Figure 4.

Analysis of PolgA and mtDNA enrichment in 5mC and 5hmC MeDIP of ESCs and somatic tissues. DNA samples from cultured CC9^mus, CC9^spretus and CC9^dunni cells; liver, spleen, heart and brain samples were immunoprecipitated using antibodies against (A) 5mC and (B) 5hmC, and analysed using real time PCR for PolgA (exon 2) enrichment. Bars represent means ± SEM. Significant differences between cell types are indicated (**P < 0.01; ***P < 0.001). (C) MeDIP (5mC and 5hmC) enrichment (Figure 4A and B) was validated against the corresponding means for bisulphite sequencing methylation levels (Figures 1L and 3G) using Pearson correlation coefficient (R²). Closed circles represent 5mC enrichment and open circles represent 5hmC enrichment. The P-value signifies the significance of the line from zero. (D) The relationship between PolgA expression and the corresponding MeDIP (5mC and 5hmC) enrichment levels using a Pearson correlation coefficient (R²). Closed circles represent 5mC enrichment and open circles represent 5hmC enrichment. The P-value signifies the significance of the line from zero. (E) Real time PCR for the enrichment of PolgA (exon 2) and mtDNA (CytB, tRNA/COXI and D-loop regions) in 5mC and 5hmC immunoprecipitated DNA. Enrichment values for CC9^mus, CC9^spretus and CC9^dunni cells; and liver, spleen, heart and brain samples were normalized against β-actin (unmethylated control). Data shown is log₁₀ 5mC against log₁₀ 5hmC. Data points are colour-coded to represent DNA sequences analysed (PolgA exon 2 and mtDNA: CytB, tRNA/COXi and D-loop regions). Data points highlighted in the red circle represent the high levels of enrichment observed for liver, spleen, heart and brain samples (cf Figure 4A and B).