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.

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

Grant support

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.