KLF4 promoter is hypermethylated in B-cell lymphomas. (A) Region of KLF4 promoter analyzed by pyrosequencing (PYRO) from −226 to −252 base pairs from start of transcription. MSP-F and MSP-R indicate the positions of methylation-specific primers for Q-MSP; N-F and N-R, locations of outer primers for nested PCR; and P-F and P-R, locations of the pyrosequencing amplification primers. The transcription start site is indicated by an arrow. (B) Pyrosequencing analysis of KLF4 promoter methylation in LCL and lymphoma cell lines. The methylation status of 7 CpG dinucleotides was analyzed. The ratio of methylated CpG is indicated by color coding. (C) Pyrosequencing analysis of KLF4 promoter methylation in peripheral blood and tonsillar CD19⁺ cells as well as in tonsillar B-cell subsets. (D) Pyrosequencing analysis of KLF4 promoter methylation in the primary cases of FL, DLBCL, and BL. (E) Methylation status of cHL samples. Bisulfite-converted genomic DNA from 7 samples of normal CD19⁺ cells isolated from peripheral blood, 8 samples of primary cHL cases, and from B-cell lymphoma cell lines L428, KM-H2, L540, Namalwa, Raji, and Ramos were analyzed for the presence of methylated KLF4 alleles by Q-MSP. Data represent the ratio of methylated to unmethylated KLF4 alleles in the sample. The ACTB template, which is amplified independently of methylation, is used as a reference. CD19⁺ normal B cells represent hypomethylated control, and B-lymphoma cell lines are used as hypermethylated control. KLF4 promoter methylation level in cHL samples is significantly higher than in normal CD19⁺ cells (2-sided t test). (F) Genomic DNA was isolated from 100 microdissected HRS or stromal and bystander cells isolated from 10 cHL samples each. Bisulfite-converted samples were amplified by nested PCR with primers located as shown in panel A, and methylation status of 7 individual CpGs was measured as described in panel B.