Structure-function analysis reveals the importance of DNA binding and an N-terminal–repressive domain in promyelocytic transformation. (A) Design of Hhex mutants tested in differentiation assay and summary results. (B) LSK cells were infected with retroviruses expressing the indicated Hhex mutants (as in panel A) and cultured in myeloid growth conditions (IMDM with 10% fetal bovine serum [FBS], 3 U/mL EPO, 10 ng/mL IL-3, and 25 ng/mL SCF). Cytocentrifuge preparations were taken at weekly intervals and the percentage of blast/promyelocyte (Blast/pro), neutrophil (G), monocyte (M), basophil (Ba), and erythroid (E) cells was determined following Wright-Giemsa staining and microscopic examination. Data are representative of 3 separate experiments. (C) Pml is not required for myeloid differentiation block by Hhex. Wild-type and Pml-knockout LSK cells were retrovirally transduced with MIG or Hhex and cultured in myeloid growth conditions as in panel B. Cytocentrifuge preparations were taken at weekly intervals and the percentage of cell types determined by microscopic analysis as in panel B. CT, C-terminus; Gro, groucho; HD, homeodomain; NT, N-terminus; TLE, transducin-like enhancer.