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We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human β-globin promoter. Upon incubation with β-globin promoter fragments containing the duplicated CACCC boxes, nuclear proteins from human erythroid cells generate complexes yielding four retarded bands in acrylamide gels; the three slowest bands are common to both erythroid and non erythroid cells. The fast band is present only in K562 erythroleukemic cells induced to differentiation and hemogloblin accumulation and in fetal and adult erythroblasts, but absent in uninduced K562 cells. Binding occurs on a short DNA region including the proximal CACCC box, and is not significantly competed by excess γ-globin fragments containing the CACCC box; the CACCC box appears to be essential for this binding, as shown by the failure of a fragment containing a natural β-thalassemic mutation (−87, C >G) to bind significantly to nuclear factors. These data suggest that the erythroid specific CACCC binding factor might play a role in the developmental activation of β-globin transcription.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human γ-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erytroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the γ-globin octamer containing fragment. The possible role of these nuclear proteins in γ-globin gene regulation and/or cell differentiation is discussed.