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

血管祖细胞中 C-G 表观遗传程序的基因组图谱

• 批准号: 7136410
• 负责人: Beverly Marie Emerson
• 金额: $ 44.85万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7136410
• 关键词: DNA; RNA; animal tissue; artificial chromosomes; bioinformatics; blood vessels; cell differentiation; chemical structure function; chromatin immunoprecipitation; computer simulation; cytosine nucleotides; developmental genetics; embryonic stem cell; epigenetics; gene expression; gene expression profiling; genetic regulation; genetic transcription; genetically modified animals; guanine nucleotides; immunofluorescence technique; intermolecular interaction; methylation; molecular site; pluripotent stem cells

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.

描述（由申请人提供）：该提案的重点是了解多能细胞如何在最早的分化阶段对特定的命运承诺。细胞身份取决于其基因组DNA的转录电位。这在很大程度上是通过对基因活性进行表观遗传编程的DNA和染色质的特定化学修饰来实现的。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为控制细胞专业化的程序来“硬系”基因组。由于这是在干细胞中最积极发生的，因此小鼠ES/EB系统对于在早期阶段捕获多能基因组的完整和有序的编程特别有价值，并且具有可用于分析的数量。我们建议净化血管血管祖细胞与分化小鼠ES细胞的离散群体，并将DNA甲基化和RNA表达谱与没有血管血管潜力的分化祖细胞的DNA甲基化和RNA表达谱进行比较。通过这种方式，我们可以区分与一般分化的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/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承诺状态，将这些信息与RNA表达分析相结合，并将其与获得的类似数据进行比较来自具有不同发育潜力的细胞。我们确定的血管血管特异性甲基化DNA区域的功能验证将在ES细胞分化测定中进行，以检查这些区域在细胞谱系投入中的生物学作用。通过采用基因组工具，我们希望为细胞如何整理其细胞命运选择的基础。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，长期替代造血系统将需要移植最不成熟的细胞，例如源自血管血管祖细胞的细胞。我们的工作将产生该重要先驱的高度纯化种群的生化量，该量将通过DNA甲基化和RNA表达分析来定义其表观遗传学特征。我们希望应用此平台明确识别人群中任何细胞的精确表观遗传特征，以评估其治疗潜力。