In vivo imaging of X inactivation

X 失活的体内成像

• 批准号: 8470606
• 负责人: John Greally
• 金额: $ 56.71万
• 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/8470606
• 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

首席研究员：GREALLY, J.M., LEVY, M. 项目摘要 X 失活的体内成像。 我们建议开发一种系统，用于 X 涉及的表观遗传调控过程的体内成像 染色体失活。 X 失活是表观遗传基因调控的一个经过充分研究的范例，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。已发现许多表观遗传调控过程有助于 失活过程，当使用免疫荧光对固定细胞进行成像时会产生信号 整个失活 X 的染色体区域。该信号的稳健性使得 X 失活成为 对于开发体内成像方法具有吸引力的系统。不活跃的 X 的特点是 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，由多梳族蛋白建立的修饰，当突变时， 与 X 失活失败有关。然而，还有其他监管调解者参与其中 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、基质附着区域 DNA 结合或与以下相关的功能 染色体结构维持基序。作为理解 X 的每个组成部分如何 失活系统在功能上相互作用，体内系统将允许观察顺序 将蛋白质介体和组蛋白修饰定位到失活的 X 染色体上，从而 在这个复杂的表观遗传过程中建立一个可能的调控层次。 为了开发这样的系统，需要汇集多个领域的专业知识。项目 从体外生成组蛋白肽（最终重建整个核小体）开始 甲基化和泛素化标记（David Allis 和 Tom Muir，洛克菲勒大学），然后用于 共同 PI Matthew Levy（爱因斯坦）进行体外选择，以创建特异性结合这些后处理的 RNA 适体 翻译修饰。然后这些适体在表达构建体中与结合的 RNA 发夹连接 通过荧光标记的噬菌体外壳蛋白，该系统由联合研究员罗伯特·辛格（爱因斯坦）首创 转录研究中追踪体内 RNA 的一种方法。该项目代表了相同技术的首次使用 表观遗传学研究系统。系统将被优化的细胞类型将是雌性小鼠 胚胎干细胞系，不仅可以进行 X 失活研究，还可以更广泛地使用该系统 当多能细胞提供给科学界时。 X失活研究将得到促进 通过开发 X 失活候选蛋白介体的荧光标签（Edith Heard， 居里研究所，巴黎，法国）。因此，该项目建立在强大且多方面的专业知识基础之上 和资源。 PHS 398/2590（修订版 11/07）继续格式页