FIG. 2.

Aiolos and Ikaros bind c-Myc promoter in pre-B cells in vivo. (A) Schematic diagram of the murine c-Myc promoter. Two major transcription starting sites, P1 and P2, are indicated. Two highly conserved Ikaros binding sites Ikaros1 and Ikaros2 are indicated with black bars. The shaded rectangle represents the first exon of c-Myc gene. (B) Sequence alignment of Ikaros1 and surrounding sequences between the human and mouse c-Myc promoters. (C and D) Aiolos and Ikaros bind to the c-Myc promoter in vivo. DKO pre-B cells were infected with control, Aiolos, or Ikaros. A ChIP assay was performed 24 h later to determine whether Aiolos and Ikaros bind to c-Myc promoter in vivo. The empty-vector-infected cells were analyzed as a control (Vector). The binding of control antibody (rabbit IgG), Aiolos antibody (Aiolos Ig), and Ikaros antibody (Ikaros Ig) to the Ikaros1 and Ikaros2 sites on c-Myc promoter was analyzed by real-time PCR. The binding of Aiolos and Ikaros to regions either 2 kb upstream or downstream of c-Myc promoter was also examined. The intensity of the signals is expressed as a percentage of the input. The values are averages and standard deviations from three independent experiments. (E) Ikaros binds to c-Myc promoter in sorted wild-type pre-B cells. Pre-B cells (B220⁺ CD43⁻ IgM⁻) were isolated via sorting from wild-type mice. A total of 10⁷ pre-B cells were used for ChIP analysis.