Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.
An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.
DeWitt Wallace Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York.
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Barnhart
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Kim
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Sheffery
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D050981:Erythroid-Specific DNA-Binding Factors; D005803:Genes, Immunoglobulin; D005914:Globins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D014157:Transcription Factors; D005914:Globins; D004247:DNA; D003850:Deoxyribonuclease I