TGFBR3 is a target gene of Gfi-1B in MEP cells. (A-B) TGF-β receptor expression at the mRNA (A) and protein (B) levels in shControl- or shGfi-1B–transduced K562 cells. Data are expressed as the ratio between TβRIII and GAPDH mRNA; error bars represent SD of 2 experiments (A). Proteins were analyzed by Western blot with antibodies indicated on the left in panel B. (C) Schematic representation of the TGFBR3 promoter. Distal and proximal regions were shown. (D) TGFBR3 promoter activity in the absence or presence of Gfi-1B expression plasmid. Transient transfections were performed into HEK293 cells. The proximal region of the TGFBR3 promoter cloned in front of luciferase gene reporter was transfected without or with 2 different doses (125 or 250 ng) of Gfi-1B expression vectors as indicated. Luciferase activity was measured 48 hours after transfection. Results are mean ± SD of 4 experiments (x on the TGFBR3 promoter indicates AATC/GATT sequence). ***P < .001, *P = .05 determined by Student t test. (E) Gfi-1B binding to the TGFBR3 promoter. Oligo-pull down experiments were performed using K562 cell extracts and increasing concentrations of oligonucleotides corresponding to a sequence surrounding the −134 Gfi-1B binding site of the wild-type TGFBR3 proximal promoter. GATT was mutated in GACC as indicated by a cross. Results are representative of 3 independent experiments. The membrane was hybridized with HMGB2 antibody to quantify the loading and to show the specificity of the Gfi-1B binding to this region of the TGFBR3 promoter. (F) Gfi-1B binding to the TGFBR3 promoters in vivo. ChIP analyses were performed with chromatin from undifferentiated shControl- or shGfi-1B–transduced K562 cells using antibodies against Gfi-1B. Quantitative PCR was performed with primers amplifying the TGFBR3 promoter. Results are fold increase (mean ± SD) of 3 independent ChIP experiments. *P < .02 determined by Student t test.