Gfi1 is a target of Mysm1. (A) Schematic diagram of Gfi1 locus encompassing the promoter and enhancer region; arrows indicate positions of the primers used for ChIP assays. (B) ChIP assays of WT Lin⁻ cells using anti-Mysm1 and anti-IgG. The precipitated DNA was analyzed by real-time PCR using primers along the Gfi1 promoter and enhancer sequence. The relative amount of immunoprecipitated DNA is presented as a percentage of input DNA. (C) In vitro Co-IP experiment in 293T cells. A flag-tagged Mysm1-encoding plasmid was cotransfected with Myc tagged Gata2, Runx1, PU.1, or Scl in 293T cells. Cell lysates were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were examined by western blotting using anti-Myc and anti-Flag antibody. 10% of cell lysates was used as input. (D) ChIP assays in WT and Mysm1^−/− Lin⁻ cells using antibodies against Gata2, Runx1, PU.1, Scl, and IgG antibodies. The precipitated DNA was analyzed by real-time PCR using primers along the −35 kb Gfi1 enhancer region. Primers along the +23 kb Runx1 regulatory sequence were used as a positive control. The relative amount of immunoprecipitated DNA is presented as a percentage of input DNA. (E) The −3.4 kb Gfi1 promoter and its minimal core region (−3.4 kb min pro) were cloned into pGL3 basic luciferase vector. Promoter activity was examined in the presence of pcDNA or pcDNA-Mysm1 48 hours after transfection in 293T cells. (F) pGL3-enhancer control (pGL3c) luciferase vector or pGL3c-(−35 kb) luciferase vector which contains the −35 kb Gfi1 enhancer was cotransfected with siRNA control (siControl), siMysm1, or vectors encoding Gata2 and Runx1 into 293T cells. Luciferase activity was recorded 48 hours after transfection. (B,D-F) Data are representative of three independent experiments. (D) Data are representative of two independent experiments. *P < .05.