title: Role of AP1/NFE2 binding sites in endogenous alpha-globin gene transcription.
abstract: High-level alpha-globin expression depends on cis-acting regulatory sequences located far upstream of the alpha-globin cluster. Sequences that contain the alpha-globin positive regulatory element (PRE) activate alpha-globin expression in transgenic mice. The alpha-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 alpha-globin gene transcription, we mutated the AP1/NFE2 sites in the alpha-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 death and alpha-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, alpha-globin expression, measured by allele-specific RNA polymerase chain reaction (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 alpha-globin PRE contribute to long-range alpha-globin gene activation. The proteins that mediate this effect remain to be determined.
authors.affiliation: Department of Biochemistry, Rm 4064, Thomas Tower, St Jude Children's Research Hospital, 332 N Lauderdale St, Memphis, TN 38105-2794, USA.
authors.forename: Melanie R
authors.initials: MR
authors.lastname: Loyd
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Mindy S
authors.initials: MS
authors.lastname: Randall
authors.forename: Paul A
authors.initials: PA
authors.lastname: Ney
mesh_terms: D000818:Animals; D019843:Archaeal Proteins; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D001853:Bone Marrow / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004622:Embryo, Mammalian; D004909:Erythrocyte Indices; D004912:Erythrocytes / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050984:NF-E2 Transcription Factor, p45 Subunit; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050984:NF-E2 Transcription Factor, p45 Subunit; C494693:Nfe2 protein, mouse; D012269:Ribosomal Proteins; D014157:Transcription Factors; C097242:ribosomal protein HS26, Haloarcula marismortui; D005914:Globins