Global Tracing of Epigenetic Marks in Early C. elegans Embryos

早期线虫胚胎表观遗传标记的全局追踪

• 批准号: 8119616
• 负责人: Robert Riehn
• 金额: $ 17.88万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119616
• 关键词: Antibodies; Caenorhabditis elegans; Caliber; Cell Line; Cell division; Cells; Chromatin; Chromosomes; Comb animal structure; Complex; Coupled; Cytolysis; DNA; Detection; Development; Devices; Embryo; Embryonic Development; Epigenetic Process; Evolution; Fiber FISH; Figs - dietary; Fluorescence; Fluorescence Microscopy; Fluorescent Antibody Technique; Gene Expression Profile; Generations; Genes; Genome; Genomics; Histones; Image; In Situ Hybridization; Individual; Individuality; Label; Malignant Neoplasms; Maps; Measurement; Mechanics; Methods; Microfluidics; Mitotic; Mitotic Chromosome; Modification; Molecular; Monitor; Organ; Organism; Organogenesis; Pathway Analysis; Pattern; Preparation; Process; Proteome; Public Health; Regulatory Element; Resolution; Signal Transduction; Staging; Stretching; Structure; Surveys; System; Tail; Techniques; Testing; Tissues; Whole Organism; Work; base; cancer stem cell; design; embryo cell; epigenomics; genome-wide; histone modification; improved; micro-total analysis system; nanochannel; nanofluidic; novel; programs; public health relevance; tool

DESCRIPTION (provided by applicant): Epigenetic factors participate in the development and organogenesis in higher organisms. For the underlying process of differentiation, the emergence of epigenetic individuality of cells within the organism is required. Our application aims at developing a method for preparing global profiles of histone modification states over whole chromosomes, from single cells. The measurement of epigenetic individuality also becomes important in understanding organs undergoing rapid change, such as cancers that include cancer stem cells. The proposed method is conceptually similar to fiber-FISH. In particular, we plan on extracting chromosomes from a single, targeted cell. We then plan on visualizing the histone modification pattern by stretching whole chromosomes and fluorescent imaging of antibodies coupled to those histone tail modifications. In order to provide homogeneous stretching of ultra-long chromatin molecules, we will utilize a novel stretching technique based on nanofluidic channels. We have recently shown an elongation of 49 kbase chromatin molecules in such a channel to about 3 micrometers, leading to an equivalent expected resolution 6 kbase under fluorescence microscopy. The use of nanochannels enables stretching of ultra-long molecules without the problem of tension- based breaking such as in molecular combing. We propose to demonstrate the utility of our approach by following the global epigenetic state of early C. elegans embryos. Since C. elegans is an organism with an extremely early commitment of some cells to specific cell lines, we anticipate individuality of cells within such embryos. On the other hand, we will also be able to probe whether pluripotent cells are identical, or are also characterized by some individuality. All preliminary work will also be performed on the embryonic C. elegans system. Our application is structured into three distinct parts. Part one investigates whether whole chromosomes derived from mitotic cells can be stretched inside nanochannels. Part two is concerned with the design, development, and testing of a microfluidic platform that enables automated lysis of embryos and cells, and introduction into nanochannels. Part three will follow the epigenetic embryonic development from the 8 to the 28 cell stage. PUBLIC HEALTH RELEVANCE: This project has the potential to improve public health by developing a new technique for monitoring the epigenetic programming state of genomic sized molecules, ideally whole chromosomes, from single cells. Epigenetic programming has broad implications critical to both diseased and healthy states, in particular cancer development. This proposed work will enable tracing the epigenetic individuality of single cells from complex tissues.

描述（由申请人提供）： 表观遗传因子参与高等生物的发育和器官发生。对于分化的基本过程，需要有机体内细胞的表观遗传个体性的出现。我们的申请旨在开发一种方法，用于从单细胞制备整个染色体上的组蛋白修饰状态的全局配置文件。测量表观遗传个体性对于理解经历快速变化的器官也很重要，例如包括癌症干细胞的癌症。 所提出的方法在概念上类似于光纤FISH。特别是，我们计划从单个靶细胞中提取染色体。然后，我们计划通过拉伸整个染色体和与这些组蛋白尾部修饰偶联的抗体的荧光成像来可视化组蛋白修饰模式。 为了提供超长染色质分子的均匀拉伸，我们将利用基于纳米流体通道的新型拉伸技术。我们最近已经显示了49 kbase染色质分子在这样的通道中延伸到约3微米，导致荧光显微镜下的等效预期分辨率6 kbase。纳米通道的使用使得能够拉伸超长分子，而没有诸如在分子梳理中的基于张力的断裂的问题。 我们建议通过遵循早期C的全局表观遗传状态来证明我们的方法的实用性。线虫胚胎自从C.线虫是一种具有极早的特定细胞系的细胞的生物体，我们预期这些胚胎中的细胞具有个体性。另一方面，我们也将能够探测多能细胞是否是相同的，或者也具有某些个性。所有的前期工作也将在胚胎C上进行。elegans系统 我们的应用程序分为三个不同的部分。第一部分研究是否来自有丝分裂细胞的整个染色体可以在纳米通道内拉伸。第二部分涉及微流体平台的设计、开发和测试，该平台能够自动裂解胚胎和细胞，并引入纳米通道。第三部分将跟踪从8到28细胞阶段的表观遗传胚胎发育。 公共卫生相关性： 该项目有可能通过开发一种新技术来监测来自单细胞的基因组大小分子（理想情况下是整个染色体）的表观遗传编程状态，从而改善公共卫生。表观遗传编程具有广泛的影响，对疾病和健康状态都至关重要，特别是癌症的发展。这项拟议的工作将使跟踪表观遗传个体的单细胞从复杂的组织。