This proposal is submitted in response to RFA-RM-13-022, entitled "Revisions to Add Single Cell Analysis to Active Research Projects (R01)." The studies represent a joint effort from the laboratories of Oleg Denisenko and Diane Krause to apply novel single cell epigenetic assays to biological questions in megakaryopoiesis addressed in the parent grant. We propose the development and application of a powerful novel technology to interrogate chromatin structure at the single cell level. Specifically, we will use the new methods that Dr. Denisenko is developing to probe changes in the epigenetic state of individual genes as primary human megakaryocyte-erythroid progenitor (MEP) cells undergo megakaryocyte lineage commitment and differentiation. The ability to analyze the epigenetic state in single cells has been a roadblock in our studies of hematopoietic specification and differentiation.
The aims represent progressive steps 1) from the development of the single cell epigenetic assay in human hematopoietic cell lines to primary human cells;2) from analysis of the EGR1 locus (which the Denisenko laboratory has already successfully imaged) to the ARH-GEF2 locus, which is the focus of the parent R01; and 3) from analysis of changes in the densit of abundant histone modifications to more specific analysis of transcription factor binding. By applying this novel single cell approach, these studies will not only advance the aims of the parent R01, but will also provide feedback to Dr. Denisenko on the further development of his approach. Analysis of single cell epigenetic changes in primary human megakaryocyte-erythroid progenitor cells and megakaryocytes at different stages of maturation will complement the aims of the parent R01 and bring us closer to the longterm goal of determining the basic molecular mechanisms underlying specification and maturation of megakaryocytes. Such analyses will provide insight into the percentage of cells undergoing specific epigenetic modifications at a given gene, will allow assessment of whether the changes are monoallelic versus biallelic, and the kinetics of such changes at the single cell level compared to other epigenetic modifications and gene expression levels, again all at the level of single cells. These studies, which will be performed over a 2 year period, will position us for future studies focused on how disruptions in these processes cause hematological diseases.

Public Health Relevance

Normal megakaryocytopoiesis is necessary for the production of platelets, which are critical for blood clotting. The parent grant is focused on how blood stem cells in the bone marrow become mature megakaryocytes and make platelets. This work is extremely important because clinically 1) there are many patients who receive an injury that causes acute blood loss (e.g. car accidents) and need to have platelet transfusions, 2) there are patients who develop diseases that affect their ability to make their own platelets, 3) there are victims of radiation exposure who cannot make their own platelets, and 4) many medications have as a side effect transient toxicity to the patient's ability to make platelets (fr example cancer chemotherapy for cancer). The goal of this collaborative effort is to advance the aims of the funded project by applying a novel approach for epigenetic analysis of single cells that has been pioneered by Dr. Oleg Denisenko at the University of Washington.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK094934-03S1
Application #
8831876
Study Section
Special Emphasis Panel (ZRG1-CB-R (50))
Program Officer
Bishop, Terry Rogers
Project Start
2012-04-01
Project End
2017-07-31
Budget Start
2014-09-23
Budget End
2015-07-31
Support Year
3
Fiscal Year
2014
Total Cost
$440,381
Indirect Cost
$116,317
Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06510
Krause, Diane S; Crispino, John D (2013) Molecular pathways: induction of polyploidy as a novel differentiation therapy for leukemia. Clin Cancer Res 19:6084-8