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

Figure 5. L3MBTL3 and DCAF5 target methylated K20 to promote EZH2 proteolysis.

(A) Mouse embryos at the indicated embryonic days were prepared and the indicated proteins in the lysates were examined with respective antibodies. (B) LacZ staining in mouse embryos carrying the LacZ gene under the L3mbtl3 promoter control (L3mbtl-LacZ) identifies the endogenous L3MBTL3 expression during mouse development. (C) Endogenous L3MBTL3 interacts with EZH2. Lysates were extracted from mouse embryos (E14.5) and the interaction between L3MBTL3 and EZH2 were analyzed by co-immunoprecipitation and blotted with respective antibodies. Input: 1/10 of the lysates for Western blotting. (D) SET7 stimulates the interaction between L3MBTL3 and EZH2. 293T cells were transiently transfected with the expression vector of Set7 wild-type, Set7H297A mutant, or the empty vector for 48 hr. Cell lysates were immunoprecipitated by anti-L3MBTL3 antibodies and blotted with antibodies against EZH2 and L3MBTL3. (E) The K20R mutant does not interact with L3MBTL3. HA-tagged EZH2 or the HA-K20R mutant expressing constructs were co-transfected with vector expressing Set7 or empty vector into 293T cells. Cell lysates were immunoprecipitated with the anti-HA antibody and the blots were immunoblotted with anti-L3MBTL3 and anti-HA antibodies. (F) The Flag-tagged Ezh2 wild-type, K20r, or S21a mutant were stably expressed in G401 cells. The cells were then transfected with 50 nM siRNAs of luciferase or Kdm1a for 48 hr. The protein levels of Flag-EZH2, H3K27me3, and indicated other proteins were analyzed by immunoblotting. 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. (G) and (H) The EGFP-tagged wild-type Ezh2 or K20r mutant were co-transfected into 293T cells together with vectors expressing HA-tagged ubiquitin (HA-Ub) and SET7 in the presence or absence of L3MBTL3 and DCAF5 expressing constructs as indicated. Proteasome inhibitor MG132 (5 μg/ml) was added for the last 6 hr to stabilize the polyubiquitinated EZH2. Proteins were immunoprecipitated with anti-GFP antibodies and Western blotted with anti-GFP and other antibodies against indicated proteins.

Figure 5—source data 1. Original blots for Figure 5.
Figure 5—source data 2. Original table sources for quantification of Figure 5 plots.

Figure 5.

Figure 5—figure supplement 1. The mRNA analysis in the mouse embryos.

Figure 5—figure supplement 1.

Mouse embryos (E12.5) at the indicated embryonic days were prepared and reverse-transcriptional quantitative PCR (RT-qPCR) analysis of the mRNA levels of Kdm1a, Ezh2, Set7, Dcat5, and L3mbtl3 in the brain. The mRNA levels were measured in triplicate by RT-qPCR. Quantifications are represented by a bar graph with mean and standard deviation (S.D.) for error bars from three replicate samples and normalized to the control E12.5 embryo mRNAs and plotted.