Id2 is a direct transcriptional target of Gfi-1. (A) Sequence alignment of mouse, rat, and human Id2 promoters. Potential Gfi-1 binding sites are boxed. Numbers indicates nucleotide position in the mouse Id2 promoter. (B) Id2 promoter reporter plasmid (Id2pro-1036 or Id2pro-1905 or Id2pro-3507) and Gfi-1 expression plasmid (pcDNA-Gfi-1 or pcDNA control) were cotransfected into 293T cells. Luciferase activities were examined 24 hours after transfection. To compare Gfi-1 repression on 3 Id2 promoter constructs, Id2 promoter activities in pcDNA-Gfi-1–transfected cells were expressed as the percentage of those in pcDNA-transfected cells. Three independent experiments were performed. (C) LightShift EMSA were performed by the use of nuclear extracts from pcDNA (lanes 1, 5, 9) or pcDNA-Gfi-1 (lanes 2, 3, 4, 6, 7, 8, 10, 11, 12)–transfected 293T cells, and biotin-labeled oligonucleotides from Id2 promoter containing the potential Gfi-1 binding sites, in the presence or absence of a 30-fold excess of nonlabeled competitors or anti–Gfi-1 antibody. Three independent experiments were performed. (D) ChIP assays were performed in EML cells. Immunoprecipitation was performed with IgG or anti–Gfi-1. PCR quantification of immunoprecipitated DNA was performed with the SYBR Green PCR Master Mix and primers designed to amplify regions covering each Gfi-1 binding site in the Id2 promoter. Three independent experiments were performed.