Mutations in the PCS affect EMSA bands and β-promoter transcription.
(A) Mutations in the PCS influence the DNA-protein complex formation. EMSA was carried out with the wild-type PCS and its mutant variants by using K562 nuclear extracts. The 20-μL reaction mixture contained 2 ng 32P-labeled double-stranded DNA (∼100 000 cpm) in the binding buffer and 5 μg nuclear extract. The sequences of the sense strand of each of the double-stranded 32P-labeled DNA probes is as follows: wild type, oligo 1; AT to CC substitution, oligo 2; Δ GACAGGTA, oligo 3; and Δ CGGCT, oligo 4 (Table 1). Sequences from the pGL3 plasmid-cloning sites were included in oligo 3 and 4 to maintain their length equivalent to the wild-type PCS oligonucleotide. The free probe lane is the mixture of all the above sequences in the binding reaction mixture without nuclear extracts. The EMSA bands are numbered and indicated by arrows. (B) Functional effects of mutations in the PCS. The wild-type β-promoter from −136 to +54 bp and its mutants were generated by PCR and cloned into the Hind III-XhoI site of the pGL3. The forward primers used in the PCR are as follows: wild-type β-promoter, oligo 5; β-promoter with AT to CC substitution in the PCS, oligo 6; β-promoter with Δ GACAGGTA in the PCS, oligo 7; and β-promoter with Δ CGGCT in the PCS, oligo 8. Oligo 9 served as the reverse primer in all the PCR reactions. A 736-bp HS2 (from nucleotides 8486 to 9222 on the human β-globin locus) was cloned at the BamHI-SalI site of the pGL3. All the values in the bar diagram are the averages of 3 to 8 separate experiments, most of which were done in duplicate. The luciferase activities are normalized as activity per 10 μg total protein. Horizontal lines above each bar indicate 1 SD in the average of the experimental values.
