Global Tracing of Epigenetic Marks in Early C. elegans Embryos

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。对于潜在的分化过程，有机体内细胞的表观遗传个性的出现是必要的。我们的应用旨在开发一种方法，用于从单个细胞中制备整个染色体上的组蛋白修饰状态的全局轮廓。表观遗传个性的测量在理解经历快速变化的器官方面也变得重要，例如包括癌症干细胞在内的癌症。提出的方法在概念上类似于光纤FISH。特别是，我们计划从单个目标细胞中提取染色体。然后，我们计划通过拉伸整个染色体和结合到这些组蛋白尾部修饰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，我们将利用一种基于纳米流体通道的新拉伸技术。我们最近在这样的通道中显示了49KBase染色质分子的伸长到约3微米，导致在荧光显微镜下获得相当的预期分辨率6KBase。纳米通道的使用使超长分子得以拉伸，而不会出现分子梳理等基于张力的断裂问题。我们建议通过跟踪早期线虫胚胎的全球表观遗传状态来证明我们的方法的实用性。由于线虫是一种生物体，某些细胞极早地结合到特定的细胞系，我们预计这些胚胎中的细胞具有个性。另一方面，我们还将能够探索多能细胞是否相同，或者是否也具有某些个性。所有的前期工作也将在线虫胚胎系统上进行。我们的应用程序被组织成三个不同的部分。第一部分研究从有丝分裂细胞中衍生出来的整个染色体是否可以在纳米通道内伸展。第二部分涉及微流控平台的设计、开发和测试，该平台能够自动裂解胚胎和细胞，并将其引入纳米通道。第三部分将跟踪表观遗传学胚胎发育从8细胞期到28细胞期。 公共卫生相关性： 该项目有可能通过开发一种新的技术来监测基因组大小的分子的表观遗传编程状态，最好是从单个细胞中监测整个染色体，从而改善公众健康。表观遗传编程具有广泛的含义，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追踪复杂组织中单个细胞的表观遗传个性成为可能。

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.