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. 2024 Feb 12;13:e86168. doi: 10.7554/eLife.86168

Figure 4. The K20 residue in EZH2 is methylated by SET7.

(A) EZH2 contains a conserved lysine residue, Lys 20 (K20), within the consensus lysine residues (K*) of the H3K4-like R/K-S/T/V-K* methylation motifs methylated by SET7. (B) K20 (red) is next to serine 21 (S21) that is phosphorylated by AKT in EZH2. The critical amino acid residues (RXRXXS) in the AKT phosphorylation consensus motif are labeled green. (C) KDM1A demethylates the methylated K20 peptide. Purified 1 μg GST control or GST-KDM1A proteins were incubated with 100 ng of mono-methylated K20 peptides for 4 hr at room temperature and the resulting peptides and input methylated peptides were spotted onto nitrocellulose membrane and blotted with affinity purified anti-methylated K20 and anti-K20 antibodies as indicated. (D) HCT116 cells were transfected with 50 nM luciferase (Luc) control or Kdm1a siRNAs for 48 hr and added 5 μg/ml MG132 or DMSO for the last 6 hr before lysing the cells for blotting. Proteins were detected by Western blotting with indicative antibodies. (E) The protein levels of EZH2, EZH2-K20me, and H3K27me3 in the brain extracts of E18.5 Nestin-Cre;Kdm1afl/+ heterozygous control or Nestin-Cre;Kdm1afl/fl homozygous Kdm1a conditional deletion mice were analyzed by Western blotting. (F) The L3mbt3 wild-type and deletion (−/−) mutant embryos on embryonic day 15.5 (E15.5) were analyzed for monomethylated K20 of EZH2. Total lysates were immunoprecipitated with EZH2 antibody and blotted with indicated antibodies. (G) HCT116 cells were transfected with control vector (pcDNA3) or SET7 expression construct for 48 hr and protein extracts were prepared. Proteins were detected by Western blotting with indicative antibodies (H) The K20-methylated EZH2 preferentially binds to L3MBTL3. The 293T cells were transfected with control vector (pcDNA3) or SET7 expression construct for 48 hr, and proteasome inhibitor MG132 (5 μg/ml) was added for the last 6 hr. Interactions between L3MBTL3 and EZH2-K20me were analyzed by co-immunoprecipitation and Western blotting analyses. (I) Silencing of SET7 re-stabilizes the protein levels of EZH2 in KDM1A deficient cells. H1299 cells expressing stably expressed Flag-EZH2 were transfected with 50 nM siRNAs of luciferase (Luc), Kdm1a (Kdm1a-1), Set7 (Set7-1) siRNAs and their combination. The indicated proteins were analyzed by Western blotting. The protein bands were quantified and normalized to that of the luciferase control. Significance was indicated as a two-tailed, unpaired, t-test. Values are expressed as the mean ± SEM. *p<0.05. **p<0.01.

Figure 4—source data 1. Original blots for Figure 4C–I.
Figure 4—source data 2. Original table sources for quantification of Figure 4 plots.

Figure 4.

Figure 4—figure supplement 1. Specificity of anti-methylated K20 peptide antibodies.

Figure 4—figure supplement 1.

The unmethylated and monomethylated K20 cognate peptides, the unmethylated K17 and the monomethylated K17 cognate peptides, di-methylated and tri-methylated K20 cognate peptides were spotted onto nitrocellulose membrane as indicated. The methylated peptides were detected by the affinity-purified anti-mono-methylated K20 peptide and EZH2 antibodies.
Figure 4—figure supplement 1—source data 1. Original dot blots for Figure 4—figure supplement 1.
Figure 4—figure supplement 2. Loss of L3mbtl3 reduces the decrease of EZH2 protein induced by Kdm1a silencing.

Figure 4—figure supplement 2.

(A) Silencing of Set7 re-stabilizes the protein levels of EZH2 in Kdm1a deficient cells. H1299 cells stably expressing Flag-EZH2 were transfected with 50 nM siRNAs of luciferase (Luc), Kdm1a (Kdm1a-2), Set7 (Set7-2) siRNAs. The indicated proteins were analyzed by Western blotting. The protein bands were quantified and normalized to that of the luciferase control. Significance was indicated as a two-tailed, unpaired, t-test. Values are expressed as the mean ± SEM. *p<0.05. **p<0.01. (B and C) Silencing of L3mbtl3 re-stabilizes the protein levels of EZH2 in Phf20l1 deficient cells. H1299 cells expressing stably Flag-EZH2 were transfected with 50 nM siRNAs of luciferase (Luc), Phf20l1 (Phf20l1-1 or Phf20l1-2), L3mbtl3-1 and Phf20l1 or Phf20l1-2, and L3mbtl3-1 siRNAs as indicated. The indicated proteins were analyzed by Western blotting. (D) T47D cells were transfected with control vector (pcDNA3) or SET7 expression construct for 48 hr and protein extracts were prepared. Proteins were detected by Western blotting with indicative antibodies.
Figure 4—figure supplement 2—source data 1. Original blots for Figure 4—figure supplement 2.