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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 334-339
Direct interaction of NF-E2 with hypersensitive site 2 of
the  -globin locus control region in living cells
E. Camilla Forsberg,
Karen M. Downs, and
Emery H. Bresnick
From the Department of Pharmacology, the Department of Anatomy, and
the Molecular and Cellular Pharmacology Program, University of
Wisconsin Medical School, Madison, WI.
The human  -globin locus control region (LCR) confers high-level,
tissue-specific expression to the -globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2)
subregion of the LCR are important for the strong enhancer activity of
the LCR. Multiple proteins are capable of interacting with these sites
in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for -globin gene expression is evident in murine erythroleukemia cells lacking the
p45 subunit of NF-E2. These CB3 cells have a severe defect in  - and
-globin gene transcription, which can be restored by expression of
NF-E2. However, mice nullizygous for p45 express nearly normal levels
of -globin. Thus, either a redundant factor(s) exists in mice that
can functionally replace NF-E2, or NF-E2 does not function through the
LCR to regulate -globin gene expression. To address this issue, we
asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact
cells. Using a chromatin immunoprecipitation assay, we provide evidence
for NF-E2 binding directly and specifically to HS2 in living
erythroleukemia cells and in mouse fetal liver. The specific
immunoisolation of HS2 sequences was dependent on the presence of p45
and on intact MAREs within HS2. These results support a direct role for
NF-E2 in the regulation of -globin gene expression through
activation of the LCR.
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