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

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

doi:10.1371/journal.pone.0142655

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

PLoS One. 2015 Nov 16;10(11):e0142655. doi: 10.1371/journal.pone.0142655. eCollection 2015.

Authors

Christina B Wölwer^{1

2}, Luke B Pase^{1

2}, Helen B Pearson^{1

2}, Nathan J Gödde^{1

2}, Kurt Lackovic^{3

4}, David C S Huang^{3

4}, Sarah M Russell^{2

5

6}, Patrick O Humbert^{1

2

7

8}

Affiliations

¹ Cell Cycle and Cancer Genetics, Peter MacCallum Cancer Centre, Melbourne, Australia.
² Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia.
³ The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.
⁴ Department of Medical Biology, University of Melbourne, Melbourne, Australia.
⁵ Immune Signaling Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia.
⁶ Centre for Micro-Photonics, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Australia.
⁷ Department of Pathology, University of Melbourne, Melbourne, Australia.
⁸ Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation
Cell Nucleus / metabolism
Cell Separation
Cyclin-Dependent Kinase 9 / metabolism
Erythroblasts / drug effects*
Erythroblasts / metabolism*
Erythropoiesis / drug effects*
Erythropoiesis / physiology*
Flow Cytometry
Histone Deacetylases / metabolism
MAP Kinase Signaling System
Mice
Mice, Inbred C57BL
Phenotype
Proteasome Endopeptidase Complex / metabolism
Proteasome Inhibitors / chemistry
Reticulocytes / cytology*
Reticulocytes / physiology
Spleen / cytology
Spleen / drug effects
Technology, Pharmaceutical / methods*

Substances

Proteasome Inhibitors
Cdk9 protein, mouse
Cyclin-Dependent Kinase 9
Proteasome Endopeptidase Complex
Histone Deacetylases

Grants and funding

CBW was supported by the Australian Postgraduate Award. DCSH is supported by grants (program 1016701 and Independent Research Institutes Infrastructure Support Scheme grant 361646) and a fellowship (1043149) from the National Health and Medical Research Council (NH&MRC) of Australia; the Leukemia Lymphoma Society (SCoR 7001-13); and a Victorian State Government Operational Infrastructure Support (OIS) grant. SMR was supported by grants and fellowships from the National Health and Medical Research Council and Australian Research Council (ARC) of Australia. POH was supported by a Senior Research Fellowship and a project grant from the National Health and Medical Research Council of Australia.