Levels of trimethylation of H3K27 in the absence of Ezh2. (A) Levels of trimethylation at H3K27 in Ezh2^Δ/Δ fetal livers. Histones were extracted from CD45⁺Lin⁻ immature hematopoietic cells purified from E12.5 Tie2-Cre (Ezh2^+/+) or Tie2-Cre;Ezh2^fl/fl (Ezh2^Δ/Δ) fetal liver cells (left 2 lanes) and from recipients' BM repopulated by E12.5 Tie2-Cre (Ezh2^+/+) or Tie2-Cre;Ezh2^fl/fl (Ezh2^Δ/Δ) fetal liver cells (right 2 lanes), and were analyzed by Western blotting using an anti–H3K27me3 antibody. Levels of trimethylated H3K27 were normalized to the amount of H3 and are indicated relative to Tie2-Cre control values. (B) A schematic representation of the Ink4a/Arf locus (left panel). ChIP assays were performed using an anti–H3K27me3 antibody. The regions amplified from the precipitated DNA by site-specific quantitative PCR are indicated by a bar (#1). Quantitative ChIP analysis of the Ink4a/Arf locus in CD45⁺Lin⁻ cells from E12.5 Tie2-Cre (Ezh2^+/+) and Tie2-Cre;Ezh2^fl/fl (Ezh2^Δ/Δ) fetal livers and CD45⁺Lin⁻c-Kit⁺ cells from recipients' BM repopulated with Cre-ERT (Ezh2^+/+) or Cre-ERT;Ezh2^fl/fl (Ezh2^Δ/Δ) BM cells at 32 weeks after tamoxifen treatment. Percentages of input DNA are shown as the mean ± SD (right panel). (C) Quantitative RT-PCR analysis of Ezh1 and Ezh2 expression in E14.5 fetal liver and BM from 8-week-old mice. mRNA levels in indicated hematopoietic fractions were normalized to Hprt1 expression. Expression levels relative to those in the BM KSL cells are shown as the mean ± SD for triplicate analyses. Lin⁺ indicates lineage marker-positive differentiated hematopoietic cells. (D) A Venn diagram depicting derepressed genes (> 4-fold) in Ezh2-deficient fetal liver MEPs and BM MEPs.