Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells.
The human beta-globin locus control region (LCR) confers high-level, tissue-specific expression to the beta-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 beta-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in alpha- and beta-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of beta-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 beta-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 beta-globin gene expression through activation of the LCR.
Forsberg
E C
EC
Department of Pharmacology, the Department of Anatomy, and the Molecular and Cellular Pharmacology Program, University of Wisconsin Medical School, Madison, WI 53706, USA.
Downs
K M
KM
Bresnick
E H
EH
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002843:Chromatin / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D005333:Fetus; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute; D008099:Liver / Q000378:metabolism; D019901:Locus Control Region; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; C494693:Nfe2 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors; D005914:Globins