Epigenetic and Transcriptional Changes Which Follow Epstein-Barr Virus Infection of Germinal Center B Cells and Their Relevance to the Pathogenesis of Hodgkin's Lymphoma

Abstract

Although Epstein-Barr virus (EBV) usually establishes an asymptomatic lifelong infection, it is also implicated in the development of germinal center (GC) B-cell-derived malignancies, including Hodgkin's lymphoma (HL). Following primary infection, EBV remains latent in the memory B-cell population, where host-driven methylation of viral DNA contributes to the repression of viral gene expression. However, it is still unclear how EBV harnesses the cell's methylation machinery in B cells, how this contributes to viral persistence, and what impact this has on the methylation of cellular genes. We show that EBV infection of GC B cells is followed by upregulation of the DNA methyltransferase DNMT3A and downregulation of DNMT3B and DNMT1. We show that the EBV latent membrane protein 1 (LMP1) oncogene downregulates DNMT1 and that DNMT3A binds to the viral promoter Wp. Genome-wide promoter arrays performed with these cells showed that EBVassociated methylation changes in cellular genes were not randomly distributed across the genome but clustered at chromosomal locations, consistent with an instructive pattern of methylation, and were in part determined by promoter CpG content. Both DNMT3B and DNMT1 were downregulated and DNMT3A was upregulated in HL cell lines, recapitulating the pattern of expression observed following EBV infection of GC B cells. We also found, by using gene expression profiling, that genes differentially expressed following EBV infection of GC B cells were significantly enriched for those reported to be differentially expressed in HL. These observations suggest that EBV-infected GC B cells are a useful model for studying virus-associated changes contributing to the pathogenesis of HL.

Contributions of CTCF and DNA Methyltransferases DNMT1 and DNMT3B to Epstein-Barr Virus Restricted Latency

“…The apparent opposite effect of EBV on expression of DNMT1 and -3B in GC B cells (repression) and epithelial cells (activation) may reflect involvement of different cellular and/or viral factors. We note that the repression of DNMT1 and -3B in GC B cells (28) appears to be consistent with the lower level of these DNMTs in BL and LCL lines that maintain latency III relative to latency I BL lines, as observed here. Interestingly, following infection of GC B cells, DNMT3A could be detected within chromatin associated with Wp (but not Cp) (28), the EBNA promoter used prior to EBNA2 transactivation of EBNA expression from Cp.…”

“…We note that the repression of DNMT1 and -3B in GC B cells (28) appears to be consistent with the lower level of these DNMTs in BL and LCL lines that maintain latency III relative to latency I BL lines, as observed here. Interestingly, following infection of GC B cells, DNMT3A could be detected within chromatin associated with Wp (but not Cp) (28), the EBNA promoter used prior to EBNA2 transactivation of EBNA expression from Cp. Most importantly, EBV-mediated induction of DNMT3A and its direct association with Wp, a latency gene promoter that undergoes methylation relatively early in infection (16) and which must ultimately be silenced to establish and maintain restricted latency, support the notion that methylation of the EBV genome is a regulated process.…”

“…In apparent contrast to the previous reports of LMP1 and LMP2A induction of DNMT1 and -3B, a recent report demonstrated that EBV infection of GC B cells leads to repression of DNMT1 and DNMT3B expression and an upregulation of the de novo DNA methyltransferase DNMT3A (28). Though repression of DNMT1 (but not DNMT3B) could be attributed to LMP1, induction of DNMT3A expression was not due to LMP1.…”

“…Though repression of DNMT1 (but not DNMT3B) could be attributed to LMP1, induction of DNMT3A expression was not due to LMP1. Moreover, the relative levels of DNMT1, -3A, and -3B observed in newly established LCLs generated from these GC B cells (which maintain latency III) matched those in cell lines derived from Hodgkin lymphoma, a tumor of GC B-cell origin (28). The apparent opposite effect of EBV on expression of DNMT1 and -3B in GC B cells (repression) and epithelial cells (activation) may reflect involvement of different cellular and/or viral factors.…”

Genome-Wide DNA Methylation as an Epigenetic Consequence of Epstein-Barr Virus Infection of Immortalized Keratinocytes

“…Promoter hypomethylation of a number of proliferative genes was consistent with the conversion of resting B cells to proliferating blasts (26,27). EBV infection of germinal center B cells not only was shown to alter DNMT expression, resulting in changed DNA methylation patterns at particular regions, but also induced the histone 3 lysine 27 demethylase, KDM6B, potentially regulating genes differentially expressed in Hodgkin's lymphoma (28,29). However, in carcinoma cells, we previously demonstrated that EBV infection led to epigenetic silencing at the PYCARD and E-cadherin gene loci involving DNA methylation and repressive chromatin modifications, respectively (30).…”