The overall goal of this project is to elucidate the factors and mechanisms that control the decision of early erythroid progenitors to undertake terminal differentiation. Much evidence suggests that a crucial aspect of differentiation decisions is a requirement for coordination with the cell proliferation program. However, our current knowledge about the connections between the cell proliferation and differentiation programs is very limited. Work from our lab and others has identified the Ets - family transcription factor PU.1 as a key regulator of the terminal differentiation decision in early erythroid progenitors. And yet, the factors that control PU.1 expression and activity in these early erythroid progenitors and the mechanisms that link the differentiation decision to the cell cycle are not known. Recently, we identified two types of factors that have a very strong potential for connecting PU.1 and the terminal differentiation decision to the cell cycle. One factor is CDK6, which we discovered inhibits erythroid differentiation, like PU.1. We also showed that: 1) CDK6 is the active G1 phase D-cyclin kinase in erythroid progenitors; 2) PU.1 controls transcription of the CDK6 gene; 3) CDK6 phosphorylates PU.1. The other factors are E2F2 and E2F4, transcriptions factors that play major roles in G1 to S phase cell cycle progression. We found that E2F2 and E2F4 occupy the promoter and upstream regulatory element (URE) of the PU.1 gene in erythroid progenitors. They also bind very close to PU.1 at many other PU.1 target genes in these cells.
In Aims 1 and 2, we propose a series of experiments to determine how CDK6 and E2F2 and E2F4 influence PU.1 expression and activity and how they collaborate with PU.1 in the erythroid terminal differentiation decision. We also propose to investigate roles for CDK6 and E2F2 in stress erythropoiesis and in ex vivo self-renewal of erythroid precursors. Work from our lab and others showed that one of the principal mechanisms used by PU.1 to regulate the erythroid differentiation decision is repression of erythroid-specific gene expression Recently, we discovered that PU.1 forms a complex with the chromatin remodeling ATPase SNF2H and the maintenance DNA methyltransferase DNMT1; two enzymes that we hypothesize help PU.1 to repress erythroid gene expression.
In Aim 3, we propose studies to elucidate the role of SNF2H and DNMT1 in PU.1-mediated repression of erythroid-specific genes and inhibition of erythroid terminal differentiation. The successful completion of the proposed studies will lead to new insights into the mechanisms connecting the proliferation and differentiation programs in hematopoietic cells and a much deeper understanding of how the hematopoietic system regulates red blood cell production, including during ontogeny and in stress conditions. The proposed work will also provide important information enabling the development of new approaches to managing defects in red blood cell production that occur in chronic and acute anemia.

Public Health Relevance

The overall goal of this project is to understand the factors and mechanisms that regulate the decision of red blood cell precursors to undertake terminal differentiation into mature red blood cells. The proposed studies are expected to provide important new information about how the blood system normally produces the proper number of red blood cells and how it responds to the need to produce more red cells in stress conditions. Some of the proposed work will involve identification of factors that are required for expansion of red blood cell precursors ex vivo, an approach that could provide a source of red blood cells for transfusion therapy. The successful completion of this work will enable the development of new approaches to managing defects in red blood cell production that occur in chronic and acute anemia due to a variety of causes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
6R01DK096266-05
Application #
9135828
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2012-08-01
Project End
2017-05-31
Budget Start
2015-09-01
Budget End
2016-05-31
Support Year
5
Fiscal Year
2015
Total Cost
$272,419
Indirect Cost
$109,294
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
079783367
City
Bronx
State
NY
Country
United States
Zip Code
10461
Limi, Saima; Senecal, Adrien; Coleman, Robert et al. (2018) Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process. J Biol Chem 293:13176-13190
Kelleher, Andrew M; Milano-Foster, Jessica; Behura, Susanta K et al. (2018) Uterine glands coordinate on-time embryo implantation and impact endometrial decidualization for pregnancy success. Nat Commun 9:2435
Kokavec, Juraj; Zikmund, Tomas; Savvulidi, Filipp et al. (2017) The ISWI ATPase Smarca5 (Snf2h) Is Required for Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells. Stem Cells 35:1614-1623
He, Shuying; Limi, Saima; McGreal, Rebecca S et al. (2016) Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation. Development 143:1937-47
Vierstra, Jeff; Rynes, Eric; Sandstrom, Richard et al. (2014) Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution. Science 346:1007-12
Yue, Feng; Cheng, Yong; Breschi, Alessandra et al. (2014) A comparative encyclopedia of DNA elements in the mouse genome. Nature 515:355-64
Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun et al. (2014) Principles of regulatory information conservation between mouse and human. Nature 515:371-375
Yu, Yiting; Mo, Yongkai; Ebenezer, David et al. (2013) High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis. J Biol Chem 288:8805-14