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.

More than 50 genes of the bacterial genetic reservoir are required for motility and chemotaxis. These genes are strictly regulated by a hierarchy of transcription controls that determine the temporal order of flagellar assembly, motility, and chemotaxis, as has been intensely studied in enterobacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium (1, 31, 56). The E. coli flagella, motility, chemotaxis, and regulatory genes map in four separate clusters referred to as the flagellar regulon. These have been assigned to three sequentially expressed classes. Class I comprises two genes, flhD and flhC, encoding the global transcriptional activator FlhD 2 FlhC 2 . This, in turn, regulates the expression of class II genes, including determinants of the flagellar basal body, the flagellin-specific type III export, the flagellar hook, and FliA, a 28 ( F ) transcription factor for class III. This ultimate class contains the fla (flagellin), mot (proton channel), and che (signal transduction) genes.The nitrogen-fixing plant symbiont Sinorhizobium meliloti, a member of the ␣ subgroup of proteobacteria (38), differs from the enterobacterial ␥ subgroup behavioral scheme in its filament structure, the mode of flagellar rotation, and steps of signal procession (49). The rigid "complex" flagellar filaments consist of four related flagellin subunits, and interflagellin bonds lock the filaments in right-handedness (7,20,48). Hence, S. meliloti cells are propelled by exclusively clockwiserotating flagella, and swimming cells respond to tactic stimuli by modulating their rotary speed (2, 47). Whereas in E. coli tactic signals are processed by a single response regulator, CheY, and a phosphatase, CheZ, signal processing in S. meliloti involves a retrophosphorylation loop with two response regulators, CheY1 and CheY2, but no phosphatase (49,53,54). In addition, a new periplasmic motor protein, MotC, controls flagellar rotary speed in an as yet enigmatic way (41). The arrangement of chemotaxis (che), flagellar (fla, flg, flh, and fli), motility (mot), and regulatory (visN and visR) genes differs from the enterobacterial pattern in that all 51 known genes are clustered in one contiguous 56-kb chromosomal region, the flagellar regulon (19,55). Two genes, visN and visR (assigned class IA), encode the LuxR-type subunits of a heterodimeric (or heterotetrameric) global transcriptional activator, VisNR (52). Inactivating deletions of visN or visR were shown to result in the loss of class II (flg, flh, fli, and mot) and class III (fla and che) gene expression, suggesting that their transcription is directly controlled by the global regulator VisNR (52). However, while VisNR is synthesized throughout growth, swimming motility is restricted to exponential growth. We describe here another master regulator, Rem (regulator of exponential growth motility) (Smc03046 [19]), that actively controls the transcription of class II genes and that confines their expression to the exponential phase of bacterial growth. The monocistroni...

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 EBVassociated 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.Epstein-Barr virus | IL-21 | lymphoma | STAT3

Epstein-Barr viral (EBV) latency-associated promotersEpstein-Barr virus (EBV) infection is the cause of infectious mononucleosis and is most closely associated with tumor diseases Burkitt's lymphoma (BL) and nasopharyngeal carcinoma. EBV infection of human B lymphocytes in vitro results in B-cell proliferation and transformation into continuously growing lymphoblastoid cell lines (LCL) (for a review, see reference 42). In latently infected cells, viral genomes are maintained as multiple circular episomal copies which are replicated once per cell cycle (2, 103). Several classes of latency have been described depending on the gene expression pattern (41,77,78). In strict type I latency, represented by BL cells, viral gene expression is restricted to the two RNA polymerase III-transcribed EBER RNA genes and the EBNA1 gene (78) that is transcribed from the Q promoter (Qp) (68). The EBNA1 protein is required for the maintenance of the viral plasmid in dividing cells (45,58). In type III latency, in addition to the EBERs, EBNA-LP, -2, -3A, -3B, -3C, and -1 are expressed from the C promoter (Cp) (6), whereas LMP-1 and -2B are expressed from the bidirectional LMP1 promoter (46), and a larger splice variant of LMP-2, LMP-2A, is expressed from the TP1 promoter (36). Qp generally is supposed to be silent in type III latency (82, 105), although there is also a different view (93). Among the viral proteins expressed in latency type III, EBNA2 plays a central role in switching EBNA transcription from Wp to Cp (W to C switch) (102,104) and in the establishment and maintenance of B-cell transformation (11, 28), as EBNA2 transcriptionally activates the expression of the six nuclear antigens from the C promoter (Cp) and the membrane proteins LMP-1 and -2B from the LMP1 promoter (LMP1p), LMP-2A from the TP1 promoter, and a number of cellular proteins associated with the LCL phenotype (1,12,18,39,44,72,76,90,95,98,99,100,101,102,104,110,111). A crucial mechanism involved in the silencing of Cp and LMP1p in type I latency has been shown to be methylation of CpG dinucleotides (3,15,35,54,60,61,70,73,74,75,84,91,94). In LCL, the EBV genome is mostly free of CpG methylation, whereas in BL cells, EBV genomes are highly methylated. An essential step in understanding the differences between latency types I and III is to elucidate the patterns of methylation and in vivo protein binding of the latency promoters of EBV at nucleotide resolution. Therefore, we decided to examine Qp, Cp, and LMP1p in cells of both latency types.(The contributions of Daniel Salamon to this work were made in partial fulfillment of the requirements for a Ph.D. from Semmelweis University, Budapest, Hungary.) MATERIALS AND METHODSCell lines and tissue culture. LCL 721 is a B95-8-transformed LCL with type III phenotype (40,52,57). Rael (15,43,61) is a group I BL cell line. Mutu BLI-C1216 is a subclone of the BL line Mutu, representative of latency type I (27). Mutu BLIII-C199 is a subclone of the BL line Mutu, representative of latency type III (27). Raji cells expre...