Role of KLF2 in erythropoiesis and globin expression

Summary

Principal Investigator: Joyce Lloyd
Abstract: [KLF2 and EKLF have compensatory and critical roles in primitive erythropoiesis.] We hypothesize that KLF2 acts in an erythroid cell autonomous manner, directly binding to globin genes. The first aim is to further elucidate the mechanistic role of KLF2 in erythropoiesis. [The stage of maturation of KLF2-/- embryonic erythroid cells will be determined with a flow cytometry assay, and the ability of these cells to differentiate in vitro with exogenous factors will be assessed.] Mice with a conditional knockout (KO) the KLF2 gene (KLF2flox allele) have been bred with Cre recombinase mice with an erythroid-specific promoter (LCR-beta pr-Cre), and >90% of the KLF2flox alleles can be deleted. This system will be used to determine whether the cell morphology, globin gene expression, differentiation and apoptotic effects of KLF2 on primitive erythroid cells are cell autonomous, as expected. Using this system, we will also test the role of KLF2 in definitive (adult) erythropoiesis, measuring the same parameters. ChIP (chromatin immunoprecipitation) assays will be used to determine if KLF2 interacts with CACCC elements in the beta-globin locus in vivo, using K562, HEL and MEL cell lines expressing an inducible, tagged construct encoding KLF2. [If possible, ChIP assays will be performed on native mouse erythroid precursors]. The second aim is to mechanistically study the compensatory roles of KLF2 and EKLF in erythropoiesis using [our established] double KO mouse model. EKLF is expressed in primitive erythropoiesis, and [EKLF-/-KLF2-/- embryos are severely anemic at embryonic day 10.5, when single KOs appear unaffected. To assess the maturation and differentiation of EKLF-/-KLF2-/- embryonic erythroid cells, flow cytometry and progenitor assays will be used.] To study the effects of gene dosage on red cell morphology and globin gene expression, EKLF-/-KLF2 and EKLF KLF2-/- will be compared to wildtype, EKLF-/-, KLF2-/- and EKLF-/-KLF2-/- embryos. We will determine whether EKLF-/-KLF2-/- embryos have reduced gamma-globin mRNA compared to wildtype, using mice with the human beta-globin locus. [EKLF-/-KLF2-/- yolk sacs have abnormal endothelial cells, which is not the case in single KO yolk sacs. We will generate embryos lacking EKLF and KLF2 in erythroid cells only, to determine whether the endothelial defects are directed by erythroid cells.] Our long-term goal is to use KLF2 in strategies to increase embryonic/fetal beta-globin, to treat patients with beta-hemoglobinopathies.
Funding Period: ----------------2007 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Krüppel-like factor 2 is required for normal mouse cardiac development
    Aditi R Chiplunkar
    Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, United States of America
    PLoS ONE 8:e54891. 2013
  2. pmc Transcription factors KLF1 and KLF2 positively regulate embryonic and fetal beta-globin genes through direct promoter binding
    Yousef N Alhashem
    Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia 23298 0035, USA
    J Biol Chem 286:24819-27. 2011

Detail Information

Publications2

  1. pmc Krüppel-like factor 2 is required for normal mouse cardiac development
    Aditi R Chiplunkar
    Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, United States of America
    PLoS ONE 8:e54891. 2013
    ..In conclusion, KLF2-/- heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important cardiovascular genes...
  2. pmc Transcription factors KLF1 and KLF2 positively regulate embryonic and fetal beta-globin genes through direct promoter binding
    Yousef N Alhashem
    Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia 23298 0035, USA
    J Biol Chem 286:24819-27. 2011
    ..Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos...