Mice deficient in the erythroid specific zinc-finger transcription factor EKLF die ~d14-15 of gestation of severe anemia, attributed to decreased expression of β-globin. The morphology of fetal-liver derived erythroid cells in EKLF-deficient mice does not mimic that seen in thalassemia, but instead shows hemolysis with uniform, nucleated erythroid progenitor cells. This has led to the hypothesis that a block in erythroid differentiation contributes to the anemia in EKLF-deficient mice. To address this, we performed microarray analyses with Affymetrix GeneChip Mouse Genome 430 2.0 arrays and RNA from d13.5 fetal livers of wild type (WT) and EKLF-deficient mice. Three independent EKLF +/+ and −/− RNA samples were analyzed. Numerous genes were down regulated including AHSP, pyruvate kinase, ankyrin, β spectrin and band 3. Verification of reduced expression of selected genes demonstrated that expression levels of many genes identified as down regulated via microarray analyses were minimally reduced in EKLF −/− RNA (<20%) compared to normal (Rh 30, protein 4.2, protein 4.9, p55, AQP1, and ALAS-E). Flow cytometry of WT d14.5 fetal liver cells using TER 119 and CD71 was performed. In WT fetal livers, this identifies 5 populations, designated R1-R5, with R1/R2 composed of primitive progenitors and proerythroblasts and R3, R4, and R5 composed of more mature erythroblasts (
Blood 102:3938, 2003
). In EKLF −/− fetal livers, R3, R4, and R5, populations involved in terminal erythroid differentiation, were completely absent, suggesting many of the genes identified by microarray analyses were differentially expressed because of a bias introduced by a differentiation block to more mature erythroid cells. Confirming this hypothesis, we demonstrated that genes with <20% difference in expression between WT and EKLF-deficient fetal liver mRNA had 4-fold or higher levels in wild type R3+R4+R5 RNA compared to R1+R2 RNA. To better understand how differentially expressed genes were integrated into specific regulatory and signaling pathway networks, we used Ingenuity Pathway Analysis. A subset of focus genes incorporated into a biological network with highly a significant scores (>40) was generated containing 35 focus genes. The biological function of this network involved cell cycle and DNA replication. At the central nodes of this network were E2F1 and E2F2, transcription factors involved in cell cycle control. Cell cycle analysis demonstrated that EKLF-deficient R1 cells exhibited a significant delay exiting G0+G1 and entering S phase and both R1 and R2 cells exhibited a defect in exiting S and entering G2+M. Colony assays with R1 and R2 cells revealed that EKLF-deficient fetal liver cells had decreased frequency of CFU-E, but similar absolute numbers of CFU-E as WT. As predicted by the cell cycle defect, EKLF−/− FL cells were severely (~10 fold) deficient in their ability to generate BFU-E. Flow cytometry with annexin V revealed no difference between WT and EKLF-deficient cells indicated that apoptosis was not contributing to the differentiation block. These results support the hypothesis that the failure of definitive erythropoiesis in EKLF deficient mice is due to decreased expression of many erythroid genes involved in erythroid differentiation, stabilization of α-globin protein, membrane stability, and glycolysis, not simply decreased transcription of the β-globin gene.
