Specific repression of beta-globin promoter activity by nuclear ferritin.
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.
Broyles
R H
RH
Departments of Biochemistry and Molecular Biology, and Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. robert-broyles@omrf.ouhsc.edu
Belegu
V
V
DeWitt
C R
CR
Shah
S N
SN
Stewart
C A
CA
Pye
Q N
QN
Floyd
R A
RA
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
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