ASXL1-MT and HHEX cooperatively upregulated Myb and Etv5. (A) c-Kit⁺ BM cells derived from control mice or ASXL1-MT KI mice were transduced with control/HHEX (coexpressing NGFR). NGFR-positive cells were sorted 48 hr after transduction. Relative mRNA level of Myb and Etv5 were analyzed by qRT-PCR. Results were normalized to Gapdh, with the relative mRNA level in vector-transduced cells set at 1 (n = 4-5). (B) Relative expression of Myb and Etv5 were assessed in vector- or sgHhex-transduced cSAM cells using qualitative reverse transcription-polymerase chain reaction (n = 4). Results were normalized to Gapdh, with the relative mRNA level in sgNT-transduced cells set at 1. (C) Expression correlation between HHEX and MYB/ETV5 mRNA expression in primary AML patients (Beat AML). (D) ChIP-seq reads across MYB and ETV5 promoter loci with 293T cells transfected with control vector (upper) or HA-HHEX (lower) against anti-HA antibody. HHEX bound to −2 kb of promoter loci of MYB and ETV5 in 293T cells. (E-G) ChIP-qPCR assay MYB and ETV5 locus in HL-60 cells. HL-60 cells were retrovirally transduced with vector/ASXL1-MT (coexpressing GFP) and vector/HA-HHEX (coexpressing NGFR). After sorting GFP⁺/NGFR⁺ cells, genomic DNA fragments from these cells were immunoprecipitated with anti-HA (E) and anti-H2AK119ub (F) antibodies (n = 4 each). (G) Genomic DNA fragments from vector/ASXL1-MT–expressing HL-60 cells were immunoprecipitated with anti-IgG or anti-HHEX antibody (n = 4 each). (H) HHEX directly increased promoter activity of MYB and ETV5 genes. 293T cells were cotransfected with a pGL4.71 vector (coexpressing firefly luciferase [FLuc]) plus either pGL4.1-MYB promoter locus or pGL4.1-ETV5 promoter locus (coexpressing Renilla luciferase [RLuc]) together with either HHEX or control vector. Luciferase assays were performed with a triplicate set. Statistical analyses were performed by 1-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.