We have shown that whereas EBV downregulates the expression of the DNA methyltransferases DNMT1 and DNMT3B in GC B cells, it upregulates that of DNMT3A; these changes are seen shortly following the onset of EBV infection. This pattern of expression recapitulates that seen in HL cell lines and is different from that associated with EBV infection of epithelial cells. These findings are also consistent with gene expression profiling performed with microdissected HRS cells, which showed a downregulation of DNMT1 and DNMT3B and an upregulation of DNMT3A (5
). We also show that LMP1, the major EBV oncogene expressed in HL, is responsible for the downregulation of DNMT1.
Although we have shown deregulation of DNMT3A and DNMT3B in HL cell lines, we were unable to reproduce this pattern of deregulation following transfection of GC B cells with viral genes usually expressed in HL. However, in these experiments, these genes were transfected singly into GC B cells, and we cannot exclude the possibility that the deregulation of DNMT3A and DNMT3B in HL may be dependent on achieving levels of expression not attained in our transfection experiments or that deregulation may be dependent on the cooperative activity of two or more of these viral genes. Such cooperation between EBV latent genes has been shown to modulate the behavior of B cells in other systems (38
Our observations also show how EBV-induced changes in the expression of the DNMT might contribute to the flexibility of latent promoter usage critical to the virus's strategy for persistence in vivo
). It has been known for some time that the Wp promoter is silenced by DNA methylation in GC-derived tumors (33
). We were able to reveal a potential role for EBV-induced upregulation of DNMT3A in maintaining viral persistence by demonstrating that the expression of Wp begins to decline when that of DNMT3A first increases and also that DNMT3A binds to Wp in the critical BSAP region in a GC B-cell-derived LCL (see Fig. S1 in the supplemental material).
EBV-induced changes in the expression of the DNMT in GC B cells were associated with widespread changes in the methylation status of cellular genes. These changes were not randomly distributed across the genome but appeared to cluster at certain chromosomal locations. Concordant methylation of adjacent CpG island gene promoters has also been reported for a number of gene clusters in cancer, and recent genome-wide analyses have identified large chromosomal regions containing several CpG islands which are often methylated and transcriptionally repressed in cancer (9
). Although these observations suggest that coordinated epigenetic control over large regions is common in cancer, this phenomenon has not previously been reported following the infection of primary cells with an oncogenic virus (10
). Why particular genes become more methylated and others less methylated following EBV infection of GC B cells has yet to be determined. However, we were able to reveal a relationship between a change in methylation status and promoter CpG content. Whereas promoters with a high CpG content were significantly more likely to have increased methylation following EBV infection, those with a low CpG content were more likely to become less methylated. Similar associations have been reported in melanoma, prostate cancer, and in hematological malignancies. In these tumors, genes with a high CpG content are more likely to be hypermethylated, whereas those with a low CpG content are more likely to be hypomethylated (11
). Not surprisingly, this association with CpG content also appears to explain in part the ontological profile of those genes in which methylation changes were observed. For example, we have shown that genes with decreased methylation following EBV infection are significantly enriched for those involved in G protein-mediated signaling. Genes involved in G protein-mediated signaling are in turn significantly enriched for those with a low CpG content (P
= 3.98E−132). Thus, it would appear that in both cancer and in primary GC B cells infected with EBV, an instructive pattern of de novo
methylation change is determined in part by cis
-acting susceptibility factors, such as promoter CpG content. Other determinants of CpG methylation in cancer under investigation, for example, local sequence context, may yet be found to determine the pattern of oncogenic virus-induced methylation changes (21
We found only one example (TP73) of a TSG which is hypermethylated following EBV infection and which is reported to be epigenetically silenced in HL. However, as there are only 22 TSG reported to be silenced by methylation in HL, and no instance of a gene is reported in the literature to be hypomethylated in this lymphoma, a final judgment on the relevance of EBV-associated methylation changes in GC B cells must be postponed until we have the results of a comparable methylation array performed on microdissected HRS cells. However, our observations offer some clues as to how EBV-induced deregulation of the DNMT might contribute to the pathogenesis of HL. EBV is known to drive the differentiation of B cells toward a post-GC stage, and we have shown that its major oncoprotein, LMP1, reprograms GC B cells toward an HRS-like phenotype by hijacking the B-cell transcriptional program and subverting normal B-cell differentiation (16
). Here, we show that LMP1 downregulates DNMT1 in GC B cells. This is of interest because DNMT1 has recently been shown to have an essential role in maintaining the progenitor state of constantly replenishing somatic tissue. DNMT1 depletion is followed by the exit of epidermal cells from the progenitor compartment and their premature differentiation (29
). This transition is associated with the downregulation of UHRF1, a component of the cell's DNA methylation machinery, and with the upregulation of GADD45, a putative DNA demethylase. Similar transcriptional changes are seen during normal B-cell differentiation, when DNMT1 and UHRF1 are downregulated and GADD45A is upregulated in both plasma cells and in memory cells compared with results for centrocytes (5
). We have confirmed not only the transcriptional downregulation of DNMT1 but also the downregulation of UHRF1 and the upregulation of GADD45A in both EBV-infected GC B cells and in HL cell lines compared with that in GC B cells (). DNMT1 and UHRF1 have also been found to be downregulated and GADD45A to be upregulated on gene expression profiling of microdissected HRS cells compared with that of centrocytes (5
). Given that the balance between renewal of progenitor cells and differentiation appears to be controlled by a dynamic antagonism between regulators of DNA methylation, it is tempting to speculate that EBV and its latent genes contribute to the pathogenesis of HL by disrupting the expression of those epigenetic regulators which control this balance.