Erythroid Krüppel-Like Factor (EKLF) is the founding member of the mammalian Krüppel subfamily of transcription factors with 3 C2H2-type zinc fingers. In mice, loss of EKLF is lethal at day 14 of gestation (dE14), due to severe anemia. To study the physiological properties of the mixed population of circulating primitive and definitive erythrocytes in dE13.5 mice, we developed an osmotic fragility assay that compares the relative amount of embryonic (primitive) and adult (definitive) hemoglobin in the supernatant and pellet of cells exposed to increasing concentrations of NaCl. We found that both primitive and definitive EKLF-deficient cells were resistant to osmotic stress, consistent with the thalassemia-like phenotype of the cells. Flow cytometric analysis of WT dE13.5 fetal liver (FL) cells with TER119 and CD71 identified 5 previously described populations: R1+R2, composed of pro- and basophilic normoblasts, and R3+R4+R5, composed of more mature polychromatic and orthochromatic normoblasts and reticulocytes, respectively. Analysis of dE13.5 EKLF-deficient FL cells showed that R3+R4+R5 were absent, and morphological examination demonstrated that R1 and R2 were homogeneous populations of pro- and basophilic normoblasts. Colony-forming assays revealed an increase in the absolute number of BFU-E and CFU-E in EKLF-deficient R1 and R2 FL cells. Although the EKLF-deficient colonies required 24–72 more hours of culture than WT colonies to mature, they were composed of definitive erythroid cells as they expressed only adult β-globin. EKLF-deficient FL also contained 2-fold more CFU-Meg colonies (p<0.01). From these data we concluded that the lack of R3+R4+R5 cells in EKLF-deficient FL is due to a block in erythroid differentiation. We compared mRNA from sorted R1+R2 WT and EKLF-deficient FL cells by microarray and used Ingenuity Pathways Analysis (IPA) to demonstrate that the levels of mRNAs encoding proteins involved in the cell cycle and DNA replication were significantly altered. Cell cycle analyses showed that EKLF-deficient R1+R2 cells are significantly delayed exiting G0/G1 (p<0.002). A central node in the cell cycle network was E2F2, a transcription factor involved in cell cycle progression and differentiation. E2F2 mRNA was decreased to 7.6±1.6% of WT levels in EKLF-deficient FL, with a comparable reduction in protein. We hypothesized that E2F2 is a direct EKLF target gene, and examined the chromatin of the E2F2 locus in a high throughput DNase-I hypersensitive site (HS) assay. The E2F2 locus is sensitive to DNase I in WT FL cells and resistant to DNase I in EKLF-deficient FL cells. Three EKLF-dependent HS were identified: in the promoter, intron 2, and the 3′ untranslated region. We used chromatin immunoprecipitation to show that EKLF associates with consensus DNA-binding sites in the E2F2 promoter HS. IPA also demonstrated that apoptosis was abnormal in EKLF-deficient R1+R2 cells. Flow cytometry with annexin V staining demonstrated that EKLF-deficient R1+R2 cells were resistant to apoptosis (p<0.01), consistent with the severe anemia, indicating that apoptosis was not contributing to the differentiation block. Our results support a model in which EKLF-deficiency leads to a block in definitive erythroid maturation associated with decreased cell cycling, resulting in the limited production of dehydrated erythrocytes.

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Disclosure: No relevant conflicts of interest to declare.