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

Raymond Liang,Kathleen Mcgrath,Genís Campreciós,Merlin Nithya Gnanapragasam,Carolina L Bigarella,Emery Bresnick,Xin Zhang,Saghi Ghaffari,James Palis,Pauline Rimmelé,Avi Ma’Ayan,James J Bieker,Yan Kou,Roberta Nowak,Dmitri Papatsenko,Velia Fowler,Seana Catherman

doi:10.1371/journal.pgen.1005526

Abstract

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.

Highlights

Erythropoiesis ensures the daily production of over 200 billion red blood cells (RBCs) whose main function is to carry oxygen
Using an integrated computational and experimental biology approach, we found that the nuclear factor FOXO3 is a crucial regulator of red blood cell production by coordinating the expression of many of the genes specific for terminal maturation of red blood cells
We found that FOXO3 can even increase the production of normal red blood cells in culture raising the possibility that enhancing FOXO3 may have a therapeutic use

Summary

Introduction

Erythropoiesis ensures the daily production of over 200 billion RBCs whose main function is to carry oxygen. Despite recent development [1] achieving efficient production of functional RBCs has been hindered by incomplete knowledge of terminal erythroblast maturation. Reticulocytes remodel their membrane and clear mitochondria and remaining organelles to transition into fully mature erythrocytes [2]. This complex process is controlled by integration of erythropoietin receptor (EpoR) signaling with the function of erythroid lineage-specific transcription factors including GATA–1, KLF–1 and TAL–1 (SCL) and their cofactors [3]. Despite recent progress [4,5,6], many questions remain unanswered regarding whether these factors function alone or together to control enucleation and/or to remove organelles, including mitochondria, during terminal erythroblast maturation. Increasing evidence suggest that FOXO3 cooperates with these factors and their requisite coregulators to control specific molecular/cellular steps that drive terminal erythroid maturation [4,5,6]

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