Fig. 4.

DPF2 recruitment to histones inhibits myeloid differentiation. (A) Expression of DPF2 in a panel of acute leukemia cell lines compared with normal CD34⁺ CB cell control. DPF2 was detected with an anti-DPF2 antibody at a concentration of 1:1,000 (sc-101943, Santa Cruz). (B) Overexpression of DPF2 wild-type (DPF2^WT) or DPF2 triple mutant (DPF2^MUT; F275A, R300A, and D346A) protein in MOLM-13 cells as assessed by Western blot analysis. (C) Occupancy of the miR-223 promoter regions in MOLM-13 cells, overexpressing DPF2^WT or DPF2^MUT protein. Blue and yellow bars represent regions 4 and 5 of the miR-223 promoter region, respectively. Data represent the mean ± SD from three independent experiments. (D) Occupancy of a non-DPF2 responsive promoter (albumin) region in MOLM-13 cells, overexpressing DPF2^WT or DPF2^MUT protein. Data represent the mean ± SD from three independent experiments. (E) Schematic representation of the miR-223 promoter region. Two black lines represent the RUNX1 binding site in the miR-223 promoter region. (F) Overexpression of DPF2^WT or DPF2^MUT protein in human CB CD34⁺ cells as assessed by Western blot analysis. (G) DPF2^MUT blocks the repressive effect of DPF2 on myeloid differentiation of HSPCs. CD34⁺ cells expressing DPF2^WT, DPF2^MUT, or a vector control were cultured in myeloid differentiation-promoting cytokines for 7 d. Myeloid differentiation was determined by FACS analysis for expression of CD11b. A representative experiment is shown. (H) Data represent the mean ± SD of percent CD11b⁺ cells from three independent experiments. *P < 0.01 by Student’s t test.