Figure 3
Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1, DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.’
