ABSTRACT
Krüppel-like factors (KLFs) have been systematically screened as potential candidates to regulate human gamma-globin gene expression through its CACCC element. Initially, 21 human proteins that have close sequence similarity to EKLF/KLF1, a known regulator of the human beta-globin gene, were identified. The phylogenetic relationship of these 22 KLF/Sp1 proteins was determined. KLF2/LKLF, KLF3/BKLF, KLF4/GKLF, KLF5/IKLF, KLF8/BKLF3, KLF11/FKLF, KLF12/AP-2rep and KLF13/FKLF2 were chosen for functional screening. Semi-quantitative RT-PCR demonstrated that all eight of these candidates are present in human erythroid cell lines, and that the expression of the KLF2, 4, 5 and 12 mRNAs changed significantly upon erythroid differentiation. Each of the eight KLF mRNAs is expressed in mouse erythroid tissues, throughout development. UV cross-linking assays suggest that multiple erythroid proteins from human cell lines and chicken primary cells interact with the gamma-globin CACCC element. In co-transfection assays in K562 cells, it was demonstrated that KLF2, 5 and 13 positively regulate, and KLF8 negatively regulates, the gamma-globin gene through the CACCC promoter element. The data collectively suggest that multiple KLFs may participate in the regulation of gamma-globin gene expression and that KLF2, 5, 8 and 13 are prime candidates for further study.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Globins/genetics , Kruppel-Like Transcription Factors/physiology , Animals , Base Sequence , Cell Cycle Proteins/genetics , Cell Cycle Proteins/pharmacology , Cell Cycle Proteins/physiology , Cell Differentiation/genetics , Chickens , DNA-Binding Proteins/genetics , DNA-Binding Proteins/pharmacology , DNA-Binding Proteins/physiology , Drug Evaluation, Preclinical/methods , Humans , K562 Cells , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/pharmacology , Mice , Phylogeny , Promoter Regions, Genetic , RNA, Messenger/analysis , Repressor Proteins/genetics , Repressor Proteins/pharmacology , Repressor Proteins/physiology , TransfectionABSTRACT
The Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the beta-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the beta-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and beta h1-globin but not zeta-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult beta(maj)- and beta(min)-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic epsilon-globin gene but not the adult beta-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death.
