Structure and bisulfite sequencing of the p18^INK4c promoter. (A) Top: Genomic structure of the p18^INK4c gene. Middle: Distribution of CpG dinucleotides in the promoter region of p18^INK4c. Each vertical bar represents a CpG dinucleotide, and the transcription start site is indicated by an arrow. The position of the primer sets used for MSP assays (M-FW, M-RV, U-FW, and U-RV) and bisulfite sequencing (BS-FW and BS-RV for cell lines; BS-FW, LCM-EXT, and LCM-INT for microdissected cells) is also indicated. FW indicates forward; RV, reverse. Bottom: Bisulfite sequencing of the 2 HL-derived cell lines showing p18^INK4c promoter hypermethylation (L-540 and HDLM-2). A 483-bp fragment comprising the region immediately upstream of the transcriptional start site and part of exon 1 was amplified and cloned from bisulfite-modified DNA. From each cell line, 6 clones were sequenced. Each row represents 1 clone, and squares represent CpG dinucleotides. Open squares indicate unmethylated CpG sites; gray squares, methylated CpG sites. (B) Bisulfite sequencing of microdissected cell populations. RS and normal cells were isolated by LCM (left). Original magnification × 400. DNA extracted from these cells was treated with sodium bisulfite and used as a template to amplify a 165-bp fragment from thep18^INK4c promoter (right), which was cloned and sequenced (5 clones from each reaction are represented as in panel A). Arrows above the electropherograms indicate differences between methylated (bottom, left) and unmethylated (bottom, right) sequences.