Genomic Mapping of C-G Epigenetic Programs in Hematovascular Progenitor Cells

描述(申请人提供)：这项建议的重点是了解多能细胞如何在分化的最早阶段致力于特定的命运。细胞的特性由其基因组DNA的转录潜力决定。这在很大程度上是通过DNA和染色质的特殊化学修饰来实现的，这些化学修饰对基因活性进行了表观遗传编程。表观遗传编程的一个关键方面是DNA甲基化，它通过将转录输出限制在那些控制细胞特化的程序中，将基因组“硬连接”起来。由于这最活跃地发生在干细胞中，小鼠的ES/EB系统对于捕获早期阶段的多潜能基因组的完整和有序的编程以及易于分析的数量特别有价值。我们建议从正在分化的小鼠ES细胞中分离纯化一组离散的血管前体细胞，并将DNA甲基化和RNA表达谱与不具有血管潜能的分化前体细胞的DNA甲基化和RNA表达进行比较。通过这种方式，我们可以区分与一般分化相关的DNA甲基化模式和特定于造血系的DNA甲基化模式。我们将使用一个混合的高通量表观遗传学/基因/生物信息学平台，在基因组从多能性转变为承诺状态时识别基因组中DNA甲基化的初始位置，将这些信息与RNA表达分析结合起来，并将其与从具有不同发育潜力的细胞获得的类似数据进行比较。我们确定的血液血管特异性甲基化DNA区域的功能验证将在ES细胞分化试验中进行，以检查这些区域在细胞谱系承诺中的生物学作用。通过使用基因组工具，我们希望为细胞如何编码它们的细胞命运选择奠定基础。我们工作的一个重要方面是它可能应用于血液病的细胞替代治疗。例如，造血系统的长期替代将需要移植最不成熟的细胞，例如那些来自血液血管前体细胞的细胞。我们的工作将产生这一重要前体的高纯度种群的生化数量，这将被用于通过DNA甲基化和RNA表达谱来定义其表观基因组特征。我们希望应用这个平台来明确地识别一个群体中任何细胞的精确表观遗传学特征，该群体将被评估其治疗潜力。

DESCRIPTION (provided by applicant): The focus of this proposal is to understand how a pluripotent cell commits to a specific fate at the earliest stages of differentiation. Cell identity is determined by the transcriptional potential of its genomic DNA. This is achieved, in large part, through specific chemical modifications of DNA and chromatin that epigenetically programs gene activity. A critical aspect of epigenetic programming is DNA methylation, which "hard-wires" the genome by limiting transcriptional output to those programs that control cellular specialization. Because this occurs most actively in stem cells, the mouse ES/EB system is especially valuable for capturing the complete and ordered programming of a pluripotent genome in its early stages and in quantities that are amenable to analyses. We propose to purify a discrete population of hematovascular progenitor cells from differentiating mouse ES cells and compare the DNA methylation and RNA expression profiles with those of differentiated progenitors that do not possess a hematovascular potential. In this way, we can discriminate between DNA methylation patterns that are associated with differentiation in general and those that are specific to the hematopoietic lineage. We will use a blended high-throughput epigenetic/genic/bioinformatic platform to identify the initial sites of DNA methylation within a genome as it transitions from pluripotency to a committed state, couple this information with RNA expression analyses, and compare it with similar data obtained from cells of distinct developmental potential. Functional verification of hematovascular-specific methylated DNA regions that we identify will be performed in ES cell differentiation assays to examine the biological role of these regions in cell lineage commitment. By employing genomic tools, we hope to establish a basis for how cells codify their cell fate choices. An important aspect of our work is its possible application to cell replacement therapy for blood diseases. For example, long-term replacement of the hematopoietic system will necessitate transplantation of the most immature cells, such as those derived from hematovascular progenitor cells. Our work will generate biochemical quantities of highly purified populations of this important precursor, which will be used to define its epigenomic signature through DNA methylation and RNA expression profiling. We hope to apply this platform to unambiguously identify the precise epigenetic characteristics of any cell within a population that is to be evaluated for its therapeutic potential.