Specific repression of beta-globin promoter activity by nuclear ferritin

Citation
Rh. Broyles et al., Specific repression of beta-globin promoter activity by nuclear ferritin, P NAS US, 98(16), 2001, pp. 9145-9150
Citations number
42
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
0027-8424 → ACNP
Volume
98
Issue
16
Year of publication
2001
Pages
9145 - 9150
Database
ISI
SICI code
0027-8424(20010731)98:16<9145:SROBPA>2.0.ZU;2-K
Abstract
Developmental hemoglobin switching involves sequential globin gene activati ons and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repress or of the adult beta -globin gene in embryonic erythroid cells. Our data sh ow that a ferritin-family protein in K562 cell nuclear extracts binds speci fically to a highly conserved CAGTGC motif in the beta -globin promoter at -153 to -148 by from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin t hat is enriched in ferritin-H chains also binds the CAGTGC promoter segment . Expression clones of ferritin-H markedly repress beta -globin promoter-dr iven reporter gene expression in cotransfected CV-1 cells in which the beta -promoter has been stimulated with the transcription activator erythroid K ruppel-like factor (EKLF). We have constructed chloramphenicol acetyltransf erase reporter plasmids containing either a wildtype or mutant beta -globin promoter for the -150 CAGTGC motif and have compared the constructs for su sceptibility 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 CAGT GC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promote r in our cell transfection assay, providing a strong link between DNA bindi ng 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.