Regulation of Chromatin Structure Dynamics in Hematopoietic and Leukemic Stem Cel

描述（由申请人提供）：我们的长期研究目标是了解调节干细胞命运决定的机制。该建议侧重于细胞身份的表观遗传调控。开放染色质被认为是干细胞的标志之一，解释了它们的多谱系潜能。使用造血干细胞（HSC）系统作为成体干细胞分化的模型，我们建议测试HSC是否具有比谱系定向祖细胞和成熟细胞更开放和动态的染色质结构。此外，我们将测试染色质凝聚的功能后果，并在HSC分化过程中操纵异染色质的形成。重要的是，我们发现高度纯化的造血干细胞和祖细胞与成熟细胞相比具有对核酸酶更敏感的染色质结构。这种核酸酶的敏感性与谱系潜力和不同的三维染色质组织相关。在这里，我们将研究这些差异背后的机制。我们将通过能够检测少量细胞差异的灵敏测定来分析DNA和组蛋白的整体分子变化。我们将测试HSC分化和命运的决定如何改变染色质修饰酶的抑制后，使用互补的分子，细胞和功能测定在体外和体内的组合。最后，我们将比较染色质 HSC和白血病干细胞之间的分析，并评估白血病转化中染色质修饰的贡献。我们预计，这些结果将提供新的见解染色质结构和组织的动态干细胞分化和转化过程中，具有重要意义的正常造血发育和白血病。

DESCRIPTION (provided by applicant): Our long-term research goals are to understand the mechanisms that regulate stem cell fate decisions. This proposal focuses on epigenetic regulation of cell identity. Open chromatin has been proposed as one of the hallmarks of stem cells, explaining their multilineage potential. Using the hematopoietic stem cell (HSC) system as a model of adult stem cell differentiation, we propose to test whether HSC have a more open and dynamic chromatin structure than lineage committed progenitors and mature cells. In addition, we will test the functional consequences of chromatin condensation, and manipulate heterochromatin formation during HSC differentiation. Importantly, we have discovered that highly purified hematopoietic stem and progenitor cells have a more nuclease-sensitive chromatin structure compared to mature cells. This nuclease sensitivity correlates with linage potential and with differential three-dimensional chromatin organization. Here, we will investigate the mechanisms behind these differences. We will analyze the global molecular changes of DNA and histone proteins by sensitive assays capable of detecting differences in small numbers of cells. We will test how HSC differentiation and fate decisions change upon inhibition of chromatin modifying enzymes using a combination of complementary molecular, cellular, and functional assays in vitro and in vivo. Lastly, we will perform comparative chromatin analyses between HSC and leukemic stem cells and assess the contribution of chromatin modifications in leukemic transformation. We anticipate that these results will provide novel insights to the dynamics of chromatin structure and organization during stem cell differentiation and transformation, with important implications for both normal hematopoietic development and leukemogenesis.