Department of Pediatrics, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan
Correspondence
Hideaki Kikuta
hide-ki{at}med.hokudai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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It has been reported that several mechanisms, including methylation and action of viral latent proteins and cellular factors, operate to regulate the promoters and control latent gene expression. Methylation of the cytosine residue of CpG dinucleotides (CpG) in eukaryote DNA is an important mechanism of promoter regulation and genetic imprinting (Singal & Ginder, 1999). EBV latent gene expression is controlled by distinct usage of three promoters (Cp, Wp and Qp). Promoter activity has been shown to be inversely correlated with the methylation status of promoters, although Qp is a remarkable exception (Robertson et al., 1995
, 1996
; Robertson & Ambinder, 1997a
, b
; Falk et al., 1998
; Salamon et al., 2001
; Tierney et al., 2000b
). When Cp is hypomethylated, it permits expression of all EBNAs during latency III. On the other hand, Qp gives rise to a selective expression of EBNA1 during latency I, while latency II is characterized by expression of EBNA1, LMP1 and LMP2 with transcripts initiated from Qp and LMP promoters (Brooks et al., 1992
; Deacon et al., 1993
). Cp was hypermethylated and inactivated in BL and HD (Robertson et al., 1996
; Tierney et al., 2000a
). Multiple usage of Cp, Wp and Qp was found in EBV-infected cells of patients with IM (Tierney et al., 1994
; Laytragoon-Lewin et al., 1997
; Niedobitek et al., 1997
). Wp was hypermethylated and Cp was completely unmethylated in most patients with IM (Tierney et al., 2000a
). Promoter usage in healthy carriers is controversial (Tierney et al., 1994
; Chen et al., 1995
). (Robertson et al., 1997b
) reported that about half of Cp was methylated in healthy carriers. Paulson & Speck (1999)
reported that Wp was hypermethylated but that Cp was only sparsely to moderately methylated in healthy carriers. The purpose of this study was to determine the EBNA promoter usage and to characterize the methylation status of the Cp and Wp regions in CAEBV.
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METHODS |
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RT-PCR.
Five spleen samples were examined for the presence of viral RNA transcripts by RT-PCR. The conditions for RT-PCR were described previously (Yoshioka et al., 2001). The primer pairs were designed in order to characterize the promoter usage of EBNA1 transcripts (Fig. 1
, Table 2
). Cp- and Wp-initiated transcripts have BamHI-C1/C2/W1/W2- and BamHI-W0/W1/W2-spliced structures, respectively. Therefore, the primer pair C1/C2-5' and W2-3' was used for Cp-initiated transcripts, and the primer pair W0/W1-5' and W2-3' was used for Wp-initiated transcripts. Since Qp-initiated transcripts have a BamHI-Q/U/K-spliced structure, the primer pair Q-5' and K-3' was used for the first amplification and the primer pair U-5' and K-3' was used for the second amplification for Qp-initiated transcripts. To evaluate the sensitivity of the RT-PCR, serial 10-fold dilutions of cDNA from the positive control for each RT-PCR analysis were subjected to PCR.
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Bisulfite PCR analysis of EBV genome methylation.
Before bisulfite PCR analysis, untreated DNA was sequenced to confirm the sequences of the CpG sites in the samples. Although several point mutations were detected in promoter regions, the primers used in this study were not affected by the mutations (data not shown). The methylation status of CpG was determined by methylation-specific bisulfite PCR analysis (Frommer et al., 1992; Herman et al., 1996
; Kubota et al., 1997
) in five spleen samples. In this method, the bisulfite treatment converts unmethylated cytosine to uracil but does not affect the methylated cytosine. PCR primers that anneal selectively to unmethylated DNA after bisulfite conversion were designed. Briefly, 10 µg aliquots of DNA were denatured in NaOH (0·2 mol l-1) for 10 min at 37 °C. Each sample was resuspended in 520 µl of freshly prepared sodium bisulfite (3·0 mol l-1; Sigma) and 30 µl of hydroquinone (10 mmol l-1, pH 5; Sigma), overlaid with mineral oil and incubated for 20 h at 50 °C. After incubation, the bisulfite-modified DNA was purified and desalted with a Wizard DNA Clean-up system (Promega), desulfonated in NaOH (0·3 mol l-1), precipitated with ammonium acetate (Wako), ethanol and 1·2 µg of carrier glycogen (Boehringer Mannheim), and resuspended in 50 µl of sterile deionized water. For each sample, 2 µl aliquots of bisulfite-modified DNA were amplified with primers specific for the regulatory regions of Cp and Wp as shown in Table 3
.
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RESULTS |
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Wp (Fig. 2B)
A total of 26 clones in samples from the five CAEBV patients was sequenced, and the methylation status at 18 CpG sites in the Wp region was analysed. Of a total of 468 sites analysed, 378 (81 %) were methylated. The transcription factors, such as YY1, BSAP/Pax5, RFX family protein and CREB family protein, are important for Wp activation in B cells (Bell et al., 1998; Kirby et al., 2000
; Tierney et al., 2000a
, b
). CpG cytosines at nucleotide positions 14085, 14101, 14143, 14259, 14261, 14288, 14290 and 14296, which are present around the binding sites of these transcription factors, were almost completely methylated in CAEBV. These results indicated that the activation of Wp by these factors might be inhibited.
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DISCUSSION |
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During latent EBV infection of human B lymphocytes, six EBNAs (EBNA1, EBNA2, 3A, 3B, 3C and LP) are expressed from a single complex transcriptional unit that spans nearly 100 kb of the viral genome by means of alternative splicing and alternative polyadenylation sites (Kieff, 1996). The first EBNA genes expressed are EBNA-LP and EBNA2, by initiation from Wp. Subsequently, transcription of EBNA2 activates Cp, LMP and a number of cellular promoters for growth response genes, and dominant promoter usage switches from Wp to Cp (Alfieri et al., 1991
; Tierney et al., 2000a
). Generally Cp-initiated EBNA1 cDNA has a Y1/Y2/Y3/U/K-spliced structure downstream of W1/W2 repeats. However, Tierney et al. (1994)
reported that C1/C2/W2-initiated transcripts were detected in all 14 IM patient blood samples, whereas Cp-initiated, Y3/U/K-spliced EBNA1 transcripts were detected in only half of the samples. We detected C1/C2/W2-initiated transcripts in all five samples but could not detect Y3/U/K-spliced EBNA1 transcripts in any samples except for that from patient 4 (data not shown). Furthermore, several unique Cp-initiated transcripts that skipped one or more exons downstream of a Cp-initiated site have been reported (Bodescot & Perricaudet, 1986
; Qu & Rowe, 1992
, 1995
). The mechanisms regulating alternative splicing and polyadenylation site usage within the EBNA transcription unit remain largely unknown. Although Cp permits the full spectrum of EBNA gene expression, EBV latent gene expression is restricted in CAEBV. Therefore, we speculate that Cp-initiated transcripts have unique splicing patterns and preferentially induce EBNA1 transcripts in most cases of CAEBV.
Methylation of CpG is an important mechanism of promoter regulation and EBV latent gene expression. EBNA promoter activity has been shown to be inversely correlated with the methylation status of the promoters Cp (Robertson et al., 1995, 1996
; Robertson & Ambinder, 1997a
, b
; Salamon et al., 2001
), Wp (Tierney et al., 2000b
) and LMP1 (Falk et al., 1998
). Methylation may inhibit transcription by a number of mechanisms, either by direct blocking of the binding of transcription factors to DNA or through mediation of methyl-CpG-binding proteins, which recruit histone deacetylases to the DNA, leading to the remodelling of chromatin into an inactive configuration (Singal & Ginder, 1999
). Wp is exclusively utilized during the initial stage of EBV infection in primary B lymphocytes, followed by a switch to Cp usage (Alfieri et al., 1991
; Tierney et al., 2000a
). Hypermethylation in the Wp region indicated that the promoter of EBNA might have already switched from Wp to Cp or Qp in CAEBV, which is consistent with the results of transcriptional analysis. The mechanism of Wp down-regulation in CAEBV might be the same as that in EBV-infected B cells, and hypermethylation of Wp might be sufficient to prevent transcription from Wp in CAEBV. Qp is a TATA-less promoter at the centre of a hypomethylated island (Schaefer et al., 1995
). Qp was found to be completely unmethylated in samples from patients (data not shown). Qp might be inactivated by several factors, such as EBNA1, E2F, interferon response factors (IRFs)-1, -2 and -7 and high-mobility group protein (HMG)-I (Y)-containing complex, under the conditions of Cp activation in CAEBV (Sung et al., 1994
; Schaefer et al., 1997
; Chen et al., 1999
).
Cp activity is the major difference between latency III and the other latency forms. Several mechanisms that may regulate Cp activity have been described. First, Cp can be activated by binding of EBNA1 to oriP, an enhancer 3 kb upstream of Cp (Puglielli et al., 1996). Second, glucocorticoid response elements (GREs) about 900 bp upstream of Cp respond to glucocorticoids and activate Cp (Evans et al., 1996
). Third, the EBNA2 response region in the Cp region is critical for Cp activity. The EBNA2 response region contains the binding sites for the cellular DNA-binding proteins CBF1 and CBF2. The methylation of CpG sequences in the CBF2-binding domain inhibits Cp activity, and CBF2 binding is crucial for EBNA2-mediated activation of Cp (Robertson et al., 1995
, 1996
). Finally, several cellular factors that control the transcription activity of Cp have been identified. NF-Y transcription factor interacts with the CCAAT box in the -71 to -63 region of Cp. Members of the C/EBP transcription factor family interact with the C/EBP consensus sequence in the -119 to -112 region of Cp. Sp1 and Egr-1 interact with a GC-rich sequence in the -99 to -91 region of Cp. NF-Y, members of the C/EBP, and Sp1 are positive regulators of Cp activity, and Egr-1 is a negative regulator of Cp activity (Nilsson et al., 2001
).
There was a discrepancy between Cp usage and the hypermethylation status of the Cp region in CAEBV. The level of methylation in the Cp region in CAEBV was, surprisingly, as high as that in BL (Robertson et al., 1996; Tierney et al., 2000a
). We cannot rule out the possibility that we could not detect cells with unmethylated Cp by bisulfite PCR analysis because the population size of such cells was very small. However, since Cp is regulated in a very complex manner as mentioned above, it could be concluded that the hypermethylation status of the Cp region might be insufficient to prevent transcription from Cp. This finding is consistent with the genetic analysis of Cp function by Evans et al. (1996)
. Furthermore, CAEBV is considered to be a T-cell LPD and only occasionally a B-cell LPD. Patient 2 was a rare case of B-cell LPD, but the methylation status was the same as that of T-cell LPD, indicating that hypermethylation in the Cp region occurs regardless of the phenotypic difference in EBV-infected cells.
In this study, we found unique EBV latent gene expression, EBNA promoter usage and methylation status of EBNA promoters in CAEBV. The unique relationships, seemingly contradictive, might offer the key for understanding the pathogenesis of CAEBV.
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ACKNOWLEDGEMENTS |
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Received 13 August 2002;
accepted 10 January 2003.
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