Promoter regions of SYK, PU.1, and CD19 are methylated in cHL cell lines. (A) Analysis of promoter methylation by amplification of genomic DNA digested with methylation-sensitive endonucleases. The positions of the forward and reverse primers are marked with F and R, respectively. Genomic DNA was digested with the methylation-sensitive enzyme HpaII or with its methylation-insensitive isoschizomer MspI followed by amplification with primers, located on both sides of the recognized sequence 5′-CCGG-3′ (shown as vertical lines). Amplification of HpaII-digested DNA is possible only when the cytosine of the CpG dinucleotide is methylated and the DNA is therefore not digested. The SYK amplicon is 264 bp in length and contains five 5′-CCGG-3′ motifs at positions -30, -107, -136, -186, and -214 bp counting from the start site of transcription (arrow) as predicted by the National Center for Biotechnology Information (NCBI) Entrez Gene program.⁶⁸  The SYK CpG island (shown as a gray horizontal bar) spreads over exon 1 (thick filled bar). Sp1 binding sites are shown as horizontal filled ovals. For PU.1 promoter methylation analysis, we used NCBI sequence (accession no. U34046). The amplified region contains two 5′-CCGG-3′ sites at positions -66 and +187, an octamer motif (•), and Sp1 and PU.1 (vertical filled oval) binding sites. The coding region of exon 1 is shown in black. The 211-bp-long amplicon of CD19 5′ untranslated region (NCBI accession no. M84371) included 2 BSAP (B-cell-specific activator protein)/Pax5 binding sites (filled hexagons) and one 5′-CCGG-3 HpaII/MspI recognition site at position +26. (B) Methylation of the SYK, PU.1, and CD19 promoter regions. The PCR amplification products were separated on 1.5% agarose gels and visualized by EtBr staining. All experiments were performed at least in triplicate.