Mechanisms of megakaryocyte maturation

 描述（由申请人提供）：本提案是为了响应标题为“将单细胞分析添加到正在进行的研究项目（R 01）的修订版”的RFA-RM-13-022而提交的。“这些研究代表了Oleg Denisenko和Diane Krause实验室的共同努力，将新的单细胞表观遗传学检测应用于母基金中提到的巨核细胞生成的生物学问题。 我们提出了一个强大的新技术的开发和应用，在单细胞水平上询问染色质结构。 具体来说，我们将使用Denisenko博士正在开发的新方法来探测单个基因的表观遗传状态的变化，因为原代人巨核细胞-红系祖细胞（MEP）细胞经历巨核细胞谱系定型和分化。 分析单细胞中表观遗传状态的能力一直是一个挑战。 这是我们研究造血特化和分化的一个障碍。这些目标代表了从1）在人造血细胞系中开发单细胞表观遗传测定到原代人细胞; 2）从分析EGFR 1基因座（Denisenko实验室已经成功成像）到ARH-GEF 2位点，这是亲本R 01的焦点; 3）从分析丰富组蛋白修饰密度的变化到更特异的转录因子结合分析。 通过 应用这种新的单细胞方法，这些研究不仅将推进亲本R 01的目标，而且还将为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R 01的目的，并使我们更接近确定巨核细胞特异性和成熟的基本分子机制的长期目标。 这样的分析将提供对在给定基因处经历特定表观遗传修饰的细胞的百分比的洞察，将允许评估所述变化是否是单等位基因的还是双等位基因的，以及与其他表观遗传修饰和基因表达水平相比，在单细胞水平上的这样的变化的动力学，同样都在单细胞水平上。 这些研究将在2年内进行，将使我们能够进行未来的研究，重点关注这些过程的中断如何导致血液病。

 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.