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J. Virol., Sep 1997, 6445-6454, Vol 71, No. 9
Copyright © 1997, American Society for Microbiology

Mapping promoter regions that are hypersensitive to methylation- mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter

KD Robertson and RF Ambinder
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

Methylation-associated transcriptional repression is recognized in many settings and may play a role in normal differentiation and in tumorigenesis. Both sequence-specific and nonspecific mechanisms have been elaborated. Recently, we have presented evidence that methylation- associated inhibition of the Epstein-Barr virus (EBV) major latency promoter (BamHI C promoter or Cp) in Burkitt's lymphoma and Hodgkin's disease may play an important role in the pathogenesis of these tumors by protecting them from CD8+ cytotoxic T-cell immunosurveillance. The mechanism of transcriptional repression may relate to specific inhibition of the binding of a cellular transcription factor by methylation. To dissect the viral promoter with regard to transcriptional sensitivity to methylation, we have devised an assay that allows the methylation of discrete regions of reporter plasmids. During the course of the assay, methylation patterns appeared to be stable; there was no evidence of either spread or reversal of the imposed methylation pattern. Application of the assay to the 3.8-kb region upstream of the major EBV latency promoter with natural Cp reporter plasmids showed that sensitivity to methylation is not homogeneously distributed but is concentrated in two discrete regions. The first of these methylation-hypersensitive regions (MHRI) is the previously identified EBNA-2 response element, which includes the methylation-sensitive CBF2 binding site. The second (MHRII) is a sequence further downstream whose potential role in methylation- mediated transcriptional repression had been previously unsuspected. In chimeric enhancer/promoter plasmids, methylation of this downstream region was sufficient to virtually abolish simian virus 40 enhancer- driven transcription. Further dissection indicated that methylation of the EBNA-2 response element (MHRI) was sufficient to abolish EBNA-2- mediated Cp activity while methylation of a region including the EBNA-2 response element and downstream sequence (MHRI and MHRII) was sufficient to abolish all Cp-mediated reporter activity, including that driven by the EBNA-1-dependent enhancer in the origin of plasmid replication, oriP.

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