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Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.
Mol Cell Biol. 1995 Nov; 15(11):6150-9.MC

Abstract

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.

Authors+Show Affiliations

Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7565767

Citation

Robertson, K D., et al. "Transcriptional Activation of the Epstein-Barr Virus Latency C Promoter After 5-azacytidine Treatment: Evidence That Demethylation at a Single CpG Site Is Crucial." Molecular and Cellular Biology, vol. 15, no. 11, 1995, pp. 6150-9.
Robertson KD, Hayward SD, Ling PD, et al. Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial. Mol Cell Biol. 1995;15(11):6150-9.
Robertson, K. D., Hayward, S. D., Ling, P. D., Samid, D., & Ambinder, R. F. (1995). Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial. Molecular and Cellular Biology, 15(11), 6150-9.
Robertson KD, et al. Transcriptional Activation of the Epstein-Barr Virus Latency C Promoter After 5-azacytidine Treatment: Evidence That Demethylation at a Single CpG Site Is Crucial. Mol Cell Biol. 1995;15(11):6150-9. PubMed PMID: 7565767.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial. AU - Robertson,K D, AU - Hayward,S D, AU - Ling,P D, AU - Samid,D, AU - Ambinder,R F, PY - 1995/11/1/pubmed PY - 1995/11/1/medline PY - 1995/11/1/entrez SP - 6150 EP - 9 JF - Molecular and cellular biology JO - Mol Cell Biol VL - 15 IS - 11 N2 - 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. SN - 0270-7306 UR - https://www.unboundmedicine.com/medline/citation/7565767/Transcriptional_activation_of_the_Epstein_Barr_virus_latency_C_promoter_after_5_azacytidine_treatment:_evidence_that_demethylation_at_a_single_CpG_site_is_crucial_ L2 - http://mcb.asm.org/cgi/pmidlookup?view=long&pmid=7565767 DB - PRIME DP - Unbound Medicine ER -