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. 2021 Sep 11;297(4):101195. doi: 10.1016/j.jbc.2021.101195

Figure 2.

Figure 2

DNMT1 mediates rDNA gene body methylation.A, knockout of DNMT1 significantly reduces gene body methylation. Genomic DNA coupled with bisulfite sequencing assays were performed to show the gene body methylation levels in control and DNMT1 KO cells. The black and white circles represent corresponding CpG sites in rDNA gene bodies, denoting methylated and unmethylated CpG sites, respectively. The average ratio of black sites for all colonies represents the relative DNA methylation level. Corresponding sequence is shown in Figure S1G. B, analysis for the results in (A) showing the percentage of methylated and unmethylated gene body CpG sites in control and DNMT1 KO cells. The relative proportion of unmethylation and methylation was calculated by counting the numbers of black circles (methylated CpG sites) and white circles (unmethylated CpG sites). C, loss of DNMT1 markedly reduces pre-rRNA synthesis. Immunostaining showing cell nuclei (blue), DNMT1 (red), and FUrd (green). HEK293T and DNMT1 KO cells were cocultured and the pre-rRNA synthesis was measured by FUrd incorporation. Bar graph shows the average FUrd fluorescence intensity per cell of DNMT1 KO or HEK293T cells (n = 4 or n = 7). A total of 155 cells were subjected to statistics. Scale bar, 5 μm. D, northern blotting shows the level of 45S pre-rRNA in DNMT1 KO or HEK293T cells. 7SL RNA served as a loading control. E, RT-qPCR results show the pre-rRNA level in DNMT1 KO and DNMT1-overexpressed DNMT1 KO cells. The cycle threshold (Ct) of 45S pre-rRNA was normalized with GAPDH. Data are presented as the mean ± SD. F, the mRNA and protein levels of Dnmt1 were decreased upon differentiation of C2C12 myoblasts as revealed by RNA-seq and western blotting, respectively. The cycle threshold (Ct) of Dnmt1 was normalized with Gapdh in RT-qPCR. GAPDH served as a loading control in western blotting. G, the occupancy of DNMT1 markedly declines upon differentiation of C2C12 myoblasts. ChIP experiments were performed using primers coated on the 28S rRNA coding region in C2C12 myoblasts and myotubes. Data are presented as the mean ± SD. H, the mRNA and protein levels of DNMT1 were reduced upon serum starvation of HEK293T cells as detected by RT-qPCR and western blotting, respectively. The cycle threshold (Ct) of DNMT1 was normalized with B2M in RT-qPCR. TUBULIN served as a loading control in western blotting. Data are presented as the mean ± SD. I, the occupancy of DNMT1 reduces in serum-deprived HEK293T cells. ChIP experiments were performed using primers coated on the 28S rRNA coding region in serum-deprived and control cells. Data are presented as the mean ± SD. ∗p < 0.1, ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed unpaired Student's t test.