A comparison of the DNA methylation levels of selected genes between RBLs and resulting LCLs. (a) Bisulfite pyrosequencing was performed for the genes selected from experiments with methylation arrays. The distribution of CpG sites (vertical black line), the transcription start site (TSS; arrow) and the exact CpGs analyzed by the array (horizontal light grey bar) and pyrosequencing (horizontal dark grey bar) are indicated above each gene. Average data from six samples is represented. (b) EBNA2 and DAPI staining of B lymphocytes at 0 and 24 hours after infection with EBV derived from 293 cells carrying a recombinant B95.8 EBV genome, and LCLs, in which high levels of infection were achieved. (c) Pyrosequencing data showing the methylation changes at selected genes at different times. (d) Analysis of the 5-hydroxymethylcytosine content/changes at CpG sites undergoing hypomethylation in selected genes. As a positive control, in vitro hydroxymethylated DNA was used. (e) Changes in expression of activation-induced deaminase (AID) during RBL conversion to LCLs at the mRNA and protein levels. For protein analysis, the Burkitt's lymphoma cell line RAJI was used as a positive control for AID expression. (f) Overlap between AID target genes and hypomethylated genes. (g) Comparison between B cells activated with IL4 and CD40L and infected with EBV. The top panel shows the proportion of activated cells (left; determined by fluorescence-activated cell sorting (FACS) analysis with CD86) and proportion of dividing cells (right; determined with bromodeoxyuridine (BrdU)). Bottom, bisulfite pyrosequencing analysis of selected CpGs in association with EBV infection and with B cell activation by IL4 and CD40L. W, week.