Regulation of Id2 by GM-CSF-responsive STAT5 and C/EBPβ. (A) Hematopoietic-Stat5^Δ/Δ mice and Stat5^+/+ control animals were treated by GM-CSF HGT or HGT pORF vector, as indicated. CDPs were isolated from BM after 2 days and analyzed for Id2 expression by quantitative PCR, using primers listed in supplemental Table 1. Id2 mRNA was normalized to Rpl13a mRNA amounts. Results represent average values from 2 independent experiments. Error bars represent SEM of average values. P values of indicated comparisons are shown. (B) Purified CDPs from hematopoietic-Stat5^Δ/Δ mice and Stat5^+/+ controls were cultured ex vivo with GM-CSF for 24 hours, 72 hours, or left untreated, as indicated. Id2 expression was analyzed by quantitative PCR as described in panel A. Results are presented as the fold induction of GM-CSF–treated samples relative to untreated samples. Results represent average values from 3 independent experiments. (C) D2SC/1 cells were transfected with the PGL3/Id2 reporter construct or empty vector, together with phRL-TK-Renilla and plasmids expressing WT STAT5A or a STAT5A mutant containing deletion of the transactivation domain (TAD). Cells were treated with GM-CSF for 2 hours. Results are expressed as normalized values (normalized values = relative light units [RLU] from GM-CSF–treated cells/RLU from untreated cells). Data represent the mean ± SEM of 3 independent experiments. (D) EMSAs were performed with nuclear extracts from D2SC/1 cells treated with or without GM-CSF for 30 minutes, using a ³²P-labeled oligonucleotide probe corresponding to the Id2 distal STATx site (region −574 to −566; Figure 3A) with or without competitor oligonucleotides (I indicates Id2 probe WT; S5, an oligonucleotide containing the STAT5 consensus binding site; m, mutant Id2 probe) or anti-STAT5 antibody (☆ represents STAT5/DNA complexes; and ★, antibody supershifted STAT5/DNA complexes), as indicated (left). Control EMSAs were performed with a consensus STAT5 probe as indicated (right). (E) ChIP assays were performed with D2SC/1 cells treated with or without GM-CSF for 1 hour, using anti-STAT5 or IgG control antibody. Results were normalized to total cell lysate (input). (D-E) Results represent data from 3 independent experiments. (F) Hematopoietic-Stat3^Δ/Δ mice and Stat3^+/+ control animals were treated by Flt3L HGT or HGT vector, as indicated. Id2 mRNA amounts were measured in CDPs after 2 days, as described in panel A. (G-H) Lin⁻ Flt3⁺ cells were cultured with GM-CSF or Flt3L for 24 hours, 72 hours, or left untreated. (G) Cebpb mRNA was analyzed by quantitative PCR. (H) C/EBPβ amounts were determined by immunoblotting. (I-J) Reporter assays were performed in D2SC/1 (I) or D2SC/mFlt3 cells (J) with the pGL3/Id2 reporter construct, in the presence of plasmids expressing STAT5A (WT), STAT5 TAD mutant (TAD), C/EBPβ (WT), or C/EBPβ T188A (T188A) as indicated, after GM-CSF (I) or Flt3L (J) stimulation for 2 hours. The fold change in RLU in GM-CSF– or Flt3L–treated cells relative to untreated cells is presented as mean ± SEM of 3 independent experiments.