Marked inter-tissue differences in global 5hmC levels. (A) Dot-blots of decreasing amounts of a PCR product of the mouse Tex19 promoter sequence in which all cytosines are either unmodified (C), methylated (5mC), or hydroxymethylated (5hmC), were probed with α-5hmC and α-5mC antibodies. The α-5hmC and α-5mC antibodies are specific for their respective marks. (B) Duplicate dot-blots of DNA from human tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal human tissues. An α-ssDNA antibody was used to control for loading; 500 ng and 100 ng were loaded in the upper and lower lanes, respectively. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA. (C) Inter-tissue differences in global 5hmC levels as determined by densitometric analysis of dot-blots shown in panel B. 5hmC values were normalized to an ssDNA loading control and scaled relative to brain. Values are the means of two independent biological replicates. Spleen and ES values represent single measurements. (D) A scatterplot showing a lack of correlation between global 5mC and 5hmC values for seven samples. 5mC values were obtained from previously published HPLC analyses of global 5mC content in human tissues (Ehrlich et al. 1982; Weisenberger et al. 2005). (E) Duplicate dot-blots of DNA from mouse tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal mouse tissues. An α-ssDNA antibody was used to control for loading. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA.
