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

描述（由申请人提供）：该提案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。细胞身份由其基因组DNA的转录潜能决定。这在很大程度上是通过DNA和染色质的特定化学修饰来实现的，这些化学修饰在表观遗传学上对基因活性进行编程。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来“硬连接”基因组。因为这在干细胞中发生得最活跃，所以小鼠ES/EB系统对于捕获多能基因组在其早期阶段的完整和有序编程以及适于分析的数量特别有价值。我们建议从分化的小鼠ES细胞中纯化一个离散的血管祖细胞群体，并将DNA甲基化和RNA表达谱与不具有血管潜能的分化祖细胞进行比较。通过这种方式，我们可以区分与一般分化相关的DNA甲基化模式和造血谱系特异性的DNA甲基化模式。我们将使用混合的高通量表观遗传/基因/生物信息学平台来识别基因组中DNA甲基化的初始位点，因为它从多能性转变为定型状态，将此信息与RNA表达分析相结合，并将其与从具有不同发育潜力的细胞中获得的类似数据进行比较。我们将在ES细胞分化试验中对我们鉴定的血管特异性甲基化DNA区域进行功能验证，以检查这些区域在细胞谱系定型中的生物学作用。通过使用基因组工具，我们希望为细胞如何编码其细胞命运选择建立基础。我们工作的一个重要方面是它可能应用于血液疾病的细胞替代疗法。例如，造血系统的长期替代将需要移植最不成熟的细胞，例如来自血管祖细胞的那些细胞。我们的工作将产生生物化学量的高度纯化的群体的这一重要的前体，这将被用来定义其表观基因组签名通过DNA甲基化和RNA表达谱。我们希望应用这个平台来明确识别群体中任何细胞的精确表观遗传特征，以评估其治疗潜力。

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.