Daniel Salamon

Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization

Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

Protein-DNA Binding and CpG Methylation at Nucleotide Resolution of Latency-Associated Promoters Qp, Cp, and LMP1p of Epstein-Barr Virus

ABSTRACT Epstein-Barr viral (EBV) latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five representative cell lines and correlated the results with the known in vitro binding data and activities of these promoters from previous transfection experiments. Promoter activity inversely correlated with the methylation state of promoters, although Qp was a remarkable exception. Novel protein binding data were obtained for all promoters. For Cp, binding correlated well with promoter activity; for LMP1p and Qp, binding patterns looked similar regardless of promoter activity.

IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter.

Epstein–Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4+ T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.

STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus–encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma

In line with the B-lymphotropic nature of Epstein-Barr virus (EBV), the virus is present in several types of B-cell lymphomas. EBV expresses a different set of latent genes in the associated tumors, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane proteins (LMPs; type II latency) in classical Hodgkin lymphomas (HLs). We previously reported that exposure of in vitro EBV-converted, HL-derived cell line KMH2-EBV to CD40-ligand and interleukin-4 (IL-4) induced the expression of LMP-1. Here, we show that exposure to IL-4 or IL-13 alone induced LMP-1 in the absence of EBNA-2. Induction of LMP-1 by IL-4 and IL-13 was mediated by the signal transducer signal transducer and activator of transcription 6 (STAT6) and a newly defined high-affinity STAT6-binding site in the LMP-1 promoter. IL-4 induced LMP-1 also in Burkitt lymphoma-derived lines and in tonsillar B cells infected with the EBNA-2-deficient EBV strain P3HR-1. Furthermore, coculture of EBV-carrying Burkitt lymphoma cells with activated CD4(+) T cells resulted in the induction of LMP-1 in the absence of EBNA-2. Because Hodgkin/Reed-Sternberg cells are known to secrete IL-13, to have constitutively activated STAT6, and to be closely surrounded by CD4(+) T cells, these mechanisms may be involved in the expression of LMP-1 in EBV-positive chronic HLs.

Epigenetics of latent Epstein-Barr virus genomes: high resolution methylation analysis of the bidirectional promoter region of latent membrane protein 1 and 2B genes.

Abstract We analysed the methylation patterns of CpG dinucleotides in a bidirectional promoter region (LRS, LMP 1 regulatory sequences) of latent EpsteinBarr virus (EBV) genomes using automated fluorescent genomic sequencing after bisulfiteinduced modification of DNA. Transcripts for two latent membrane proteins, LMP 1 (a transforming protein) and LMP 2B, are initiated in this region in opposite directions. We found that B cell lines and a clone expressing LMP 1 carried EBV genomes with unmethylated or hypomethylated LRS, while highly methylated CpG dinucleotides were present at each position or at discrete sites and within hypermethylated regions in LMP 1 negative cells. Comparison of high resolution methylation maps suggests that CpG methylationmediated direct interference with binding of nuclear factors LBF 2, 3, 7, AML1/LBF1, LBF5 and LBF6 or methylation of CpGs within an Ebox sequence (where activators as well as repressors can bind) is not the major mechanism in silencing of the LMP 1 promoter. Although a role for CpG methylation within binding sites of Sp1 and 3, ATF/CRE and a sisinducible factor (SIF) cannot be excluded, hypermethylation of LRS or regions within LRS in LMP 1 negative cells suggests a role for an indirect mechanism, via methylcytosine binding proteins, in silencing of the LMP 1 promoter.

Soluble factors produced by activated CD4+ T cells modulate EBV latency.

Following infection with Epstein–Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4+ T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4+ T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4+ T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4+ T cells can control the EBV-induced B-cell proliferation.

Acetylated histone H3 and H4 mark the upregulated LMP2A promoter of Epstein-Barr virus in lymphoid cells.

ABSTRACT We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.

Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein-Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein-Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.

High-resolution methylation analysis and in vivo protein-DNA binding at the promoter of the viral oncogene LMP2A in B cell lines carrying latent Epstein-Barr virus genomes.

Latency protein LMP2A of Epstein–Barr virus (EBV) has been implicated in EBV related tumorigenesis. To understand the host cell dependent expression of the LMP2A gene, it is necessary to analyse the regulatory mechanisms of the LMP2A promoter (LMP2Ap). By transient transfection and in vitro binding analyses two CBF1 sites have previously been shown to be involved in the regulation of LMP2Ap. However, the promoter structure has not been examined at the nucleotide level in vivo. Therefore we undertook a comprehensive analysis of in vivo protein binding and of CpG-methylation patterns at LMP2Ap in a panel of B cell lines carrying latent EBV genomes. The presence of characteristic footprints on two CBF1 and further binding-sites, together with overall hypomethylation of CpG dinucleotides correlated well with promoter activity. In contrast, the absence of several genomic footprints, as well as the presence of patches of highly methylated CpG dinucleotides were characteristic of silent LMP2Aps.

T Cells Modulate Epstein-Barr Virus Latency Phenotypes During Infection of Humanized Mice

ABSTRACT Human B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cells in vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ−/− mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferated in vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that the in vitro growth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8+ cell-depleted mice, and it was absent from CD4+ cell-depleted mice. These results indicate that CD4+ T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4+ T cells contributed to this process through their CD40L expression. IMPORTANCE At primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4+ T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.