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很重要，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。许多组织是由不同类型的细胞组成的，表面相同的细胞之间表观遗传状态的差异影响了它们的发育和病理潜力。我们建议开发能够可视化活动物复杂组织中单个细胞的表观遗传状态的技术。表观遗传状态包括分子标记和作用于这些标记的多蛋白复合体的组合。不同的表观遗传标记组合的结果由组合机制决定。表观遗传调节蛋白的功能由存在于这些蛋白形成的复合体中的相互作用伙伴的组合决定。我们建议开发能够可视化组合表观遗传标记、由表观遗传调控蛋白结合的染色质以及由动物中不同组合的相互作用伙伴形成的表观遗传调控复合体的技术。在动物体内的组合表观遗传标记和表观遗传调控蛋白复合体的可视化技术将基于荧光蛋白片段互补。当荧光蛋白的片段在分子上相互接近时，它们可以相互结合以产生荧光复合体。基于这一原理的技术非常适合于动物表观遗传状态的成像，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)高空间分辨率使亚核定位分析成为可能，以及(3)单个荧光蛋白片段的缺乏产生高信号背景比。 公共卫生相关性： 项目叙事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开启或关闭的基因。在这个项目中，我们开发的分子使研究人员能够看到活着的动物单个细胞中标签的组合。

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.