e 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 mob ility shift analyses, competition binding analyses, and DNase I footpr inting; and (iii) a single C --> T transition mutation at this site is associated with a marked reduction (> 50-fold) of transcriptional act ivity in a reporter plasmid, Thus, the CBF2 binding activity is showm to be methylation sensitive and crucial to EBNA-2-mediated activation of the C-p.