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

Figure 3.

Figure 3

DNMT1 antagonizes H4K20me3 to regulate rDNA transcription.A, the enrichment of H4K20me3 is elevated in DNMT1 KO cells. ChIP experiments were performed using primers coated on the 28S rRNA coding region in DNMT1 KO and control cells. Data are presented as the mean ± SD. B, knockout of SUV4-20H1/H2 using the CRISPR/Cas9 system as shown by western blotting. C, loss of SUV4-20H1/H2 leads to a marked reduction in H4K20me3 enrichment at gene bodies as detected by ChIP-qPCR. Data are presented as the mean ± SD. D, depletion of SUV4-20H1/H2 leads to a marked increase in pre-rRNA synthesis. Immunostaining showing cell nuclei (blue) and FUrd (green). The pre-rRNA synthesis was measured by FUrd incorporation. Bar graph shows the average FUrd fluorescence intensity per cell of SUV4-20H KO or HEK293T cells (n = 7 or n = 8). A total of 245 cells were subjected to statistics. Scale bar, 5 μm. E, pre-rRNA synthesis increases significantly upon SUV4-20H depletion compared with that in HEK293T cells. RT-qPCR assays showing levels of pre-rRNA in SUV4-20H DKO and wild-type HEK293T cells. The cycle threshold (Ct) of 45S pre-rRNA was normalized with GAPDH. Data are presented as the mean ± SD. F, knockout (KO) of SUV4-20H does not affect the rDNA gene body methylation level. Genomic DNA coupled with bisulfite sequencing assays were performed to show the gene body methylation level in SUV4-20H DKO and control 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. G, analysis for the results in (F) showing the percentage of methylated and unmethylated gene body CpG sites in SUV4-20H DKO and control cells. The relative proportion of unmethylation and methylation were calculated by counting the numbers of black circles (methylated CpG sites) and white circles (unmethylated CpG sites). H, pre-rRNA synthesis is markedly reduced in DNMT1/SUV4-20H TKO cells. Immunostaining showing cell nuclei (blue), DNMT1 (red), and FUrd (green). The pre-rRNA synthesis was measured by FUrd incorporation. Bar graph shows the average FUrd fluorescence intensity per cell of DNMT1/SUV4-20H TKO or HEK293T cells (n = 6). A total of 227 cells were subjected to statistics. Scale bar, 5 μm. I, pre-rRNA synthesis increases significantly upon triple knockout of DNMT1 and SUV4-20H1/H2 (designated as DNMT1/SUV4-20H−/−) compared with that in DNMT1 KO cells. RT-qPCR assays showing levels of pre-rRNA in DNMT1−/− and DNMT1/SUV4-20H−/− cells. The cycle threshold (Ct) of 45S pre-rRNA was normalized with GAPDH. Data are presented as the mean ± SD. ∗p < 0.1, ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed unpaired Student's t test.