Figure 4.

The TAF_II130 binding domain of NF-E2 is essential for enhancer-dependent activation of the β-globin gene. (A) Wild-type NF-E2 and NF-E2Δ80 expression in selected CB3 clones as demonstrated in a gel mobility shift assay. CB3 cells were stably transfected with pCINeoHA vector alone or pCINeoHA vectors in which the human NF-E2 cDNA or NF-E2Δ80 was fused in-frame with the HA tag. Nuclear extract was prepared from individual clones and analyzed by gel mobility shift assay, dimethyl sulfoxide-induced MEL extract serving as a control. ³²P-labeled DNA fragments containing the tandem NF-E2 binding sites in HS2 were incubated with individual extracts and electrophoresed on a 4% polyacrylamide gel. Lanes 1 and 2 show MEL cell and CB3 control extracts, the latter lacking the characteristic NF-E2 complex. Lane 3 shows a representative CB3 clone transfected with pCINeoHANF-E2 demonstrating a complex that comigrates with the NF-E2 complex observed in MEL cells. Lane 4 verified the presence of HA-tagged NF-E2 with specific ablation of the NF-E2 complex by the addition of anti-HA antibody (anti-HA). Lanes 5 and 6 show similar results for a clone expressing pCINeoHANF-E2Δ80. (B) High-level expression of a transiently transfected β-globin promoter containing construct is dependent on wild-type NF-E2 expression in CB3 cells. CB3 clones expressing wild-type NF-E2 or NF-E2Δ80 or CB3 cells transfected with the pCINeo vector (control) were transiently transfected with a plasmid containing HS2 linked to a β-promoter/luciferase gene hybrid. Whole cell extracts were assayed for luciferase activity. The results shown are representative of the three transfection experiments. MEL cells were transfected as a positive control. The fold activation observed was calculated relative to the expression of the HS2βLuciferase construct in CB3 cells.