title: Specific repression of beta-globin promoter activity by nuclear ferritin.
abstract: Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult beta-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the beta-globin promoter at -153 to -148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress beta-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the beta-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant beta-globin promoter for the -150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the -150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human beta-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.
authors.affiliation: Departments of Biochemistry and Molecular Biology, and Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. robert-broyles@omrf.ouhsc.edu
authors.forename: R H
authors.initials: RH
authors.lastname: Broyles
authors.forename: V
authors.initials: V
authors.lastname: Belegu
authors.forename: C R
authors.initials: CR
authors.lastname: DeWitt
authors.forename: S N
authors.initials: SN
authors.lastname: Shah
authors.forename: C A
authors.initials: CA
authors.lastname: Stewart
authors.forename: Q N
authors.initials: QN
authors.lastname: Pye
authors.forename: R A
authors.initials: RA
authors.lastname: Floyd
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005293:Ferritins / Q000378:metabolism / Q000502:physiology*; D005914:Globins / Q000235:genetics*; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins; D004247:DNA; D005293:Ferritins