In vitro binding of Fli-1 to the SHIP-1 promoter. (A) The 988-bp promoter region before the start codon of murine SHIP-1 was cloned into the pGL3-enhancer vector. Three possible Ets binding sites (EBS) are highlighted. S1 and S2 indicate the locations of the primers used to amplify the 294-bp region of the SHIP-1 promoter. (B) The SHIP-1 promoter regions of human, mouse, and rat were compared for conservation of nucleotides using BLAST (National Center for Biotechnology Information). The underlined regions (EBS1 and EBS2) are fully conserved in all 3 species. (C) Binding of Fli-1 to the promoters of SHIP-1 (lane 4) and MDM2 (lane 1) as determined by ChIP in CB3, HB60-5, and KH16 erythroleukemic cells using either 2 μg Fli-1 or control rabbit IgG antibody. The MDM2 promoter was used as a positive control, because it is a known Fli-1 target gene.27 (D) ChIP quantitative-PCR in CB3 cells using Fli-1 and normal rabbit IgG antibodies illustrating Fli-1 chromatin occupancy of the indicated gene promoters, as well as a region 3 kb upstream the SHIP-1 promoter (UP-SHIP-1). The β-actin and upstream SHIP-1 promoters were used as negative controls. Results are based on the relative proportions of the input and chromatin precipitated by the Fli-1 and control IgG antibodies, where the input is equal to 1.
