In vivo imaging of X inactivation

描述(由申请人提供)： 主要研究人员：Gally，J.M.，Levy，M.项目摘要X失活的活体成像。我们建议开发一个体内成像系统，用于X染色体失活涉及的表观遗传调控过程的体内成像。X失活是一种被广泛研究的表观遗传基因调控模式，它涉及雌性细胞中一条X染色体上的大多数基因沉默，这是哺乳动物剂量补偿过程的一部分。许多表观遗传调控过程被发现有助于失活过程，当使用免疫荧光对固定细胞进行成像时，该过程会产生一个遍及失活X染色体区域的信号。该信号的稳健性使X失活成为开发体内成像方法的一个有吸引力的系统。非活性X的特征是存在组蛋白翻译后抑制修饰，如组蛋白H3赖氨酸9三甲基化(H3K9me3)和H3K27me3，这些修饰由多梳状蛋白建立，当突变时，与X失活的失败有关。然而，还有其他与这些染色质状态的建立不太明显相关的功能，如解旋酶活性、RNA结合、基质附着区DNA结合等功能，或与染色体结构维持基序相关的功能。作为一种了解X失活系统的每个组件如何在功能上相互作用的手段，体内系统将允许观察失活X染色体上蛋白质介体和组蛋白修饰的顺序定位，从而在这一复杂的表观遗传过程中建立一个可能的调控层次。为了开发这样一个系统，需要汇集多个领域的专业知识。该项目首先在体外产生具有甲基化和泛素化标记的组蛋白多肽(最终是整个重组核小体)(洛克菲勒大学的David Allis和Tom Muir)，然后由共同的Pi Matthew Levy(爱因斯坦)用于体外选择，以创建与这些翻译后修饰特异结合的RNA适配子。然后，这些适配子在表达结构中连接到由荧光标记的噬菌体外壳蛋白结合的RNA发夹，这是一种由合作者罗伯特·辛格(爱因斯坦)首创的系统，作为在转录研究中跟踪体内RNA的一种手段。该项目是首次使用相同的系统进行表观遗传学研究。该系统将优化的细胞类型将是雌性小鼠胚胎干细胞系，不仅允许X失活研究，而且当该系统提供给科学界时，还可以在多潜能细胞中更广泛地使用该系统。X失活的研究将通过开发X失活的候选蛋白质介体的荧光标签来促进(伊迪丝·赫德，居里研究所，法国，巴黎)。因此，该项目建立在强大和多方面的专门知识和资源基础之上。 公共卫生相关性： 主要研究人员：Gally，J.M.，Levy，Matthew Project，X失活的活体成像。在这个项目中，我们建议开发一个系统，使我们能够在细胞核内看到对打开和关闭基因至关重要的过程是如何在物理上相互作用的。我们正在使用一个戏剧性的基因调控的例子，女性细胞中的一条X染色体的失活。我们已经知道，有许多蛋白质与X染色体的失活有关，我们认识到，不活跃的X染色体的标志是在该染色体内的蛋白质中添加了某些化学基团。不明显的是，这些监管调解人中的每一个如何控制或被参与其中的其他人控制。目前，我们需要杀死细胞来了解这些调节介质在细胞内的位置，但如果我们能够在活细胞中观察它们，我们就可以确定这些调节因子发挥作用的顺序，从而获得这一过程中调节机制的指示。我们建议将一些以前没有结合在一起的技术结合在一起，以便能够观察这些调节器如何在活细胞中相互作用。我们将使用小鼠胚胎干细胞，这是一种可以转化为体内大多数细胞类型的细胞类型，因此我们开发的资源将更普遍地对科学界有用，而不会局限于X失活的研究。我们还将使用一种名为适配子的涉及核酸结构的新技术，根据它们与感兴趣的化学基团或蛋白质的结合能力进行选择。参与的研究人员代表了X染色体失活、染色质生物学、活细胞成像和适体技术领域的领先者，他们的目标不仅是深入了解X染色体失活，而且还开发可供更广泛的科学界使用的资源。

DESCRIPTION (provided by applicant): Principal Investigators: GREALLY, J.M., LEVY, M. Project abstract IN VIVO IMAGING OF X INACTIVATION. We propose to develop a system for in vivo imaging of the epigenetic regulatory processes involved in X chromosome inactivation. X inactivation is a well-studied paradigm of epigenetic gene regulation, involving the silencing of the majority of the genes on one X chromosome in female cells, part of the process of dosage compensation in mammals. A number of epigenetic regulatory processes have been found to contribute to the inactivation process, which when imaged using immunofluorescence on fixed cells generate a signal throughout the chromosome territory of the inactive X. The robustness of this signal makes X inactivation an attractive system for the development of in vivo imaging approaches. The inactive X is characterized by the presence of repressive post-translational histone modifications such as histone H3 lysine 9 trimethylation (H3K9me3) and H3K27me3, modifications established by polycomb group proteins which, when mutated, are associated with the failure of X inactivation. There are, however, other regulatory mediators implicated with functions that are less obviously related to the establishment of these chromatin states, functions such as helicase activity, RNA-binding, matrix-attachment region DNA-binding, or those functions associated with chromosomal structural maintenance motifs. As a means of understanding how each component of the X inactivation system interacts functionally, an in vivo system would allow the observation of sequential localization of the protein mediators and histone modifications to the inactivating X chromosome, thus establishing a likely hierarchy of regulation in this complex epigenetic process. In order to develop such a system, a number of areas of expertise need to be assembled. The project starts with the in vitro generation of histone peptides (and eventually entire reconstituted nucleosomes) with methylation and ubiquitination marks (David Allis and Tom Muir, Rockefeller University) that are then used for in vitro selection by co-PI Matthew Levy (Einstein) to create RNA aptamers specifically binding to these post- translational modifications. These aptamers are then linked in an expression construct to RNA hairpins bound by fluorescently-tagged phage coat proteins, a system pioneered by co-investigator Robert Singer (Einstein) as a means of tracking RNA in vivo in transcription studies. This project represents the first use of the same system for epigenetic studies. The cell type in which the system will be optimized will be a female mouse embryonic stem cell line, allowing not only X inactivation studies but also the broader use of this system in pluripotent cells when made available to the scientific community. The X inactivation studies will be facilitated by the development of fluorescent tags for the candidate protein mediators of X inactivation (Edith Heard, Institut Curie, Paris, France). The project is thus based on a strong and multifaceted foundation of expertise and resources. PUBLIC HEALTH RELEVANCE: Principal Investigators: GREALLY, J.M., LEVY, Matthew Project narrative IN VIVO IMAGING OF X INACTIVATION. In this project, we propose to develop a system that will allow us to see within the cell nucleus how processes that are important for switching genes on and off are physically interacting. We are using a dramatic example of gene regulation, the inactivation of one of the X chromosomes in female cells. We already know that there are numerous proteins that are involved with inactivation of the X chromosome, and we recognize that the inactive X is marked by the addition of certain chemical groups to the proteins contained within that chromosome. What is not apparent is how each of these regulatory mediators controls or is controlled by the others involved. At present, we need to kill cells to see where these regulatory mediators are located within the cell, but if we were able to watch them in living cells we could determine the order in which these regulators exert their effects, thus getting an indication of the regulatory mechanism in this process. We propose to combine a number of technologies that have not been brought together previously in order to be able to watch how these regulators interact in the living cell. We will use mouse embryonic stem cells, a cell type that can turn into most cell types in the body, so the resources we develop will be more generally useful to the scientific community and will not be restricted to the study of X inactivation. We will also use a new technology involving nucleic acid structures called aptamers, selecting them for their ability to bind specifically to the chemical groups or the proteins of interest. The investigators involved represent leaders in the fields of X chromosome inactivation, chromatin biology, live cell imaging and aptamer technology, and the goal is not only to gain insights into X chromosome inactivation but also to develop resources that can be used by the broader scientific community.