Visualization of Combinatorial Epigenetic Marks and Complexes in Animals Using Fl

描述（由申请人提供）： 摘要复杂组织中细胞的表观遗传状态的可视化是重要的，因为许多细胞类型不能在体外培养，而且细胞的表观遗传状态在分离和培养过程中会发生变化。许多组织由异质细胞类型组成，表面相同的细胞之间表观遗传状态的差异影响它们的发育和病理潜能。我们建议开发技术，使活动物复杂组织中单个细胞的表观遗传状态可视化。 表观遗传状态包括分子标记与作用于这些标记的多蛋白复合物的组合。表观遗传标记的不同组合的后果由组合机制决定。表观遗传调节蛋白的功能由这些蛋白形成的复合物中存在的相互作用伴侣的组合决定。我们建议开发的技术，使可视化的组合表观遗传标记，染色质结合的表观遗传调控蛋白，和表观遗传调控复合物形成的不同组合的相互作用伙伴在动物中。 动物中的组合表观遗传标记和表观遗传调节蛋白复合物的可视化技术将基于荧光蛋白片段互补。当荧光蛋白的片段以分子接近的方式彼此连接时，它们可以彼此缔合以产生荧光复合物。基于该原理的技术非常适合于对动物中的表观遗传状态进行成像，因为（1）直接荧光读出在动物组织中具有挑战性的成像条件下是稳健的，（2）高空间分辨率使得能够分析亚核定位，以及（3）单个荧光蛋白片段的荧光的缺乏产生高的信号与背景比。 公共卫生相关性： 项目叙述 动物细胞使用不可见标签的组合来标记在不同环境中生长的不同类型细胞中应该打开或关闭的基因。在这个项目中，我们开发的分子使研究人员能够看到活动物单个细胞中的标签组合。

DESCRIPTION (provided by applicant): Abstract Visualization of the epigenetic states of cells in complex tissues is important since many cell types cannot be cultured in vitro and since the epigenetic states of cells can change during isolation and culture. Many tissues are composed of heterogeneous cell types and differences in epigenetic states among superficially identical cells affect their developmental and pathological potentials. We propose to develop technologies that enable visualization of the epigenetic states of individual cells in complex tissues of living animals. Epigenetic states consist of combinations of molecular marks in conjunction with multiprotein complexes that act on those marks. The consequences of different combinations of epigenetic marks are determined by combinatorial mechanisms. The functions of epigenetic regulatory proteins are determined by the combinations of interaction partners that are present in complexes formed by these proteins. We propose to develop technologies that enable visualization of combinatorial epigenetic marks, chromatin binding by epigenetic regulatory proteins, and epigenetic regulatory complexes formed by different combinations of interaction partners in animals. The technologies for visualization of combinatorial epigenetic marks and of epigenetic regulatory protein complexes in animals are to be based on fluorescent protein fragment complementation. Fragments of fluorescent proteins can associate with each other to produce a fluorescent complex when they are tethered in molecular proximity to each other. Technologies based on this principle are well suited for imaging epigenetic states in animals because (1) the direct fluorescence read-out is robust under challenging imaging conditions in animal tissues, (2) the high spatial resolution enables analysis of subnuclear localization, and (3) the lack of fluorescence of the individual fluorescent protein fragments produces a high signal to background ratio. PUBLIC HEALTH RELEVANCE: Project Narrative Animal cells use combinations of invisible tags to label genes that should be on or off in different kinds of cells growing in different environments. In this project, we develop molecules that enable researchers to see the combinations of tags in individual cells in living animals.