A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis

Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production in vitro. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated Foxo3-/- erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued Foxo3-/- erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.

循环红细胞（red blood cells, RBCs）对于组织氧合与内环境稳态至关重要。多种疾病中，终末红细胞生成（terminal erythropoiesis）缺陷会导致红细胞生成量减少。具体而言，终末成熟阶段的无效核排出（去核，enucleation）过程，是体外治疗性红细胞生产的一大障碍。为深入解析终末红细胞生成的分子机制，我们将研究焦点聚焦于叉头框O3（FOXO3）——一种与红细胞疾病相关的转录因子（transcription factor）。通过整合计算与实验系统生物学方法，我们证实FOXO3在终末红细胞生成过程的时序基因表达调控中发挥不可或缺的作用。我们证明，依赖FOXO3的遗传网络在终末红细胞生成的关键步骤中具有重要生理功能，包括去核与线粒体清除过程。FOXO3缺失会导致与细胞极性、核小体组装及DNA包装相关的基因转录失调，并损害红细胞系的去核过程。借助高分辨率共聚焦显微镜（high-resolution confocal microscopy）与成像流式细胞术（imaging flow cytometry），我们发现细胞极性受损会导致Foxo3基因敲除（Foxo3-/-）的成红细胞出现多叶形态，进而无法正常完成核排出。异位表达FOXO3可挽救Foxo3-/-成红细胞的去核相关基因转录缺陷、去核障碍及终末成熟异常。值得注意的是，在野生型成红细胞中异位表达FOXO3可进一步促进其成熟与去核过程，这提示增强FOXO3活性或可提升红细胞的生产效率。综上，本研究揭示FOXO3是调控成红细胞去核与终末成熟的全新调节因子，表明靶向调控FOXO3或可用于治疗红细胞系成熟缺陷相关疾病。