title: Restoration of the CCAAT box or insertion of the CACCC motif activates [corrected] delta-globin gene expression.
abstract: Hemoglobin A2 (HbA2), which contains delta-globin as its non-alpha-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the delta-globin gene in adult erythroid cells, we first compared promoter sequences and found that the delta-globin gene differs from the beta-globin gene in the absence of an erythroid Krüppel-like factor (EKLF) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human delta-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5' flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the delta promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type delta promoter. A set of substitutions that create an EKLF binding site centered at -85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase delta-globin gene expression, with potential future clinical benefit.
authors.affiliation: Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1822, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Tang
authors.forename: D
authors.initials: D
authors.lastname: Ebb
authors.forename: R C
authors.initials: RC
authors.lastname: Hardison
authors.forename: G P
authors.initials: GP
authors.lastname: Rodgers
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006443:Hemoglobin A2 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D004251:DNA Transposable Elements; D005914:Globins; D006443:Hemoglobin A2