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. 2015 Jun 19;35(14):2436–2447. doi: 10.1128/MCB.00029-15

FIG 5.

FIG 5

Epigenetic modifications of the CAG promoter in differentiating ESCs. (A) Time schedule of induction experiments in differentiating ESCs. Dashed lines, times that cells were grown in the absence of doxycycline; red lines, times that cells were grown in the presence of doxycycline; gray dots, time points of ChIP analysis; black dots, time points of bisulfite sequencing. (B) ChIP-qPCR analysis of the region encompassing the CAG and ptet promoters in differentiating clone M2-3 under noninduced (−DOX), induced (+DOX), and recovered (+DOX recovered) conditions, as outlined in panel A and the text. Antibodies against H3K36me and H3K4me3 and against whole IgG as a mock-treated control were used, as indicated. The loci analyzed, including the H3K36me3- and H3K4me3-positive controls, are indicated. Values are plotted as the ratio of the original amount of template DNA for the pulldown and input fractions. The mean and SD from two independent experiments are shown. (C) Direct comparison of H3K36me3 levels at the CAG promoter between undifferentiated and differentiating cells. Values for undifferentiated cells (Fig. 3A) and differentiating cells (B) were divided by the corresponding values for the H3K36me3-positive controls for normalization. The mean and SD from two (differentiated) or three (undifferentiated) independent experiments are shown. rec., recovered. (D) Bisulfite sequencing of the CAG promoter in several differentiating clones treated with doxycycline or not treated with doxycycline for 2 days, as outlined in panel A. For the independent clones represented in the separate panels, filled circles represent methylated CpGs and empty circles represent unmethylated CpGs. Average percentages for each condition and clone are indicated. (E) Mean EGFP FI of induced and noninduced M2-3 cells (undifferentiated and differentiating) after 2 days of doxycycline treatment and after 2 days of doxycycline treatment followed by 1 day of recovery (recovered). All cells were treated with 2,4-PDCA. Error bars represent the SDs from three independent experiments.