Figure 6
Overexpression of Egr1, one of the direct targets of Ezh2, promotes the differentiation of AML. (A) Expression of Egr1 in Lin−Sca-1−c-Kit+FcγRII/IIIhi BM leukemic cells isolated from primary recipient mice. mRNA levels of Egr1Z were evaluated by qRT-PCR and normalized to Hprt1 expression. Data are shown as the means ± SD for analyses from 3 different mice. (B) The H3K27me3 signal map at the Egr1 locus detected by the ChIP-seq analysis using an anti-H3K27me3 Ab. (C) Schematic diagram of the Egr1 and Ink4a/Arf loci indicating their genomic structures. Exons are demarcated by black boxes. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. (D) Q-ChIP analyses of CD45.2+ BM leukemic cells from recipient mice of primary transplantation. Black and gray bars represent Ezh2+/+ and Ezh2Δ/Δ cells, respectively. Abs specific to the H3K27me3 (top panel) or Ezh2 (bottom panel) were used. There were no detectable or very low levels of background signals with IgG isotype controls at all amplified regions. Percentages of input DNA are shown as the means ± SD for triplicate analyses. Data shown are representative of 2 independent experiments. **P < .01, *P < .05. (E) Colony-forming capacity of MLL-AF9–transformed GMPs overexpressing Egr1. MLL-AF9–transformed Cre-ERT;Ezh2+/+ GMPs were transduced with Egr1 or control retroviruses and purified by cell sorting using green fluorescent protein as a marker. Sorted cells (3000 cells each) were plated in methylcellulose medium containing 50 ng/mL of SCF, 10 ng/mL of FP6, 10 ng/mL of GM-CSF, and 10 ng/mL of IL-3. The black and gray bars represent compact and dispersed colonies, respectively. The data are shown as the means ± SD for triplicate analyses. **P < .01. (F) Morphology of colonies generated in panel E. Representative colonies observed under an inverted microscope. Magnification ×200. (G) Typical cell morphology of MLL-AF9–transformed GMPs overexpressing Egr1 after 3 days of culture in methylcellulose medium in panel E. Cells were cytospun onto glass slides and observed after Wright-Giemsa staining. Magnified images of the cells boxed are depicted in the insets. Magnification ×400 and ×1000 (insets).

Overexpression of Egr1, one of the direct targets of Ezh2, promotes the differentiation of AML. (A) Expression of Egr1 in Lin−Sca-1−c-Kit+FcγRII/IIIhi BM leukemic cells isolated from primary recipient mice. mRNA levels of Egr1Z were evaluated by qRT-PCR and normalized to Hprt1 expression. Data are shown as the means ± SD for analyses from 3 different mice. (B) The H3K27me3 signal map at the Egr1 locus detected by the ChIP-seq analysis using an anti-H3K27me3 Ab. (C) Schematic diagram of the Egr1 and Ink4a/Arf loci indicating their genomic structures. Exons are demarcated by black boxes. Regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by arrows. (D) Q-ChIP analyses of CD45.2+ BM leukemic cells from recipient mice of primary transplantation. Black and gray bars represent Ezh2+/+ and Ezh2Δ/Δ cells, respectively. Abs specific to the H3K27me3 (top panel) or Ezh2 (bottom panel) were used. There were no detectable or very low levels of background signals with IgG isotype controls at all amplified regions. Percentages of input DNA are shown as the means ± SD for triplicate analyses. Data shown are representative of 2 independent experiments. **P < .01, *P < .05. (E) Colony-forming capacity of MLL-AF9–transformed GMPs overexpressing Egr1. MLL-AF9–transformed Cre-ERT;Ezh2+/+ GMPs were transduced with Egr1 or control retroviruses and purified by cell sorting using green fluorescent protein as a marker. Sorted cells (3000 cells each) were plated in methylcellulose medium containing 50 ng/mL of SCF, 10 ng/mL of FP6, 10 ng/mL of GM-CSF, and 10 ng/mL of IL-3. The black and gray bars represent compact and dispersed colonies, respectively. The data are shown as the means ± SD for triplicate analyses. **P < .01. (F) Morphology of colonies generated in panel E. Representative colonies observed under an inverted microscope. Magnification ×200. (G) Typical cell morphology of MLL-AF9–transformed GMPs overexpressing Egr1 after 3 days of culture in methylcellulose medium in panel E. Cells were cytospun onto glass slides and observed after Wright-Giemsa staining. Magnified images of the cells boxed are depicted in the insets. Magnification ×400 and ×1000 (insets).

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