title: Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer.
abstract: High-level, tissue-specific expression of the beta-globin genes requires the presence of an upstream locus control region (LCR). The overall enhancer activity of the beta-globin complex LCR (beta-LCR) is dependent on the integrity of the tandem NF-E2 sites of HS-2. The NF-E2 protein which binds these sites is a heterodimeric basic leucine zipper protein composed of a tissue-specific subunit, p45 NF-E2, and a smaller subunit, p18 NF-E2, that is widely expressed. In these studies, we sought to investigate the role of NF-E2 in globin expression. We show that expression of a dominant-negative mutant p18 greatly reduces the amount of functional NF-E2 complex in the cell. Reduced levels of both alpha- and beta-globin were associated with the lower levels of NF-E2 activity in this cell line. Globin expression was fully restored upon the introduction of a tethered p45-p18 heterodimer. We also examined CB3 cells, a mouse erythroleukemia (MEL) cell line that does not express endogenous p45 NF-E2, and demonstrated that the restoration of globin gene expression was dependent upon the levels of expressed tethered NF-E2 heterodimer. Results of DNase I hypersensitivity mapping and in vivo footprinting assays showed no detectable chromatin alterations in beta-LCR HS-2 due to loss of NF-E2. Finally, we examined the specificity of NF-E2 for globin gene expression in MEL cells. These experiments indicate a critical role for the amino-terminal domain of p45 NF-E2 and show that a related protein, LCRF1, is unable to restore globin gene expression in p45 NF-E2-deficient cells. From these results, we conclude that NF-E2 is specifically required for high level goblin gene expression in MEL cells.
authors.affiliation: Division of Hematology, Children's Hospital, Boston, MA 02115, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Kotkow
authors.forename: S H
authors.initials: SH
authors.lastname: Orkin
mesh_terms: D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D015972:Gene Expression Regulation, Neoplastic; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D016350:Leucine Zippers; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics*; D050985:MafK Transcription Factor; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050985:MafK Transcription Factor; C494696:Mafk protein, mouse; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494693:Nfe2 protein, mouse; D011994:Recombinant Proteins; D014157:Transcription Factors; D005914:Globins