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

描述（由申请人提供）：本提案的重点是了解多能细胞如何在分化的早期阶段承诺特定的命运。细胞身份是由其基因组DNA的转录潜力决定的。这在很大程度上是通过DNA和染色质的特定化学修饰实现的，这些修饰在表观遗传学上编程了基因活性。表观遗传编程的一个关键方面是DNA甲基化，它通过限制转录输出到控制细胞特化的程序来“硬连接”基因组。由于这种情况在干细胞中最活跃，因此小鼠ES/EB系统在捕获多能基因组早期阶段的完整有序编程和可用于分析的数量方面特别有价值。我们建议从分化的小鼠胚胎干细胞中纯化一个离散的血管祖细胞群体，并将其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甲基化模式和特定于造血谱系的DNA甲基化模式。我们将使用混合的高通量表观遗传学/遗传学/生物信息学平台来确定基因组中从多能性到承诺状态转变时DNA甲基化的初始位点，将这些信息与RNA表达分析相结合，并将其与从不同发育潜力的细胞中获得的类似数据进行比较。我们鉴定的血管特异性甲基化DNA区域的功能验证将在胚胎干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纂它们的细胞命运选择建立一个基础。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，长期替换造血系统将需要移植最不成熟的细胞，例如来自血管祖细胞的细胞。我们的工作将产生生化数量的这种重要前体的高度纯化群体，这将用于通过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.