Hypermethylation of <i>Lxn</i> promoter CpG sites in leukemic cell lines.

<p>(<b>A</b>) CpG-enriched region in <i>Lxn</i> promoter. Sequence from 1500 base pairs upstream regulatory region to the first three exons was analyzed for CpG-enriched sites (top). A CpG island with 252 base pairs (bp_s) length was identified. The transcription starting site is indicated. Each filled circle represents a CpG dinucleotide, and there are 15. (<b>B</b>) Bisulfite sequencing for <i>Lxn</i> promoter CpG island methylation analysis in leukemic cell lines. Files are each of the 15 CpGs and ranks are replicate clones sequenced for each CpG (n = 3). Open circles indicate unmethylated CpG (n = 3) dinucleotides. Filled circles indicate methylated CpG dinucleotides. The methylation content was quantified by dividing the number of methylated CpGs with total numbers of analyzed CpGs. (<b>C</b>) Restoration or up-regulation of <i>Lxn</i> expression by 5-aza-2′-deoxycytidine. <i>Lxn</i> mRNA was measured by quantitative real-time PCR in leukemic and lymphoma cell lines exposed with or without 2 uM 5-aza-2′-deoxycytidine for 4 days. Expression levels were normalized to endogenous control, <i>Gapdh</i>. Results shown are mean± SD of 12 replicates from 3 independent experiments.