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

Figure 2. Loss of L3MBTL3 stabilizes EZH2 protein.

(A) Left: The mouse L3mbt3 wild-type (+/+) and L3mbtl3 null (-/-, KO) mutant embryos on embryonic day 17.5 (E17.5) after breeding. Right: Total lysates from the heads of mouse L3mbt3 (+/+) wild-type and L3mbt3 homozygous deletion (-/-, KO) mutant embryos (equal total proteins) were analyzed by Western blotting with antibodies for the indicated proteins. (B) Mouse embryonic fibroblasts from the wild-type and L3mbtl3 deletion mutant embryos (E13.5) were examined for EZH2 and H3K27me3 proteins by Western blotting. (C) Western blot analysis of EZH2 and SUZ12 proteins in the head extracts of Nestin-Cre;L3mbtl3fl/+ control and Nestin-Cre;L3mbtl3fl/fl conditional deletion (cKO) embryos (E14.5) using the indicative antibodies. (D) Coronal sections of the developing mouse brain at E15.5 were stained with anti-EZH2 and H3K27me3 antibodies in wild-type control and Nestin-Cre;L3mbtl3fl/fl conditional deletion mice. Scale bars, 100 μm. Arrows and arrowheads indicate the regions of EZH2 and H3K27me3 expression, respectively. LV: lateral ventricle. (E) Deletion of both Kdm1a and L3mbtl3 by 4-OH-TAM restores the protein levels of EZH2 and H3K27me3. MEFs from the Nestin-Cre;Kdm1afl/fl and CAGGCre-ER;Kdm1afl/fl;L3mbtl3fl/fl mouse embryos (E13.5) were treated with 4-hydroxytamoxifen (4-OH-TAM, 20 μg/ml) for 12 hr to delete Kdm1a and L3mbtl3. (F) Silencing of L3mbtl3 re-stabilizes the protein levels of EZH2 in Kdm1a deficient cells. The Flag-EZH2 under the retroviral LTR promoter control were ectopically and stably expressed in H1299 cells and the cells were transfected with 50 nM siRNAs of luciferase (Luc), Kdm1a (Kdm1a-1), and L3mbtl3 (L3mbtl-1) siRNAs. (G) Silencing of L3mbtl3 stabilizes EZH2 protein in Kdm1a deficient cells. The Flag-EZH2 under the retroviral LTR promoter control were ectopically and stably expressed in H1299 cells and the cells were transfected with 50 nM siRNAs of luciferase (Luc), Kdm1a (Kdm1a-2), Kdm1a and L3mbtl3 (L3mbtl3-2), and L3mbtl3 siRNAs. The indicated proteins were analyzed by Western blotting. For (A–C), (F), and (G), band intensities were quantified and normalized to that of the luciferase or actin control. Significance was indicated as a two-tailed, unpaired, t-test. Values are expressed as the mean ± SEM. *p<0.05. **p<0.01. ***p<0.001.

Figure 2—source data 1. Original blots for Figure 2A–C, original photos for Figure 2D, and original blots for Figure 2E–G.
Figure 2—source data 2. Original table sources for quantification of Figure 2 plots.

Figure 2.

Figure 2—figure supplement 1. Schematic illustration to generate a conditional flox allele in L3mbtl3fl/fl with the FRT-loxP sites at the exon 5 of the L3mbtl3 locus.

Figure 2—figure supplement 1.

The LacZ-neo fragment was removed by breeding with the FLPo-10 mice to obtain the conditional L3mbtl3fl/+ mice. The loxP-neo fragment was removed by breeding with the Sox2-Cre mice to obtain L3mbtl3-LacZ mice. Green triangles are FRT sites and the red arrows are loxP sites. F and R in the figure are PCR primers for genotyping.
Figure 2—figure supplement 2. The EZH2 protein is regulated by L3MBTL3.

Figure 2—figure supplement 2.

(A) The genomic L3mbtl3 alleles were deleted in HCT116 cells by the CRISPR-Cas9 gene editing. The protein levels of EZH2 in wild-type and L3MBTL3 deleted (KO) HCT116 cells were analyzed by western blotting. (B). The Flag-L3mbt3 under the retroviral LTR promoter control were ectopically and stably expressed in HCT116 cells. The change in the EZH2 protein level was measured by western blotting.
Figure 2—figure supplement 2—source data 1. Original blots for Figure 2—figure supplement 2.