Gamma-Globin Gene Promoter Elements Required for Interaction With
Globin Enhancers
Scott D. Langdon and
Russel E. Kaufman
From the Departments of Medicine and Biochemistry, Duke University
Medical Center, Durham, NC.
Normal expression of the human 
-globin domain genes is dependent
on at least three types of regulatory elements located within the
-globin domain: the locus control region (LCR), globin enhancer elements (3
and 3A
), and the individual globin gene
promoter and upstream regions. It has been postulated that regulation
occurs through physical interactions between factors bound to these
elements, which are located at considerable distances from each other.
To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated -globin promoters linked to the 5HS2 enhancer. We show that in K562 cells, 5HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 
+25) promoter and that truncation or site-directed
mutagenesis of the tandem CCAAT boxes eliminated the enhancement by
5HS2. Mutation of the -globin gene promoter GATA-1 binding
sites did not decrease either promoter strength or enhancement of
activity by 5HS2. To determine if enhanced expression of
-globin gene promoters carrying mutations associated with hereditary
persistence of fetal hemoglobin (HPFH) was due to
greater interactions with enhancers, we linked these HPFH -globin
gene promoters to 5HS2 and demonstrated a twofold to threefold
higher expression than the corresponding wild-type promoter plus
enhancer in MEL cells. Addition of the A-globin gene 3
enhancer to a plasmid containing the -globin gene promoter and
5HS2 did not further enhance promoter strength. Furthermore, we
have demonstrated that the previously identified core 5HS2
enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when
located 5, but not 3, to the -globin-luciferase
reporter gene, suggesting that its enhancer effect is not by DNA
looping. Our results suggest that CCAAT boxes, but not GATA or CACCC
binding sites, are required for interaction between the -globin
promoter and the LCR/5HS2 and that regulatory elements in
addition to the core enhancer may be required for the enhancer to act
from a distance.
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 309-318
© 1998 by The American Society of Hematology.
