Among the processes comprising terminal red cell development including decrease in cell size, accumulation of hemoglobin, and chromatin condensation culminating in enucleation, the discrete process of irreversible chromatin condensation is still poorly understood. Our incomplete understanding of this critical process hinders the development of improved treatments for certain erythropoietin-refractory anemias characterized by defects in late erythroid maturation. Knocking down the highly erythroid-specific nuclear exportin Xpo7 in primary murine erythroblasts resulted in severe disruption of chromatin condensation and enucleation but had little effect on hemoglobin accumulation or the erythroid expression program (manuscript in review), providing evidence that export of nuclear proteins is essential to erythroid chromatin condensation and enucleation. The work outlined in this proposal aims to understand which proteins Xpo7 exports from the erythroblast nucleus and how they work together to regulate the process of erythroid chromatin condensation. Based on proteomic examination of the composition of the erythroid precursor nucleus from early erythroblast to extrusion, it was also noted that extruded nuclei are largely depleted of protein-i particular, core structural proteins and histones-while nuclei lacking the exportin Xpo7 accumulated almost all nuclear proteins nonspecifically. Strikingly, DNA binding proteins such as histones H2A and H3 accumulate in the cytoplasm of normal late erythroblasts prior to and during enucleation but not in cells lacking Xpo7. In order to clarify the hypothesis that Xpo7 removes either inhibitors of chromatin condensation or all erythroid nuclear proteins nonspecifically in order to allow adequate condensation for proper enucleation, this proposal aims to understand (1) which proteins Xpo7 exports from the erythroid precursor nucleus, and (2) how these proteins help to regulate erythroid chromatin condensation. Because the few remaining proteins in pycnotic, extruded nuclei likely facilitate the process of condensation itsel, the proposed work will also shed light on how histone proteins are replaced in the erythroid nucleus prior to extrusion. The proposed studies will rely on more detailed proteomics and chromatography, followed by state of the art simultaneous microscopy and flow cytometric evaluation to quantify chromatin condensation and enucleation, respectively, after in vitro knockdown of putative regulators. This study will also examine the conservation of these findings in a primary human cell culture model and an in vivo mouse knockout model. Results from experiments proposed in this study will also uncover the function of Xpo7 in more detail- determining which motifs specific to Xpo7 are required for its function will help explain how a nuclear export protein can export nuclear proteins without substrate specificity. This knowledge will increase our understanding of such important basic cell biological concepts as nuclear export and nuclear condensation during terminal erythroid maturation.

Public Health Relevance

Anemia (low red blood cell count) is the most common hematological disease across the world, affecting over 3 million Americans alone. Many types of anemia in disorders such as myelodysplasia, leukemia, thalassemia, sideroblastic anemia, and anemia of inflammation are due to defects in red cell maturation and do not respond to treatment with the red cell hormone erythropoietin (Epo). The development of novel alternative treatments for these Epo-refractory anemias is limited by our incomplete understanding of crucial processes of late red cell development. In our preliminary work, we found that the removal of proteins from the nucleus is essential for one of these processes-the compaction of DNA-and the proposed experiments in this study will help us understand how this process occurs and how it is controlled.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01DK102889-01
Application #
8754807
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510