A Systems Approach Identifies Essential FOXO3 Functions in Erythroblast Enucleation Process

Elucidating mechanisms that regulate terminal erythroblast maturation may serve to improve the treatment of anemias as well as the generation of red blood cells in vitro. Key steps in red blood cell maturation are chromatin condensation and expulsion of the nucleus (or enucleation) from late-stage erythroblasts. The transcriptional program that coordinates these steps is poorly defined. The expression and transcriptional activity of FOXO3 increase with terminal erythroblast maturation suggesting a potential function for FOXO3 in this process. Using comparative transcriptomic analysis of primary freshly isolated wild type and Foxo3^-/- erythroblasts at distinct stages of maturation, we found that many genes involved in DNA packaging-related pathways were highly downregulated in Foxo3^-/- erythroblasts, raising the possibility that FOXO3 might be involved in regulating chromatin condensation and/or the enucleation process. Foxo3^-/- enucleating cells were also significantly fewer than wild type bone marrow erythroblasts according to in vivo DNA staining by DRAQ5. qRT-PCR analysis confirmed the reduced expression of genes implicated in the control of chromatin condensation and/or enucleation including Mxi1, Riok3, Trim58, Rac GTPase I and II at most if not all stages of maturation in Foxo3^-/- erythroblasts. Immunoprecipitation of endogenous FOXO3 in wild type but not in Foxo3^-/- bone marrow erythroblasts recovered the regulatory regions of both Mxi1 and Riok3 in vivo suggesting that FOXO3 controls directly the expression of these genes in erythroblasts. To investigate more directly the potential impact of loss of FOXO3 on the enucleation process, we examined bone marrow erythroblasts at high magnification by confocal fluorescence microscopy. Wild-type enucleating erythroblasts were identified by a gap in the bright TER119 membrane staining and displayed a dumbbell-shaped nucleus, with a neck located at the TER119 sorting boundary of the nascent reticulocyte, so that the cells used a single direction for nuclear extrusion. However, Foxo3 mutant erythroblasts exhibited multiple nuclear necks accompanied by multiple sorting boundaries with each lobe extruding in a different direction away from the nascent reticulocyte, suggesting defective polarization of Foxo3 mutant erythroblasts during enucleation. As a result 48% of the Foxo3^-/-enucleating erythroblasts displayed abnormal enucleation morphologies. Next using imaging flow cytometry we further characterized the potential enucleation defect in Foxo3^-/- erythroblasts. Segregation of TER119⁺ erythroblasts according to their cell body and nuclear size into distinct stages of maturation (pro-, basophilic, polychromatic and orthochromatic) showed a relative increase in late erythroid precursors in Foxo3^-/- bone marrow. It also revealed that the Foxo3 mutant erythroblasts were specifically accumulated at the orthochromatic stage of maturation. The percentage of orthochromatic erythroblasts containing an asymmetric nucleus was quantified using the delta centroid, a measure of the distance between the center of the cell body and center of the DRAQ5-stained nucleus. This analysis revealed a lower percentage of asymmetric enucleating erythroblasts in the Foxo3^-/- as compared to wild-type bone marrow. Using this approach lobular nuclei were distinguished from circular nuclei and demonstrated that odd nuclei were present with remarkably greater frequency in Foxo3^-/- than in wild type orthochromatic erythroblasts. These combined analyses support the notion that FOXO3 is critical for the erythroblast enucleation process and may be implicated in polarization of erythroblasts leading to enucleation. The direct ability of FOXO3 to rescue the defective Foxo3 mutant erythroblast enucleation was shown by ectopic expression MSCV-IRES-GFP (MIG)-FOXO3 as compared to empty control vector in wild type and Foxo3^-/- bone marrow-derived erythroid progenitors. Restoration of FOXO3 in GFP-positive Foxo3^-/-erythroblasts rescued the enucleation rate to similar levels as controls after 72 hours of retroviral transduction and significantly enhanced the levels of expression of Mxi1, Riok3 and Trim58 in Foxo3^-/-erythroblasts. Collectively, these findings show that FOXO3 is an essential component of the transcriptional program that regulates terminal erythroblast maturation and is implicated in the enucleation process.