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Selective overexpression (50- to 100-fold) in adult erythroid cells of either Gγor Aγfetal globin gene is observed in hereditary conditions known as δβ°-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C → T change at position −196 of an overexpressed Aγglobin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed Aγ-globin gene from a Sardinian patient with a different syndrome (δβ°-thalassemia). The Sardinian Aγglobin gene differs from both the HPFH and the normal Aγglobin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant −196 Aγ-globin gene is linked to a normal βglobin gene in HPFH, and to a β-thalassemic gene (β39CAG → TAG) in δβ°-thalassemia. These data strengthen the suggestion that −196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the −196 mutation in different syndromes.

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.

The -175 T▸C mutation in the promoter of the Aγ- Gor γ-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a γ-CAT reporter plasmid transfected into the erythroid cells K562, but not that of the same plasmid in non erythroid cells. The overexpression of the mutant is abolished by the mutation of the binding site for the erythroid specific factor NFE1; inactivation of the adjacent binding site for the ubiquitous factor OTF1 does not cause overexpression of the normal γ-globin promoter. Previous results demonstrated that the -175 mutation slightly increases the in vitro binding of NFE1 and almost abolishes that of OTF1; the present functional data indicate that altered binding of NFE1, but not of OTF1, is responsible for the observed overexpression of the mutated promoter.