Figure 2

Quantification of cytosine modifications in Tet1 and Tet2 knockout ESCs and EpiLCs. (a–f) Global levels of (a, b) 5mC, (c, d) 5hmC, and (e, f) 5fC, in wild-type (WT), Tet1 KO (T1KO), Tet2 KO (T2KO), and Tet1/Tet2 DKO (T12KO) ESCs and EpiLCs as determined by mass spectrometry (UHPLC-MS/MS). DNA modification levels are expressed as a percentage (%) or parts per million (ppm: 1 ppm = 0.0001%) of total cytosine (dC) and shown as the mean ± SD of biological replicates as follows: WT (ESCs: n = 18; EpiLCs: n = 24), T1KO (ESCs: n = 18; EpiLCs: n = 12), T2KO (ESCs: n = 12; EpiLCs: n = 12), and T12KO (ESCs: n = 12; EpiLCs: n = 12). * p < 0.005 to wt as determined using a one-way ANOVA followed by a post-hoc Tukey HSD test. (g–h) Correlations between 5hmC and 5fC levels in wt and Tet KO (g) ESCs and (h) EpiLCs. The dashed regression line was generated using the full data set, the solid regression line was generated by excluding Tet2 KO data. Depicted are values from the individual replicates presented in c–f. R²: coefficient of determination; r: Pearson correlation coefficient. (i) Box plots of the ratio of 5fC to 5hmC in wt, Tet1 KO and Tet2 KO ESCs and EpiLCs. Unlike the Tet1 KO, Tet2 KO drastically affects the 5fC/5hmC ratio. The median is represented by the central bold line. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper and lower whisker extend from the hinge to the largest and lowest value, respectively, no further than 1.5 * interquartile range (IQR).