Functional analysis of GATA-binding sites within

fur P1-Kpn1 promoter region. Dami cells were incubated overnight with 100 nM PMA and transiently transfected with the fur P1-KpnI mutant promoter constructs illustrated on the left side of the figure. (A) Measure of basal promoter activity (cotransfection with pMGS control vector); data are expressed as percentage of nonmutated P1-KpnI construct. (B) GATA-1–induced activity (cotransfection with pMGS or pMGS–GATA-1 vectors). Data in panels A and B are expressed as percentage of the nonmutated P1-KpnI construct. Results are expressed as the mean ± SEM; n = 4. **P < .001, compared with nonmutated P1-KpnI promoter construct. (C) Binding of GATA-1 to the −66 P1 GATA site. Nuclear extracts (1.5 μg) from PMA-differentiated Dami cells were incubated (1 hour at 4°C) in binding buffer containing 0.8 μg poly (dI-dC) before further incubation (10 minutes at room temperature) with a ³²P-labeled oligonucleotide spanning the −66 GATA motif (bp −74 to −39) of the fur P1 promoter. The specificity of complex formation was tested by the inclusion of unlabeled competitors in the binding buffer (cold probe, lanes 6, 7; cold P1 probe with mutated −66 GATA sites, lanes 8, 9; or unlabeled consensus GATA probe, lanes 10, 11), or by including antibodies to GATA-1 (antibodies N6, sc-265 and C-20, sc-1233; Santa Cruz Biotechnology), lanes 2 and 4, respectively) or isotype-matched controls (rat IgG or goat IgG, lanes 3 and 5, respectively). SS indicates supershift complex; ns, nonspecific band; I, cluster of slowly migrating bands; II and III, faster migrating complexes.