Figure 3.
Cell type specific binding of Ikaros with PU.1 at Sfpi1 cis-regulatory elements. (a) Ikaros binds the URE region in RAW264.7 and NFS-25 cells, but binds other regions with cell-type-specificity. Ikaros ChIP assays were performed using antibodies Santa Cruz sc-13039 and Active Motif #39291. Data are presented as in Fig. 2. (b,c) The CE5 region nucleates cell-type-specific myeloid complexes in vitro that contain PU.1 and Ikaros proteins. (b) Probe CE5-P2 (see Fig. 1d) and CE5-P2m2 (Ikaros site mutated; see Fig. 4a for mutated sequence) were radiolabeled and incubated with nuclear extracts from RAW264.7 cells (M) or NFS-25 cells (B). Preparation of nuclear extracts and binding conditions were described in (14). Complexes were resolved by 6% SDS PAGE, dried, and then exposed to film. Myeloid specific complexes are labeled M1, M2, and M3. Anti-PU.1 and anti-Ikaros antibodies used in gel shift experiments are sc-352 and Active Motif #39291, respectively. The overlapping PU.1/Ikaros binding site was mutated in probe CE5-P2m1. The exact sequences mutated for m1 and m2 are shown in red in the schematic in Fig. 4a. Competitor DNA probes were used at 250 fold molar excess; 4 μg of antibodies were used as labeled. (c) PU.1 can direct formation of a PU.1 and Ikaros containing complex on probe CE5-P2. TnT Couple Quick Transcription/Translation system (Promega # L1171 and L2081) was used to generate PU.1, Ikaros, or Plastic proteins. RAW264.7 nuclear extract was included (lane 1) for comparison. All other lanes contained equal volumes of reticulocyte lysate (treated or untreated). For treated lysates, 1 μg of plasmid DNA was incubated with lysates to generate proteins following manufacturer’s protocol. Ten μg of total lysate were run per lane. (d) Western blot of Ikaros and Sp1 protein. Nuclear extracts (2-8 μg) from Adh.2C2 and Raw264.7 cells were separated on an 8% SDS-PAGE gel. This blot had been previously probed for Sp1 and was shown in (14). The blot was stripped and reprobed here with anti-Ikaros antibody (sc-13039). (e) Adh.2C2 cells transduced with PU.1 retroviral vector express high levels of PU.1 mRNA. Retroviral vector supernatants were prepared by transfection of pMX-PU.1-IRES-hCD8 plasmid into Phoenix packaging cells with FUGENE 6 reagent. Virus particle-containing medium was collected at 48-72 hours post transfection. Adh.2C2 cells were subsequently transduced to express PU.1 using the TAKARA RetroNectin method. RNA was prepared using Trizol and manufacturer’s protocol. cDNA were prepared from RNA using the Superscript III system (Invitrogen #18080-400), then analyzed for PU.1 sequence by quantitative real-time PCR. PU.1 levels were normalized against GAPDH. Data are plotted on a log10 scale. (f) Forced expression of PU.1 in immature T-cells permits site-restricted PU.1 binding. PU.1 ChIP experiments are shown as described previously. Data shown are from five independent transduction and ChIP experiments. (g and h) Ectopic PU.1 expression facilitates restricted recruitment of Ikaros (g) and Pol II (h) to the PU.1 target regions CE9 and IL7r (black arrows). Data shown are from two independent experiments. *p<0.0005. **p<0.002.