Restoration of H3K27me3 levels at part of Ezh2 target gene promoters in Ezh2^Δ/Δ HSPCs. (A) Scatter plots showing the correlation of the fold enrichment values (ChIP/input) (transcription start site ± 2.0 kb) of H3K27me3 against the input signals of RefSeq genes between WT and Ezh2^Δ/Δ LSK cells from WT and Ezh2^Δ/Δ mice at 1 and 9 months postdeletion of Ezh2. (B) A scatter plot showing the correlation of H3K27me3 levels in Ezh2^Δ/Δ LSK cells against WT LSK cells (Ezh2^Δ/Δ/WT) between 1 and 9 months postdeletion of Ezh2. The diagonal light gray lines represent the boundaries for twofold increase and twofold decrease, respectively. The vertical line represents the boundary for twofold reduction in fold enrichment of H3K27me3 in Ezh2^Δ/Δ LSK cells at 1 month postdeletion of Ezh2. The genes with ChIP signals >1 over the input signals were categorized depending on the changes in H3K27me3 levels at 1 and 9 months postdeletion of Ezh2. The genes that lost H3K27me3 levels at least twofold at 1 month postdeletion of Ezh2 were defined as “Ezh2 targets.” Among these, the genes that gained H3K27me3 levels at least twofold at 9 months compared with 1 month postdeletion of Ezh2 were defined as “Ezh2 target/Ezh1 Comp.” In contrast, the genes that did not gain H3K27me3 levels were defined as “Ezh2 target/Ezh1 no Comp.” The genes that did not lose H3K27me3 levels twofold at 1 month postdeletion of Ezh2 and did not show any significant changes thereafter were defined as “Ezh1 target genes.” Each category was boxed. (C) Visualization of ChIP-sequence data of H3K27me3 levels of representative genes that belong to the indicated categories defined in panel A in WT and Ezh2^Δ/Δ LSK cells at 1 and 9 months postdeletion of Ezh2 using the Integrative Genomics Viewer (IGV). Schematic diagrams of these gene loci indicate their genomic structures. Exons and untranslated regions are demarcated by large and small black boxes, respectively. (D) Box-and-whisker plots showing the expression changes of indicated genes in Ezh2^Δ/Δ LSK cells relative to WT LSK cells (Ezh2^Δ/Δ/WT) at 9 to 12 months compared with 3 months postdeletion of Ezh2. Boxes represent 25 to 75 percentile ranges. Vertical lines represent 10 to 90 percentile ranges. Horizontal bars represent median. Mean values are indicated by red dots. Microarray data in Figure 3 were applied to this analysis. ***P < .001 (Student t test). (E) Venn diagram showing the overlap between Ezh2 target/Ezh1 Comp genes in panel B and bivalent genes. P < 1.0 × 10⁻¹⁶. (F) Box-and-whisker plots showing the changes in H3K27me3 levels of indicated genes in Ezh2^Δ/Δ LSK cells relative to WT LSK cells (Ezh2^Δ/Δ/WT) at 1 and 9 months postdeletion of Ezh2. Mean values are indicated by red dots. *P < .05, ***P < .001 (Student t test). (G) Box-and-whisker plots showing the expression changes of indicated genes in Ezh2^Δ/Δ LSK cells relative to WT LSK cells (Ezh2^Δ/Δ/WT) at 9 to 12 months compared with 3 months postdeletion of Ezh2. Mean values are indicated by red dots. Microarray data in Figure 3 was applied to this analysis. ***P < .001 (Student t test). ns, not significant.