Correlation of NF-Y-USF1/2 multiple protein complex with HOXB4 promoter activity. (A) Proper spacing between HxRE-1 and HxRE-2 is required for full HOXB4 promoter activity. K562 cells were transfected with 10 μg each indicated reporter plasmid, and luciferase activities were measured 48 hours later (i). (ii) HxRE-INS was constructed by inserting a 14-bp sequence (underlined) within the interval between HxRE-1 and HxRE-2, without altering the flanking sequences (italicized). The data presented are means ± SDs of triplicate measurements, from 1 of 3 similar experiments. (B) This insertion mutation between HxRE-1 and HxRE-2 (HxRE-INS) disrupts the cooperative in vitro binding of USF1/2 and NF-Y to the HOXB4 promoter, as detected by EMSA. Wild-type HOXB4 promoter probe was used in lanes 1 through 4; HOXB4 promoter with a mutation at either the HxRE-1 or the HxRE-2 site was used in lanes 5 and 6; and HxRE-INS was used in lane 7. K562 NE was added in lanes 2 through 7. In lanes 3 and 4, the NEs were pretreated with USF1/USF2 antibodies and NF-Ya/NF-Yc antibodies, respectively.