A Systems Approach Identifies Essential FOXO3 Functions in Erythroblast Enucleation Process

• Published in: Blood 2014-12, Vol.124 (21), p.445-445
• Main Authors: Liang, Raymond, Campreciós, Genís, Kou, Yan, McGrath, Kathleen E, Novak, Roberta, Catherman, Seana C, Rimmele, Pauline, Bigarella, Carolina L., Bresnick, Emery H, Papatsenko, Dmitri, Ma’ayan, Avi, Fowler, Velia M., Palis, James, Ghaffari, Saghi
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.445.445

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 p