TY - JOUR
T1 - Transcriptional activation of the Epstein-Barr Virus latency C promoter after 5-azacytidine treatment
T2 - Evidence that demethylation at a single CpG site is crucial
AU - Robertson, Keith D.
AU - Hayward, S. Diane
AU - Ling, Paul D.
AU - Samid, Dvorit
AU - Ambinder, Richard F.
PY - 1995/11
Y1 - 1995/11
N2 - 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).
AB - 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).
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U2 - 10.1128/MCB.15.11.6150
DO - 10.1128/MCB.15.11.6150
M3 - Article
C2 - 7565767
AN - SCOPUS:0028840682
SN - 0270-7306
VL - 15
SP - 6150
EP - 6159
JO - Molecular and cellular biology
JF - Molecular and cellular biology
IS - 11
ER -