Restoration of the CCAAT Box or Insertion of the CACCC Motif Activate 
-Globin Gene Expression
Delia C. Tang,
David Ebb,
Ross C. Hardison, and
Griffin P. Rodgers
From the Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD; Department of Pediatrics, Massachusetts General Hospital, Boston, MA, and Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA.
Hemoglobin A2 (HbA2 ), which contains 
-globin as its non-
-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 -globin gene in adult erythroid cells, we first compared promoter sequences and found that the -globin gene differs from the 
-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 -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 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 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 -globin gene expression, with potential future clinical benefit.
Blood, Vol. 90 No. 1 (July 1), 1997:
pp. 421-427
© 1997 by The American Society of Hematology.
