Submitted February 21, 2003
Accepted August 6, 2003
Role of AP1/NFE2 binding sites in endogenous 
-globin gene transcription
Melanie R Loyd, Yasuhiro Okamoto, Mindy S Randall, and Paul A Ney*
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN, USA
* Corresponding author; email: paul.ney{at}stjude.org.
High-level 
-globin expression depends on cis-acting regulatory sequences located far upstream of the -globin cluster. Sequences that contain the -globin positive regulatory element (PRE) have been shown to activate -globin expression in transgenic mice. The -globin PRE contains a pair of composite binding sites for the transcription factors Activating Protein 1 and Nuclear Factor Erythroid 2 (AP1/NFE2). To determine the role of these binding sites in -globin gene transcription, we mutated the AP1/NFE2 sites in the -globin PRE in mice. We replaced the AP1/NFE2 sites with a neomycin resistance gene (neo) that is flanked by LoxP sites (floxed). Mice with this mutation exhibited increased embryonic mortality and -thalassemia intermedia. Next, we removed the neo gene by Cre-mediated recombination, leaving a single LoxP site in place of the AP1/NFE2 sites. These mice were phenotypically normal. However, -globin expression, measured by allele-specific RNA PCR, was decreased 25%. We examined the role of the hematopoietic-restricted transcription factor p45Nfe2 in activating expression through these sites and found that it is not required. Thus, we have demonstrated that AP1/NFE2 binding sites in the murine -globin PRE contribute to long-range -globin gene activation. The proteins that mediate this effect remain to be determined.
