In vivo imaging of X inactivation

描述（由申请人提供）： 主要研究者：GREALLY，J.M.，莱维，M. X射线灭活的体内成像。 我们建议开发一个系统，在体内成像的表观遗传调控过程中涉及X染色体失活。X染色体失活是一个研究广泛的表观遗传基因调控范例，涉及雌性细胞中一条X染色体上大部分基因的沉默，这是哺乳动物剂量补偿过程的一部分。已经发现许多表观遗传调控过程有助于失活过程，当使用固定细胞上的免疫荧光成像时，在整个失活X染色体区域产生信号。该信号的鲁棒性使得X失活成为用于开发体内成像方法的有吸引力的系统。失活的X的特征在于存在抑制性翻译后组蛋白修饰，例如组蛋白H3赖氨酸9三甲基化（H3K9me3）和H3K27me3，由多梳组蛋白建立的修饰，当突变时，与X失活的失败相关。然而，还有其他调节介质与这些染色质状态的建立不太明显相关的功能有关，如解旋酶活性、RNA结合、基质附着区DNA结合或与染色体结构维持基序相关的功能。作为理解X失活系统的每个组分如何在功能上相互作用的一种手段，体内系统将允许观察蛋白质介质和组蛋白修饰对失活X染色体的顺序定位，从而在这个复杂的表观遗传过程中建立可能的调节层次。 为了建立这样一个系统，需要汇集若干领域的专门知识。该项目首先在体外产生具有甲基化和遍在蛋白化标记的组蛋白肽（并最终产生完整的重组核小体）（洛克菲勒大学的大卫·阿利斯和汤姆·缪尔），然后由共同研究者马修·利维（Matthew Levy）用于体外选择（爱因斯坦）创建与这些翻译后修饰特异性结合的RNA适体。然后，这些适体在表达构建体中连接到由荧光标记的噬菌体外壳蛋白结合的RNA发夹，这是由共同研究者Robert Singer（Einstein）开创的一种系统，作为在转录研究中体内跟踪RNA的手段。该项目代表了第一次使用相同的系统进行表观遗传学研究。该系统将被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，而且当提供给科学界时，该系统还可以在多能细胞中更广泛地使用。X失活研究将通过开发X失活的候选蛋白质介体的荧光标签来促进（Edith Heard，Institut Curie，巴黎，法国）。因此，该项目是建立在强大和多方面的专门知识和资源基础之上的。 公共卫生相关性： 主要研究者：GREALLY，J.M.，Levy，Matthew项目叙述X射线失活的体内成像。 在这个项目中，我们建议开发一个系统，使我们能够看到细胞核内对打开和关闭基因很重要的过程是如何物理相互作用的。我们正在使用一个戏剧性的基因调控的例子，女性细胞中X染色体之一的失活。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，失活的X是通过向该染色体内所含的蛋白质添加某些化学基团来标记的。目前尚不清楚的是，这些调节介质中的每一个是如何控制或被其他相关介质控制的。目前，我们需要杀死细胞来观察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剂发挥作用的顺序，从而了解这一过程中的调节机制。 我们建议将联合收割机与以前没有结合在一起的许多技术结合起来，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，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对科学界更广泛地有用，而不仅仅局限于X灭活的研究。我们还将使用一种涉及核酸结构的新技术，称为适体，选择它们与化学基团或感兴趣的蛋白质特异性结合的能力。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导者，其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.