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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CB-R (50))
Program Officer
Bishop, Terry Rogers
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Yale University
Schools of Medicine
New Haven
United States
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Krause, Diane S (2016) An oxidase road to platelet adhesion. Blood 127:1386
Denisenko, Oleg; Lucas, Emma S; Sun, Congshan et al. (2016) Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation. Biochim Biophys Acta 1859:906-13
Sanada, Chad; Xavier-Ferrucio, Juliana; Lu, Yi-Chien et al. (2016) Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction. Blood 128:923-33
Mar, Daniel; Gharib, Sina A; Zager, Richard A et al. (2015) Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes. Kidney Int 88:734-44
Sui, Zhenhua; Nowak, Roberta B; Sanada, Chad et al. (2015) Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis. Blood 126:520-30
Halene, Stephanie; Krause, Diane S (2015) Stem cell maintenance: aMPLe splicing choices. Blood 125:891-2
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
Smith, Elenoe C; Teixeira, Alexandra M; Chen, Rachel C et al. (2013) Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation. Blood 121:1094-101