Devising New Technologies for Live Imaging of Epigenetic Processes

描述（由申请人提供）： 自30多年前核小体假说提出以来，大量的工作已经证明了染色质在基因调控中的关键作用。来自不同模型系统的体外和体内实验已经明确显示，共价组蛋白修饰提供了用于招募染色质修饰蛋白的信号，所述染色质修饰蛋白激活或沉默基因表达。高通量方法认识到这些共价标记具有不同的基因组分布，一些在启动子序列上富集，另一些标记增强子，而少数修饰我们基因组的转录非活性和重复部分。此外，遗传和药理学实验表明，这些修饰尽管被称为表观遗传，但似乎非常动态;应用它们的酶与消除它们的酶不断竞争。因此，每个细胞核甚至每个DNA位点在空间和时间上都采用不同的表观遗传状态。表观基因组的动态性质对理解发育和分化过程中最终导致基因表达或沉默的事件的确切顺序构成了重大挑战。认识到表观基因组对人类健康有着深远的影响，其干扰会导致疾病，克服这一挑战变得更加关键。因此，需要开发新的技术来监测活细胞中单细胞分辨率的表观基因组。在这里，我们建议使用强大的双分子荧光互补技术对哺乳动物表观基因组的实时成像。我们的实验旨在可视化不同组蛋白标记在分化细胞中的延伸和核分布，并将这些标记的位置与特异性标记的DNA位点相关联。此外，我们将使用一种新的成像系统，软X射线断层扫描，结合显着的空间分辨率与荧光显微镜的好处。通过这项技术，我们将获得在完全水合的哺乳动物细胞核中不同表观遗传结构域的结构洞察力。 这些创新方法的组合所产生的信息将允许更好地理解模式生物中的表观遗传调节，并将促进治疗性表观遗传化合物的开发。此外，这些实验将奠定基础的表观基因组分析的遗传难治性的人类，并将提供敏感的工具，为未来的诊断，甚至预后的疾病与表观遗传扰动。 公共卫生相关性： 人类细胞的表观遗传状态对该细胞的转录程序、细胞所属器官的正常功能以及人类的整体健康具有深远的影响。因此，我们提出的技术，这将允许在体内的表观遗传调节的实时成像，将有助于更好地理解表观遗传过程以及这些过程如何导致疾病，将允许开发新的治疗化合物，并将为“表观遗传”疾病提供强大的未来诊断工具。

DESCRIPTION (provided by applicant): Project Summary and Relevance Since the proposal of the nucleosome hypothesis, more than 30 years ago, an extensive body of work has demonstrated the critical role of chromatin in gene regulation. In vitro and in vivo experiments from different model systems have unequivocally shown that covalent histone modifications provide a signal for the recruitment of chromatin modifying proteins that activate or silence gene expression. High throughput approaches recognized that these covalent marks have different genomic distribution, some are enriched on promoter sequences, others mark enhancers, whereas a few decorate the transcriptionally inactive and repetitive portion of our genome. Moreover, genetic and pharmacological experiments showed that these modifications, despite being termed epigenetic, appear extremely dynamic; the enzymes that apply them are in constant competition with the enzymes that erase them. Therefore, each nucleus and even each DNA locus adopts distinct epigenetic states in space and time. The dynamic nature of the epigenome poses a significant challenge towards the understanding of the exact order of events that culminate in gene expression or silencing during development and differentiation. Overcoming this challenge becomes even more critical by the realization that the epigenome has a profound effect in human health and that perturbations thereof result in disease. Therefore, new technologies need to be developed for the monitoring of the epigenome in a single cell resolution in living cells. Here, we propose the use of the powerful bimolecular Fluorescence Complementation technology towards the live imaging of the mammalian epigenome. Our experiments aim to visualize the extend, and nuclear distribution of different histone marks in differentiating cells and to assign the position of these marks in relation to specifically labeled DNA loci. Moreover, we will use a novel imaging system, soft X-ray tomography that combines remarkable spatial resolution with the benefits of fluorescent microscopy. With this technology we will gain structural insight of different epigenetic domains in the fully hydrated mammalian nucleus. The information generated by the combination of these innovative approaches will allow a better understanding of epigenetic regulation in model organisms and will promote the development of therapeutic epigenetic compounds. Moreover, these experiments will lay the foundation of an epigenomic analysis of the genetically intractable human being and will provide sensitive tools for the future for the diagnosis or even prognosis of diseases linked to epigenetic perturbations. PUBLIC HEALTH RELEVANCE: Project Narrative The epigenetic state of a human cell has profound consequences in the transcriptional program of this cell, the proper function of the organ at which the cell belongs to, and the overall health of the human being. Therefore, our proposed technologies, which will allow for the live imaging of the epigenetic regulation in vivo, will assist the better understanding of epigenetic processes and how these result in disease, will allow the development of novel therapeutic compounds and will provide powerful future diagnostic tools for "epigenetic" diseases.