Methylation of the TSC-22 promoter in mouse T or NK LGL leukemia. (A) Examination of sequences within the murine TSC-22 promoter shows a CpG island located from −300 to +100 (G + C, > 50%; length, > 200; observed/expected ratio, > 0.6). The vertical lines are indicative of CG dinucleotides; TSS indicates transcription start site. (B) Leukemic cells were enriched by flow cytometry (≥ 95%) from spleens of the representative mice with T or NK LGL leukemia. Data of the L1210 T-cell lymphoma cell line and of nonleukemic cells (enriched from WT mice or IL-15tg mice with polyclonal T- and NK-cell expansion) were included for comparison. Combined bisulfite restriction analysis (COBRA) of DNA from these malignant and nonmalignant populations provided evidence of TSC-22 promoter methylation (arrow) in L1210 cell line and in the T or NK LGL leukemia cells (Leu) but not in control WT cells (WT) or in cells from IL-15tg mice with polyclonal T- and NK-cell expansion (Tg). Numbers below the agarose gel represent the intensity of the methylated band, normalized by the upper unmethylated band. (C) Bisulfite sequencing of the TSC-22 promoter region from WT, IL-15tg polyclonal, and T or NK LGL leukemia from mouse spleens. Each row of ovals represents the sequence of an individual clone. Unshaded ovals indicate unmethylated CpG site; shaded ovals, methylated CpG site; TSS, transcription start site, which is designed as +1. (D) COBRA analysis of DNA from the TSC-22 promoter region in 5 murine cell lines derived from hematologic malignancies. The 2 digested fragments (arrows) correspond to methylated DNA separated on a PAGE gel.