A Chemical Screening Approach to Identify Novel Key Mediators of Erythroid Enucleation.

Christina B Wölwer,Patrick O Humbert,Nathan J Gödde,Sarah M Russell,David C S Huang,Kurt Lackovic,Helen B Pearson,Luke B Pase,Andrew C Wilber

doi:10.1371/journal.pone.0142655

Abstract

Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs. Using this approach, we have confirmed the role of HDACs, proteasomal regulators and MAPK in erythroid enucleation and introduce a new role for Cyclin-dependent kinases, in particular CDK9, in this process. Importantly, we demonstrate that when coupled with imaging analysis, this approach provides a powerful means to identify and characterize rate limiting steps involved in the erythroid enucleation process.

Highlights

Erythropoiesis involves the gradual progression of hematopoietic stem cells into mature red blood cells
The process of erythroblast enucleation occurs within a 10 minute period [5] and is not synchronized, making it challenging to interpret effects of drugs and shRNA studies
Orthochromatic erythroblasts were isolated from the spleen based on expression of Ter119 and CD44 [6] (Fig 1A)

Summary

Introduction

Erythropoiesis involves the gradual progression of hematopoietic stem cells into mature red blood cells. Proerythroblasts undergo several differentiationlinked cell divisions producing basophilic, polychromatic and orthochromatic daughter erythroblasts. The orthochromatic cells exit from the cell cycle and extrude their nuclei in a process termed erythroid enucleation. The enucleation event involves multiple pathways and shares some similarities with cytokinesis and apoptosis (for reviews see Keerthivasan et al [1] and Ji et al [2]); experimental results have been variable due to the difficulty of synchronizing erythroblasts and the inability to exclude indirect effects of inhibitors on the proliferation of earlier erythroblasts. In vivo investigations have been hindered by the PLOS ONE | DOI:10.1371/journal.pone.0142655. In vivo investigations have been hindered by the PLOS ONE | DOI:10.1371/journal.pone.0142655 November 16, 2015

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Conclusion