Figure 3

High-resolution DNA methylation mapping of individual CpG dinucleotides within highly conserved regions of the DES LCR. Sodium bisulphite conversion of Hind III-digested human unfused myoblast (UF), fused myotube (F) and PBMC DNA followed by PCR amplification, cloning and sequencing was used to obtain a high-resolution map of the methylation status across GC-rich regions of high sequence homology between mouse, rat and human within the DES LCR. (A) Top line: illustration of the DES and DES LCR HS 1–5 and positions of the evolutionarily conserved elements (vertical arrows). Middle line: CpG density map. Each vertical line represents a single CpG dinucleotide residue. Bottom line: position of PCR amplicons following genomic DNA bisulphate conversion and nChIP (Figure 2). (B) Methylation status of individual CpG sites. Each line represents the result within independent clones of PCR amplified regions. Open/white circles indicate an unmethylated CpG site; closed/black circles, methylated CpG sites. At certain sites circles are missing, sequence data was not available for these individual CpG sites. Amplicons used for nChIP analysis are shown (A-G) by a horizontal double-ended arrow. Positions of sequenced regions relative to the TSS of DES are indicated in kb. Regions of homology within the LCR are marked by a horizontal grey line.