Derepression of genes through alterations in H3K27me3 levels by Ezh2 deletion in Jak2V617F-positive hematopoietic cells. (A) Scatter plots showing H3K27me3 enrichment in Jak2^VF/+ Ezh2^−/− LSK cells compared with control or Jak2^VF/+ LSK. Genes with lower H3K27me3 enrichment in Jak2^VF/+ Ezh2^−/− LSK cells are highlighted with blue color. (B) Venn diagram showing genes with significantly more H3K27me3 enrichment in control and Jak2^VF/+ LSK compared with Jak2^VF/+ Ezh2^−/− LSK cells, as well as genes with significantly more H3K27me3 enrichment in control compared with Jak2^VF/+ LSK cells. Genes with significant H3K27me3 enrichment in the region from 5 kb upstream to 0.5 kb downstream of the transcriptional start site are detected (P < 1.0 × 10⁻⁵). (C) H3K27me3 ChIP followed by RT-qPCR showed binding of H3K27me3 in the promoters of S100a8, S100a9, Ifi27l2a, and Hmga2 genes in control and Jak2^VF/+ LSK cells. Deletion of Ezh2 significantly reduced the H3K27me3 enrichment in the promoters of these genes in Jak2^VF/+ Ezh2^−/− LSK cells. Results from 3 independent experiments are presented as mean ± SEM in bar graphs (*P < .05). The real-time PCR products were loaded onto 2% agarose gel. Representative pictures from agarose gel are shown in the bottom panels. (D) H3K27me3 ChIP followed by RT-qPCR showed binding of H3K27me3 in the promoters of S100A8, S100A9, IFI27, and HMGA2 genes in control and EZH2 knocked down SET-2 cells. Note that knockdown of EZH2 significantly reduced the H3K27me3 enrichment in the promoters of S100A8, S100A9, IFI27, and HMGA2 in JAK2V617F-positive SET-2 cells. The real-time PCR products were loaded onto a 2% agarose gel, and the representative pictures are shown. (E) Ectopic expression of S100a8, S100a9, Ifi27l2a, or Hmga2 significantly increased CFU-Mk colonies in the BM of Jak2^VF/+ mice (n = 4-5 for each construct; *P < .05).