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Spanish delta beta zero-thalassemia, a mild thalassemic condition characterized by increased level of hemoglobin (Hb) F production during adult life, is known to be due to a large deletion starting within the beta globin gene cluster and extending beyond the 3' breakpoint of any other similar deletional defects so far identified. By molecular cloning and by genomic mapping we now demonstrate that the deletion of Spanish delta beta zero-thalassemia ends at approximately 11 and 17 kilobases (kb) downstream to the 3' endpoints of black hereditary persistence of fetal hemoglobin (HPFH) type 1 and 2, respectively. As suggested by the complete characterization of this and other deletional defects involving the beta globin gene cluster, the 5' and 3' breakpoints of several deletions cluster in rather restricted DNA areas, further strengthening the idea that common molecular mechanisms may operate in causing these deletions.

We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human beta-globin promoter. Upon incubation with beta-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 hemoglobin 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 gamma-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 beta-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 beta-globin transcription.