Mechanisms of megakaryocyte maturation

 描述(由申请人提供)：本提案是针对RFA-RM-13-022号文件提出的，标题为“对现行研究项目增加单细胞分析的修订(R01)”。这些研究代表了奥列格·丹尼森科和黛安·克劳斯的实验室共同努力，将新颖的单细胞表观遗传学分析应用于父母拨款中解决的巨核生成中的生物学问题。我们提出了一种在单细胞水平上询问染色质结构的强大的新技术的开发和应用。具体地说，我们将使用Denisenko博士正在开发的新方法来探索原始人类巨核细胞-红系祖细胞(MEP)经历巨核细胞谱系承诺和分化时单个基因表观遗传状态的变化。分析单细胞表观遗传状态的能力一直是一种 在我们的造血规范和分化研究中遇到了障碍。这些目标代表了以下进步步骤：1)从人类造血细胞系的单细胞表观遗传学分析发展到原代人类细胞；2)从Egr1基因座的分析(Denisenko实验室已经成功成像)到ARH-GEF2基因座，这是亲本R01的焦点；以及3)从丰富的组蛋白修饰密度的变化分析到更具特异性的转录因子结合分析。通过 应用这种新颖的单细胞方法，这些研究不仅将推进母体R01的目标，还将为Denisenko博士提供关于其方法进一步发展的反馈。分析原代人类巨核细胞-红系祖细胞和巨核细胞在不同成熟阶段的单细胞表观遗传学变化将补充亲本R01的目标，并使我们更接近于确定巨核细胞规格和成熟的基本分子机制的长期目标。这样的分析将深入了解在给定基因上经历特定表观遗传修饰的细胞的百分比，将允许评估这些变化是否是单等位基因与双等位基因的变化，以及与其他表观遗传修饰和基因表达水平相比，单细胞水平上这种变化的动力学，同样都是在单细胞水平上。这些研究将在两年的时间内进行，将使我们为未来专注于这些过程的中断如何导致血液病的研究奠定基础。

 DESCRIPTION (provided by applicant): 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.