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. 2000 Nov;74(22):10468-79.
doi: 10.1128/jvi.74.22.10468-10479.2000.

Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation

R J Tierney  1 , H E Kirby, J K Nagra, J Desmond, A I Bell, A B Rickinson
Affiliations

Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation

R J Tierney et al. J Virol. 2000 Nov.

Abstract

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

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Figures

FIG. 1
FIG. 1
Analysis of EBV transcription in a representative BL biopsy specimen, SAV; a recently established BL cell line, EZE; and four long-established LCLs, WBW, XMW, JY, and B95.8. Corresponding data for experimentally infected B cells (2 days postinfection) are included as a reference. Shown are the results of RT-PCR analysis using primer-probe combinations specific for Wp-initiated transcripts, Cp-initiated transcripts, Wp- or Cp-initiated Y3/U/K-spliced EBNA1 transcripts, Fp- or Qp-initiated Q/U/K-spliced EBNA1 transcripts, and Fp-initiated FQ/U/K-spliced EBNA1 transcripts. Note that Wp, Cp, and Qp are latent cycle promoters and Fp is a lytic cycle promoter.
FIG. 2
FIG. 2
Methylation status of Cp and Wp regulatory regions in EBV-positive cells. (A) Diagram showing the main regulatory elements of Cp and Wp and the relative positions of CpG dinucleotides analyzed. (B, C, and D) Results of bisulfite sequencing of these regions in BL biopsy specimens (B), in BL cell lines (C), and in long-established LCLs (D). Several clones of the relevant PCR products were sequenced for each cell sample, and the individual CpG dinucleotides were identified as either methylated (+, shaded) or nonmethylated (−); the overall percentage of methylated CpGs per sample is shown.
FIG. 3
FIG. 3
Methylation sensitivity of transcription factor binding to Wp sequences in band shift assays. Shown are the results obtained for YY1 binding to the −308 to −279 probe (A), BSAP binding to the −242 to −215 probe (B), RFX binding to the −140 to −99 probe (C), BSAP binding to the −115 to −86 probe (D), and CREB binding to the −102 to −77 probe (E). In each case, the assays included a wild-type competitor, a mutant competitor sequence known to have lost transcription factor binding (7, 27, 57a), and derivatives of the wild-type sequence methylated at the CpG sites shown (●). The lanes showing assays conducted with labeled probes in the absence of the DG75 cell nuclear extract served as controls.
FIG. 4
FIG. 4
Cassette replacement experiments to determine the effect of methylating individual regions of Wp sequence in the context of a Wp reporter. The methylated regions were nucleotides −440 to +173 (A), −440 to −264 (B), −264 to −170 (C), and −170 to −16 (D), in each case relative to the Wp transcription start site. The results are shown as histograms of mock-methylated (MOCK; set at 100% activity) versus cassette-methylated (METH.) reporter activities observed in the DG75 B-cell line and in the Jurkat T-cell line.
FIG. 5
FIG. 5
Analysis of Wp and Cp transcription in recently-infected B cells. Gels showing Wp- and Cp-specific RT-PCR signals, in each case obtained by amplifying neat (N) and 10 (−1)-, 100 (−2)-, and 1,000 (−3)-fold dilutions of W2-primed cDNA. Results are shown for B cells 2, 7, 11, 15, 18, 21, and 28 days postinfection, as well as for the B95.8 LCL and Rael-BL lines as controls. Shown below the gel are the quantitative results of signal intensity phosphorimage analysis.
FIG. 6
FIG. 6
Methylation status of Cp and Wp regulatory regions in B cells 4 to 21 days following B95.8 EBV infection. (A) Diagram showing the main regulatory elements of Cp and Wp and the relative positions of CpG dinucleotides analyzed. (B) Representative sequencing gels of bisulfite-modified DNA for Cp in Rael BL cells versus B cells at 21 days postinfection (p.i.) and for Wp in B cells at 2, 7, 11, 15, 18, and 21 days p.i. The diagrams on the left and right of the gels indicate the expected positions of methylated cytosine in the C sequencing track. (C) Summarized results of bisulfite sequencing of the Cp and Wp regions presented as in Fig. 2.
FIG. 7
FIG. 7
Methylation status of Cp and Wp regulatory regions in EBV-positive cells from the blood of seven individual IM patients. The data are presented as in Fig. 2.
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