Epigenetic silencing of proapoptotic BIM gene in Burkitt lymphoma. (A) Standard MSP analysis of the BIM gene in BL-derived cell lines and patient samples (P1 and P2, unmethylated BIM; P3 and P4, methylated BIM; top). Quantitative analysis of methylation using quantitative MSP (bottom), including cell lines with complete methylation according to standard MSP analysis (light gray), cell lines with partial methylation (dark gray), and unmethylated tumors (DG75, Tanoue, and BALM9). PB indicates peripheral blood. (B) Pyrosequencing analysis of the BIM promoter area after bisulfite modification showing quantitative methylation profiles of GpG islands (expressed in percentage), which correlate with the data obtained in quantitative MSP. (C) Homozygous deletion of the BIM gene in the EBV- P32 and BL2 cell lines, as revealed by comparative genomic hybridization to BAC microarrays and PCR on tumor genomic DNA, resulting in null expression of BIM as shown by quantitative RT-PCR and Western blot analyses. (D) Quantitative ChIP assay of AcH3 binding to canonical CpG island sequences of BIM promoter in vorinostat-treated and untreated cells. An increase of AcH3 levels after treatment with vorinostat was detected in the hypermethylated Jijoye and Seraphina cell lines, but not in the unmethylated BALM9 cell line. (E) BIM epigenetic silencing was correlated with absent or low expression at RNA levels measured by quantitative RT-PCR, which correlate with BIM epigenetic status. (F) Accordingly, Western blot analysis shows protein down-regulation in BL-derived cell lines and patient samples with hypermethylation of BIM (P1 and P2, unmethylated BIM; P3, P4, and P5, methylated BIM). (G) Treatment with 5-Aza and with vorinostat restored BIM expression in methylated tumors at the RNA and protein levels.