Fig 1: Level of H3K27me3 is antagonistically regulated by Kdm6b and Ezh2 during palatogenesis.(A–E) Contribution of H3K27me3 in the palatal shelf is evaluated using immunostaining and Western blot at E13.5. Dotted lines in (A, C) indicate palatal shelf region. (B, D) are magnified images of boxes in (A, C). Asterisk in (B) indicates no accumulation of H3K27me3 observed in control mice. Arrowheads in (D) indicate accumulation of H3K27me3 observed in Wnt1Cre;Kdm6bfl/fl mice. Scale bar: 50 µm. (F–J) Contribution of Ezh1 in the palatal shelf is evaluated using RNAscope in situ hybridization and Western blot at E13.5. Dotted lines in (F, H) indicate palatal shelf region. (G, I) are magnified images of boxes in (F, H). Arrowheads in (G, I) indicate representative Ezh1+ cells. Scale bar: 50 µm. (K–O) Contribution of EZH2 in the palatal shelf is evaluated using immunostaining and Western blot at E13.5. Dotted lines in (K, M) indicate palatal shelf region. (L, N) are magnified images of boxes in (K, M). Arrowheads in (L, N) indicate representative EZH2+ cells. Scale bar: 50 µm. (P–V) Contribution of H3K27me3 in the palatal shelf of control mice, Kdm6b mutant mice, and EZH2 haploinsufficient model is evaluated using immunostaining and Western blot at E13.5. Dotted lines in (P, R, T) indicate palatal shelf region. (Q, S, U) are magnified images of boxes in (P, R, T), respectively. Asterisks in (Q, U) indicate no accumulation of H3K27me3 observed in control (Q) and Wnt1Cre;Kdm6bfl/fl;Ezh2fl/+ mice (U). White arrowheads in (S) indicate accumulation of H3K27me3 observed in Wnt1Cre;Kdm6bfl/fl mice. Scale bar: 50 µm. Figure 5—source data 1.Source data for Figure 5E. Figure 5—source data 2.Source data for Figure 5J. Figure 5—source data 3.Source data for Figure 5O. Figure 5—source data 4.Source data for Figure 5V.
Fig 2: Histone demethylation enhanced DLX5 expression and inhibited SKM-1 cell proliferation. SKM-1 cells transfected with si-EZH1, pcDNA3.1-EZH2, and pcDNA3.1-EHMT2 were used as the control group, and then treated with EPZ-6438 and (or) BIX-01294. (A) Histone methylation and protein level were measured using Western blot analysis; (B) DLX5 mRNA expression was detected using RT-qPCR; (C) Proliferation ability of SKM-1 cells under different treatments was measured using colony formation assay. The cell experiments were repeated three times. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test, *p < 0.05, vs. Control group, ***p < 0.001 vs. Control group; #p < 0.05 vs. adjacent groups, ##p < 0.01 vs. adjacent groups, ###p < 0.001 vs. adjacent groups.
Fig 3: EZH2 and EHMT2 synergistically inhibited DLX5 gene transcription. (A) DLX5 mRNA expression in SKM-1 cells was detected using RT-qPCR; (B) DLX5 mRNA expression in NHD13 mice and C57BL/6 mice was detected using RT-qPCR; (C) DLX5 mRNA expression was increased to the highest level when EZH2 and EHMT2 were at low expression at the same time, and decreased to the lowest level when EZH2 and EHMT2 were at high expression at the same time, with the X-axis representing DLX5 mRNA expression, the Y-axis representing EZH2 and EHMT2 mRNA expressions; (D) Binding levels of DLX5 promoter region with H3K27me3, H3K9me1, and H3K9me2 were detected using ChIP, with the histogram showing the binding content of DLX5 promoter in DNA IP detected using RT-qPCR after ChIP experiment, in the presence of the relative content of control DNA input; (E) Expressions of EZH1, EZH2, and EHMT2 in SKM-1 cells were detected using RT-qPCR; (F) Expressions of H3K27me3 and H3K9me2 in SKM-1 cells were detected using Western blot analysis; (G) DLX5 mRNA expression in SKM-1 cells was detected using RT-qPCR. The cell experiments were repeated three times. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test, ***p < 0.001 vs. si-NC-1 group; #p < 0.05 vs. adjacent groups, ###p < 0.001 vs. adjacent groups.
Fig 4: EZH2 regulated H3K27me3 level and EZH1 compensated for the effect of EZH2 deficiency. SKM-1 cells were transfected with si-EZH1, pcDNA3.1-EZH2, or si-EZH2. (A) H3K27me3 level in SKM-1 cells was measured using Western blot analysis; (B) EZH1 protein level in SKM-1 cells was detected using Western blot analysis. Image J was used for the gray analysis of the Western blot bands. The cell experiments were repeated three times. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001.
Fig 5: Mechanism diagram. EZH2 and EHMT2 synergistically regulated DLX5 transcription and promoted the transformation from MDS to AML. NHD13 mice with high EZH2 expression in peripheral blood had earlier AML transformation. EZH1 and EZH2 regulated the level of histone H3K27me3, and EHMT2 catalyzed H3K9me1 and H3K9me2. EZH2 and EHMT2 regulated the histone methylation level of DLX5 promoter region, synergistically inhibited the transcription of DLX5, and then promote the transformation of MDS into AML.
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