Analysis of the Multiple Roles of the Erythroid Krüppel-Like Factor (EKLF) in Erythropoiesis
Open AccessErythroid Krüppel-Like Factor (EKLF) is the founding member of the mammalian Krüppel family of zinc finger transcription factors, first identified as an important activator of human β-globin expression. In mice, loss of EKLF is embryonic lethal due to severe anemia, initially attributed to decreased β-globin expression. While the erythroid phenotype of EKLF-deficient mice is reminiscent of human β-thalassemia, EKLF-deficiency causes pleiotropic defects of gene expression. We hypothesized that only a subset of genes were direct targets of EKLF, responsible for the lethal phenotype. Two complementary models were employed to identify direct EKLF target genes: an EKLF knockout (Klf1-/-) mouse, and an HA-epitope-tagged (HA-EKLF) mouse. Through microarray analyses, transcriptional profiling, and chromatin immunoprecipitation (ChIP), we confirmed two novel targets of EKLF activation in vivo: the erythroid-specific molecular chaperone, α-hemoglobin stabilizing protein (Ahsp), a proposed modifier of the severity of β-thalassemia; and the broadly expressed cell cycle control factor, E2f2. mRNA and protein expression of both of these genes were significantly decreased in Klf1-/- fetal liver cells, and EKLF was required to remodel chromatin at both loci, including acetylation of histones and formation of DNase sensitive domains. In vitro studies demonstrated that formation of cells of other hematopoietic lineages was not compromised by loss of EKLF, and that Klf1-/- erythroid progenitor cells were able to produce mature β-globin protein. Taken together, these data support the hypothesis that the β-thalassemic phenotype experienced by Klf1-/- mice is secondary to a primary failure of definitive erythropoiesis resulting from arrested erythroid maturation. Furthermore, this study demonstrates the utility of the complementary HA-EKLF mouse model for identifying direct genetic targets of EKLF in vivo, otherwise unattainable using only the Klf1-/- system.
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