In vivo imaging of X inactivation

主要调查人员：Gally，J.M.，Levy，M. 项目摘要 X灭活的活体成像。 我们建议开发一个体内成像系统，用于对X基因涉及的表观遗传调控过程进行体内成像 染色体失活。X失活是表观遗传基因调控的一个研究很好的范例，涉及 雌性细胞中一条X染色体上的大多数基因沉默，这是剂量过程的一部分 哺乳动物的补偿。许多表观遗传调控过程已被发现有助于 失活过程，当使用固定细胞上的免疫荧光成像时，会产生一个信号 这一信号的健壮性使X失活成为一种 具有吸引力的系统，可用于体内成像方法的开发。不活跃的X的特征是 组蛋白翻译后抑制修饰的存在，如组蛋白H3赖氨酸9三甲基化 (H3K9me3)和H3K27me3，由多梳族蛋白建立的修饰，当突变时， 与X失活失败相关。然而，还有其他监管调解人与 与这些染色质状态的建立不太明显相关的功能，例如 解旋酶活性、RNA结合、基质附着区DNA结合或与以下相关的功能 染色体结构维护模体。作为一种了解X的每个组件如何 灭活系统在功能上相互作用，活体系统将允许观察序列 失活X染色体的蛋白质介体和组蛋白修饰的定位，从而 在这个复杂的表观遗传过程中建立一个可能的监管等级。 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 从组蛋白多肽(最终是整个重组核小体)的体外生成开始， 甲基化和泛素化标记(洛克菲勒大学的David Allis和Tom Muir)，然后用于 由共同的Pi Matthew Levy(爱因斯坦)在体外选择创建与这些POST-2特异结合的RNA适配子- 翻译修饰语。然后将这些适配子连接到与RNA发夹结合的表达结构中 通过荧光标记的噬菌体外壳蛋白，该系统由共同研究员罗伯特·辛格(爱因斯坦)开创， 在转录研究中跟踪体内核糖核酸的一种方法。这个项目代表了相同的第一次使用 表观遗传学研究系统。系统将进行优化的细胞类型将是雌性老鼠 胚胎干细胞株，不仅允许X失活研究，而且允许该系统在 当科学界获得多能细胞时。X失活研究将得到促进 通过开发用于X失活的候选蛋白质介体的荧光标签(Edith Head， 居里夫人学院，法国巴黎)。因此，该项目以强大和多方面的专业知识为基础。 和资源。 PHS 398/2590(11/07版)延续格式页面

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