In vivo imaging of X inactivation

主要研究者：GREALLY，J.M.，莱维，M. 项目摘要 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的X 染色体失活X染色体失活是表观遗传基因调控的一个研究很好的范例，包括： 在雌性细胞中，一条X染色体上的大多数基因沉默，这是给药过程的一部分。 哺乳动物的补偿已经发现许多表观遗传调节过程有助于 灭活过程，当在固定细胞上使用免疫荧光成像时产生信号 在不活跃的X染色体的整个区域。该信号的鲁棒性使得X失活成为可能。 有吸引力的系统在体内成像方法的发展。不活跃的X的特征在于 存在抑制性翻译后组蛋白修饰，如组蛋白H3赖氨酸9三甲基化 （H3 K9 me 3）和H3 K27 me 3，由多梳组蛋白建立的修饰，当突变时， 与X染色体失活失败有关。然而，还有其他调节介质与 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结合或与 染色体结构维持基序。作为理解X的每个组成部分 由于灭活系统在功能上相互作用，体内系统将允许观察顺序的 定位的蛋白质介质和组蛋白修饰失活的X染色体，从而 在这个复杂的表观遗传过程中建立了一个可能的调节层次。 为了建立这样一个系统，需要汇集若干领域的专门知识。项目 开始于体外产生组蛋白肽（并最终产生整个重建的核小体）， 甲基化和泛素化标记（大卫阿利斯和汤姆缪尔，洛克菲勒大学），然后用于 通过co-PI Matthew Levy（Einstein）进行体外选择以产生特异性结合这些后- 翻译修饰然后将这些适体在表达构建体中连接到结合RNA发夹的载体上。 由荧光标记的噬菌体外壳蛋白，一个系统开创了共同研究者罗伯特辛格（爱因斯坦）， 一种在转录研究中体内追踪RNA的方法。该项目代表了第一次使用相同的 表观遗传学研究系统。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，而且更广泛地使用该系统， 多能性细胞的研究。将促进X灭活研究 通过开发X灭活的候选蛋白质介体的荧光标记（EdithHeard， 法国巴黎居里研究所）。因此，该项目是建立在强大和多方面的专业知识基础之上的 和资源 PHS 398/2590（Rev.11/07）

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. PHS 398/2590 (Rev. 11/07) Continuation Format Page