Figure 2
Figure 2. Y641 mutations in B-cell lines and tumors increase steady state of H3K27me3. (A) Steady-state H3K27me3 levels in DLBCL. Nuclear lysates from DLCBCLs with either wild-type EZH2 (Pfeiffer, MD903, NU-DHL-1, NU-DUL-1, DOHH2, and Toledo) or heterozygous for EZH2 (SU-DHL-6 (+/Y641N), Karpas 422 (+/Y641N), DB (+/Y641N), WSU-DLCL-2 (+/Y641F)) were probed with the respective antibodies to the following epitopes: H4K20me3 (arrowed lower band), H3K27me3, H3K27me2, H3K9me3, or EZH2. Levels of H3 were used as a loading control for histones. (B) Whole cell lysates from frozen tumor sections (each 5 × 20-μm slices) of 10 patients with either wild-type EZH2 (IDs 396, 839, 315, 085, or 900) or heterozygous for EZH2 (IDs 694 (+/Y641F), 178 (+/Y641H), 883 (+/Y641N), 940 (+/Y641F), or 353 (+/Y641S)) were probed with the respective antibodies. This a composite figure assembled to reflect similar levels of H3 from the 2 blots. (C) Nuclear lysates from HEK293T cells stably expressing GFP-tagged EZH2 and Y641 mutants were probed with anti-Ezh2 to assess ectopically expressed levels (top band) or endogenous (lower band) Ezh2 levels and anti-H3K27me3. Anti-H3 was used to assess histones as a loading control. (D) Respective plasmids encoding GFP, EZH2, or mutants (as indicated) were transfected in HEK293T cells; the lysates were probed with the antibodies (anti-FLAG M2, anti-GFP, anti-EZH2 (top band shows GFP-tagged EZH2, lower band shows endogenous EZH2 and FLAG-tagged EZH2), monomethyl, dimethyl, or trimethyl specific H3K27. These antibodies are specific for the respective methylation states (supplemental Figure 3). (E) GFP-tagged proteins from nuclear lysates of HEK293T lines stably expressing GFP-EZH2 wild-type and mutants were immunoprecipitated with GFP-trap (“Western blotting and immunoprecipitation”) and probed with anti-EZH2 to show precipitation of GFP-tagged EZH2 (top band) with copurification of endogenous EZH2 (lower band) and EED and SUZ12 (lower band, arrowed). *Nonspecific band. (F) Mouse bone marrow stem cells were infected with the respective retroviruses expressing wild-type or Y641F EZH2 and differentiated in vitro into B cells before whole cell lysates were probed with the respective antibodies. The top band (arrowed) in the EZH2 blot represents ectopic EZH2-HA and endogenous EZH2. *The lower band indicates degradation products. (G) Densitometry measurements of H3K27me3 to total H3 ratio in wild-type and mutant cell lines. The boxplots represent the distribution of ratios in mutant and wild-type. Significance testing by unpaired Student t test. (H) Densitometry measurements of H3K27me3 to total H3 ratio in wild-type and lymphoma samples. The boxplots represent the distribution of ratios in mutant and wild-type. Significance testing by unpaired Student t test.

Y641 mutations in B-cell lines and tumors increase steady state of H3K27me3. (A) Steady-state H3K27me3 levels in DLBCL. Nuclear lysates from DLCBCLs with either wild-type EZH2 (Pfeiffer, MD903, NU-DHL-1, NU-DUL-1, DOHH2, and Toledo) or heterozygous for EZH2 (SU-DHL-6 (+/Y641N), Karpas 422 (+/Y641N), DB (+/Y641N), WSU-DLCL-2 (+/Y641F)) were probed with the respective antibodies to the following epitopes: H4K20me3 (arrowed lower band), H3K27me3, H3K27me2, H3K9me3, or EZH2. Levels of H3 were used as a loading control for histones. (B) Whole cell lysates from frozen tumor sections (each 5 × 20-μm slices) of 10 patients with either wild-type EZH2 (IDs 396, 839, 315, 085, or 900) or heterozygous for EZH2 (IDs 694 (+/Y641F), 178 (+/Y641H), 883 (+/Y641N), 940 (+/Y641F), or 353 (+/Y641S)) were probed with the respective antibodies. This a composite figure assembled to reflect similar levels of H3 from the 2 blots. (C) Nuclear lysates from HEK293T cells stably expressing GFP-tagged EZH2 and Y641 mutants were probed with anti-Ezh2 to assess ectopically expressed levels (top band) or endogenous (lower band) Ezh2 levels and anti-H3K27me3. Anti-H3 was used to assess histones as a loading control. (D) Respective plasmids encoding GFP, EZH2, or mutants (as indicated) were transfected in HEK293T cells; the lysates were probed with the antibodies (anti-FLAG M2, anti-GFP, anti-EZH2 (top band shows GFP-tagged EZH2, lower band shows endogenous EZH2 and FLAG-tagged EZH2), monomethyl, dimethyl, or trimethyl specific H3K27. These antibodies are specific for the respective methylation states (supplemental Figure 3). (E) GFP-tagged proteins from nuclear lysates of HEK293T lines stably expressing GFP-EZH2 wild-type and mutants were immunoprecipitated with GFP-trap (“Western blotting and immunoprecipitation”) and probed with anti-EZH2 to show precipitation of GFP-tagged EZH2 (top band) with copurification of endogenous EZH2 (lower band) and EED and SUZ12 (lower band, arrowed). *Nonspecific band. (F) Mouse bone marrow stem cells were infected with the respective retroviruses expressing wild-type or Y641F EZH2 and differentiated in vitro into B cells before whole cell lysates were probed with the respective antibodies. The top band (arrowed) in the EZH2 blot represents ectopic EZH2-HA and endogenous EZH2. *The lower band indicates degradation products. (G) Densitometry measurements of H3K27me3 to total H3 ratio in wild-type and mutant cell lines. The boxplots represent the distribution of ratios in mutant and wild-type. Significance testing by unpaired Student t test. (H) Densitometry measurements of H3K27me3 to total H3 ratio in wild-type and lymphoma samples. The boxplots represent the distribution of ratios in mutant and wild-type. Significance testing by unpaired Student t test.

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