The Epstein-Barr Virus Major Latent Promoter Qp Is Constitutively Active, Hypomethylated, and Methylation Sensitive

Methylation of the cytosine residue of CpG dinucleotides in vertebrate DNA is an important mechanism of promoter regulation and genetic imprinting (4). Approximately 70% of all CpG sites in the human genome are methylated. Overall, there is an inverse relationship between the methylation status and the transcriptional activity of a promoter. Methylation of CpG sites in a promoter may cause changes in chromatin that interfere with the binding of general and sequence-specific transcription factors (25, 60). Moreover, methylation-specific transcription repressors such as MeCP1 and MeCP2 may compete with transcription factors for binding sites and result in transcriptional repression (6, 7). Transcriptional silencing of tumor suppressor genes such as p15 and p16 by aberrant methylation has been implicated in tumorigenesis (3, 14). CpG methylation is also important in the gene regulation of Epstein-Barr virus (EBV), a ubiquitous human gammaherpesvirus implicated in the pathogenesis of a variety of malignancies. Among six viral latent nuclear proteins, EBV nuclear antigen 1 (EBNA1) is the only one constitutively expressed in all types of latent infection (26). EBNA1 binds to the viral latent origin of replication (oriP) and is required for viral DNA replication and episomal maintenance (2, 20, 65). EBNA1 transcripts are known to originate from four different promoters (Fig. ​(Fig.1A).1A). Following the initial infection of B lymphocytes, EBNA1 transcripts originate from the W promoter (Wp). Shortly thereafter, a promoter switch occurs as the C promoter (Cp) is activated and Wp is silenced (55). EBNA1 transcripts from either promoter include exons derived from the internal repeat region (W exons), Y and U exons, and the coding K exon (Fig. ​(Fig.1A).1A). In other forms of latency (24, 46), first recognized in tumors and tumor-derived cell lines, EBNA1 transcripts contain a Q-U-K splice (Fig. ​(Fig.1A)1A) and Wp and Cp are silent (47, 54, 56). Although initially identified as a promoter located at the junction of the F and Q fragments (47, 54), subsequent studies demonstrated that there were actually two adjacent promoters located on either side of the F-Q junction. The promoter located in the F fragment, Fp, is a lytic promoter driving EBNA1 and perhaps other transcripts (28, 51). The promoter located at the beginning of the Q fragment, Qp, is a TATA-less promoter that drives EBNA1 transcription in latency I (40, 50, 63). Positive and negative regulatory elements have been identified in the Fp and Qp regions (37, 48, 58). Recent investigations have suggested that interferon response factors (IRFs) are involved in the regulation of Qp transcription (38, 39, 49, 67). FIG. 1 (A) Promoter usage and splicing pattern of EBNA1 transcripts in different types of EBV latency. The diagram at the top shows the structure of the EBV genome, locations of the four different EBNA1 promoters, and coding exons (shadowed boxes) of EBNAs. ... An inverse relationship between methylation and transcriptional activity has been demonstrated for several EBV promoters (Cp, Wp, and promoters for LMP1 and BHRF1) (16, 23, 33, 41, 44). In latency III cell lines expressing the full spectrum of EBV latency proteins (e.g., lymphoblastoid cell lines [LCL] like B95-8), there is little if any methylation of the viral genome (1, 36). EBER promoters are hypomethylated and constitutively active in all settings except in oral hairy leukoplakia in patients with AIDS (19, 35). However, Cp, Wp, and LMP1 promoters are methylated and silent in more restricted forms of latency and in tumors (1, 22, 23, 44, 45). The LMP1 promoter is unmethylated and active in latency II tumors (9, 11–13, 15, 34). Methylation of the EBNA2 response region of Cp abolishes the binding of a transcription factor CBF2 and silences the promoter (44). In addition, 5-azacytidine treatment results in demethylation and transcriptional activation of the Cp (30, 44). Qp is a TATA-less promoter, structurally similar to housekeeping gene promoters (50). TATA-less promoters tend to be regulated by upstream CG-rich regions (5), and methylation is one of the mechanisms. A 5-kb region which includes both Qp and Fp has been reported to be hypomethylated (53). In the study reported here, we analyzed EBNA1 promoter usage in a variety of EBV-associated tumors and tumor-derived cell lines, characterized CpG methylation in the Qp and Fp regions by genomic sequencing, and used the methylation cassette assay to evaluate the effect of methylation on the transcriptional activity of Qp.