Figure 3

Global loss of 5hmC and locus-specific gains in 5mC suggest loss of demethylase activity in culture. (a) Histogram of genes showing a significant change (Mann-Whitney U-test, P <0.01) in gene body 5mC and 5hmC content upon adaptation of primary MEF cells to culture. (b) Schematic representation of the mean (N = 4) 5hmC content of two gene (Klf5 and Vwa2) bodies before and after adaptation to culture. Bar chart of hMeDIP-qPCR showing decreased gene body 5hmC content in cultured MEFs (lower panel). Values are the average of four biological replicates ± standard deviation. Multiple primer pairs were used to determine values across gene bodies of Klf5, Vwa2, LRRC30 and 1700054A03Rik (black bars). (c) A histogram of the distribution of genes showing a significant change (Mann-Whitney U-test, P <0.01) in promoter 5mC (red) and 5hmC (blue) content upon adaptation of MEF cells to culture. (d) A schematic representation of the mean (N = 4) promoter 5mC profiles at the Hoxc-12, -11 and -4 genes before (blue) and after (orange) adaptation to culture (upper panel). Bar chart of hMeDIP-qPCR showing increased promoter 5mC content at the Hoxc-12, -11 and -4 gene regions in cultured MEFs (lower panel). Values are the average of four biological replicates ± standard deviation. The location of hMeDIP-qPCR primers for each Hoxc region is shown in black above gene maps (top). (e) The imprinted locus, H19, maintains high levels of 5hmC in culture. A schematic representation is shown for the 5hmC profile across the imprinted locus, H19, for all eight samples (four male (♂) and four female (♀)). The location of hMeDIP-qPCR primers is shown in black. (f) Bar-chart of hMeDIP-qPCR showing retention of 5mC and 5hmC at the imprinted loci H19 and Gnas (lower panel) in cultured MEFs. Values are the average of four biological replicates ± standard deviation.