Identification of HHEX target genes by RNA-seq and ChIP-seq analyses. (A) Venn diagrams comparing differentially expressed genes by HHEX expression with or without ASXL1-MT in RNA-seq analysis using RUNX1-ETO–expressing CB cells (P < .05; Student t-test). Overlapped genes in Venn diagrams are defined as upregulated genes (left; 1424 genes) and downregulated genes (right; 1433 genes) by HHEX, respectively. (B) GO for upregulated and downregulated genes by HHEX expression in RUNX1-ETO–expressing cells. The x-axis shows the P value (−log₁₀). Expression of HHEX upregulated genes related to the proliferation of leukocytes and cell cycle (top), and downregulated genes related to apoptosis and differentiation (bottom). (C) GSEA revealed that genes related to leukemia stem cells were more highly expressed in cells expressing ASXL1-MT and HHEX than control cells (upper). Genes enriched in monocyte/macrophage compared with LSK cells were more highly expressed in control cells than in cells expressing ASXL1-MT and HHEX (lower). (D) The pie chart showing distribution of binding sites identified by ChIP-seq analysis. (E) HHEX binding motif of identified by ChIP-seq analysis. (F) Venn diagrams showing overlapping between genes to which HHEX bound at promoter loci and genes upregulated (left) or downregulated (right) by HHEX. A total of 301 genes were identified as candidate genes directly upregulated by HHEX, whereas 400 genes were identified as ones directly downregulated by HHEX.