Figure 5
Figure 5. Ezh2 regulates genes relevant to the development and differentiation processes. (A) Levels of H3K27me3 in MLL-AF9–transformed GMPs and BM leukemic cells. Histones were extracted from Ezh2+/+ or Ezh2Δ/Δ–transformed GMPs or CD45.2+ leukemic cells isolated from primary recipient mice and analyzed by Western blotting using an anti-H3K27me3 Ab. Levels of H3K27me3 were normalized to the amount of H3 and are indicated relative to Ezh2+/+ control values. The levels of H3K27me3 in Ezh2+/+ cells were arbitrarily set to 1. (B) Summary of H3K27me3 enrichment detected by ChIP-seq analysis. CD45.2+ Ezh2+/+ and Ezh2Δ/Δ leukemic cells isolated from primary recipient mice were subjected to ChIP-seq analysis using an anti-H3K27me3 Ab. The fold enrichment values of H3K27me3 signals were calculated against the input signals (IP/input) from 5.0 kb upstream to 3.0 kb downstream of the TSS of RefSeq genes. (C) Pie graphs illustrating the proportion of categories of GO terms showing significant differences. GO analysis was performed using 1021 genes with the H3K27me3 enrichment (WT IP/WT input) greater than 3-fold in the control AML cells (left) and 584 genes that showed a reduction in H3K27me3 enrichment (WT IP/KO IP) more than 2-fold on deletion of Ezh2 (right). (D) A scatter diagram of microarray analysis. Lin−Sca-1−c-Kit+FcγRII/IIIhi BM leukemic cells were isolated from primary recipient mice and analyzed by microarray-based expression analysis. The average signal levels of Ezh2Δ/Δ cells compared with those of Ezh2+/+ cells are plotted. The light and dark gray lines represent the borderline for a 2-fold increase and a 2-fold decrease, respectively. Egr1 is indicated with a diamond. (E) Correlation of derepression in expression in Ezh2Δ/Δ leukemic cells in terms of the degree of H3K27me3 enrichment. The genes up-regulated on deletion of Ezh2 (Ezh2Δ/Δ/control leukemic cells > 1.0 in microarray analysis) were analyzed in terms of the degree of H3K27me3 enrichment in the control leukemic cells detected by ChIP-seq. The fold enrichment values for H3K27me3 were binned (each bin containing 1.0-fold enrichment). The number of genes in a bin and the average degree of up-regulation are indicated as bars and circles, respectively.

Ezh2 regulates genes relevant to the development and differentiation processes. (A) Levels of H3K27me3 in MLL-AF9–transformed GMPs and BM leukemic cells. Histones were extracted from Ezh2+/+ or Ezh2Δ/Δ–transformed GMPs or CD45.2+ leukemic cells isolated from primary recipient mice and analyzed by Western blotting using an anti-H3K27me3 Ab. Levels of H3K27me3 were normalized to the amount of H3 and are indicated relative to Ezh2+/+ control values. The levels of H3K27me3 in Ezh2+/+ cells were arbitrarily set to 1. (B) Summary of H3K27me3 enrichment detected by ChIP-seq analysis. CD45.2+Ezh2+/+ and Ezh2Δ/Δ leukemic cells isolated from primary recipient mice were subjected to ChIP-seq analysis using an anti-H3K27me3 Ab. The fold enrichment values of H3K27me3 signals were calculated against the input signals (IP/input) from 5.0 kb upstream to 3.0 kb downstream of the TSS of RefSeq genes. (C) Pie graphs illustrating the proportion of categories of GO terms showing significant differences. GO analysis was performed using 1021 genes with the H3K27me3 enrichment (WT IP/WT input) greater than 3-fold in the control AML cells (left) and 584 genes that showed a reduction in H3K27me3 enrichment (WT IP/KO IP) more than 2-fold on deletion of Ezh2 (right). (D) A scatter diagram of microarray analysis. Lin−Sca-1−c-Kit+FcγRII/IIIhi BM leukemic cells were isolated from primary recipient mice and analyzed by microarray-based expression analysis. The average signal levels of Ezh2Δ/Δ cells compared with those of Ezh2+/+ cells are plotted. The light and dark gray lines represent the borderline for a 2-fold increase and a 2-fold decrease, respectively. Egr1 is indicated with a diamond. (E) Correlation of derepression in expression in Ezh2Δ/Δ leukemic cells in terms of the degree of H3K27me3 enrichment. The genes up-regulated on deletion of Ezh2 (Ezh2Δ/Δ/control leukemic cells > 1.0 in microarray analysis) were analyzed in terms of the degree of H3K27me3 enrichment in the control leukemic cells detected by ChIP-seq. The fold enrichment values for H3K27me3 were binned (each bin containing 1.0-fold enrichment). The number of genes in a bin and the average degree of up-regulation are indicated as bars and circles, respectively.

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