In vivo imaging of X inactivation

X 失活的体内成像

• 批准号: 8267685
• 负责人: John Greally
• 金额: $ 59.63万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8267685
• 关键词: ATP phosphohydrolase; Aptamer Technology; Area; Bacteriophages; Binding; Biochemistry; Biological Assay; Biology; Capsid Proteins; Cell Nucleus; Cells; Chromatin; Chromosome Territory; Chromosomes; Communities; Complex; Cytosine; DNA; DNA Binding; Development; Dosage Compensation (Genetics); ES Cell Line; Embryonic Development; Epigenetic Process; Failure; Female; Foundations; Functional RNA; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Goals; Hearing; Heterogeneous-Nuclear Ribonucleoprotein U; Histone H3; Histones; Image; Imaging technology; Immunofluorescence Immunologic; In Vitro; Life; Link; Location; Lysine; Maintenance; Mammals; Matrix Attachment Regions; Mediator of activation protein; Methylation; Modification; Mus; Mutate; Nucleosomes; Paris, France; Pattern; Peptides; Polycomb; Positioning Attribute; Post-Translational Protein Processing; Principal Investigator; Process; Protein Binding; Proteins; RNA; RNA Binding; RNA Phages; Random Allocation; Reagent; Regulation; Relative (related person); Reporter; Reporting; Research Personnel; Resources; Signal Transduction; Structural Protein; Switch Genes; System; Systems Development; Technology; Transcript; Ubiquitination; Universities; X Chromosome; X Inactivation; abstracting; aptamer; base; cell fixing; cell killing; cell type; cellular imaging; chemical group; chromatin modification; embryonic stem cell; epigenomics; experience; genetic regulatory protein; helicase; histone modification; in vivo; insight; interest; meetings; new technology; nucleic acid structure; reconstitution; ubiquitin-protein ligase

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

主要研究人员：Greally，J.M。，Levy，M。 项目摘要 X灭活的体内成像。 我们建议开发一个用于体内成像的系统，以X的表观遗传调节过程 染色体灭活。 X失活是表观遗传基因调节的范式，涉及 在雌性细胞中一个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。已经发现许多表观遗传调节过程有助于 灭活过程，当使用固定细胞上的免疫荧光成像时，该过程会产生信号 在整个无活动X的染色体领域中 用于开发体内成像方法的有吸引力的系统。非活动X的特征是 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）的存在 （H3K9Me3）和H3K27ME3，由Polycomb组蛋白建立的修饰，当突变时，它们是 与X失活的失败有关。但是，还有其他监管调解员与 与这些染色质状态的建立不太明显相关的功能，诸如 解旋酶活性，RNA结合，基质跟踪区域DNA结合或与之相关的功能 染色体结构维护图案。作为理解x的每个组成部分的一种手段 灭活系统在功能上进行交互，体内系统将允许观察顺序 蛋白质介质的定位和组蛋白对灭活X染色体的修饰，因此 在这个复杂的表观遗传过程中建立可能的调节层次结构。 为了开发这样的系统，需要组装许多专业领域。项目 首先从体外生成组蛋白肽（最终是整个重构核小体）开始 然后将甲基化和泛素化标记（David Allis和Tom Muir，Rockefeller University）用于 Co-Pi Matthew Levy（Einstein）的体外选择，以创建与这些后这些后结合的RNA适体 翻译修改。然后将这些适体在表达构建体中链接到RNA发夹结合 通过荧光标记的噬菌体涂层蛋白，该系统由共同投资者Robert Singer（Einstein）开创为 在转录研究中跟踪RNA的一种手段。该项目代表了同一项目的首次使用 表观遗传研究系统。系统要优化的细胞类型将是女鼠 胚胎干细胞系，不仅允许X灭活研究，还允许该系统在 多能细胞提供给科学界。 X灭活研究将得到促进 通过开发X灭活的候选蛋白介质的荧光标签（Edith Heard， 法国巴黎的居里研究所）。因此，该项目基于强大而多方面的专业知识基础 和资源。 PHS 398/2590（Rev. 11/07）延续格式页面