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The spatiotemporal mapping of the RSC and SWI/SNF chromatin remodeler complexes on the nucleosome in living cells.

活细胞核小体上 RSC 和 SWI/SNF 染色质重塑复合物的时空图谱。

基本信息

批准号：
9377747
负责人：
Bryan Jason Wilkins
金额：
$ 36.77万
依托单位：
MANHATTAN COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9377747
关键词：
ATP phosphohydrolase Address Amino Acids Architecture Arts Behavior Binding Biochemical Biochemical Pathway Biological Biological Assay Biology Biomedical Engineering Cell Cycle Cell Nucleus Cells Chromatin Chromatin Fiber Chromosome Structures Chromosomes Complex Crosslinker DNA DNA Damage Data Disadvantaged Disease Environment Enzymes Event Fiber Foundations Future Gene Expression Genetic Genetic Code Grant Hereditary Disease Histones Human Genetics Immunoprecipitation Impairment In Vitro Individual Institutes Lead Learning Maintenance Mass Spectrum Analysis Methods Molecular Monitor Multiprotein Complexes Mutation Nuclear Nucleosomes Pathway interactions Phase Physiological Plant Roots Play Post-Translational Protein Processing Protein Family Proteins Public Health Recruitment Activity Regulation Research Research Training Resolution Role Site Stream Structural Protein Structure Students Surface Techniques Technology Time Training Translations United States National Institutes of Health Work Yeasts base chromatin protein chromatin remodeling chromosomal location cohesion college crosslink design developmental disease experience experimental study fitness in vivo insight molecular dynamics peer reconstitution repaired response spatiotemporal success synthetic biology temporal measurement undergraduate student

项目摘要

Chromatin remodeler (CR) complexes play crucial roles in regulating chromosomal architectural and act to modify nucleosomal DNA contacts by repositioning nucleosomes. The mechanistic details of the CR family of proteins are of importance because each remodeler complex contributes to unique chromatin structural maintenance. A mechanistic understanding of these proteins requires resolving how they interact and influence the chromosomal fiber. CRs are comprised of an ATPase active subunit and numerous auxiliary components that are involved in elaborate protein-chromatin stabilizing interactions. This makes it a challenging task to resolve completely their structures and how each subunit may interact individually with the nucleosome. To date, there remains no high-resolution structural data for a remodeler complex bound to the nucleosome. A disadvantage to remodeler studies is that they often rely on the reconstitution of nucleosomal arrays in solution that cannot recapitulate the true nuclear environment. Therefore, to enhance the understanding of CR behavior a technique is required that illuminates the molecular contacts that occur between the nucleosome and CR proteins inside the living nucleus. This proposal will define an approach to resolve the molecular dynamics of CR proteins revealing a nucleosomal-CR interactome in living yeast. This will be achieved using a method that allows for the covalent trapping of histone-protein interactions in the nucleus by employing site-specific, UV-inducible crosslinker amino acids that are genetically incorporated into the nucleosome. Spatial details will be achieved in two ways. First, specific placement of the crosslinker within a histone protein will provide insights into the nucleosomal surface contacts made by CR subunits. Second, when this approach is paired with chromosome immunoprecipitation (CHiP) technologies it will be possible to determine the chromosomal positioning of the crosslinking event, detailing the occupancy of the interaction along the chromatin fiber. Temporal resolution will be obtained with the aid of synchronous cells and UV-control of the crosslinking event. Time-resolved in vivo crosslinking with genetically encoded UV-crosslinkers is ideally suited to address many of the open questions in chromosome structure, composition and formation. Most critically it has the potential to reveal the network of interactions within chromosomal pathways and identify the sequence of events involved in those mechanisms. Particularly, this approach will help define how histone posttranslational modification events influence CR binding and dynamics at the nucleosomal surface. This work will be the first to institute the technique to assess structural and dynamic crosslink mapping of chromosomal remodeler complexes, in vivo. This work will bridge an extensive gap that spans in vitro versus in vivo experimentation of CRs and assign biologically relevant structure/function dynamics to these large intricate complexes. The results will greatly advance the chromatin biology field. Furthermore, CRs are medicinal targets for disease and developmental disorders highlighting their relevance to public health. 1

染色质重塑（CR）复合物在调节染色体结构中起着至关重要的作用，通过重新定位核小体来修饰核小体DNA接触。CR系列的机械细节蛋白质是重要的，因为每个重塑复合物有助于独特的染色质结构上维护对这些蛋白质的机械理解需要解决它们如何相互作用和影响染色体纤维CRs由ATP酶活性亚基和许多辅助成分组成参与了复杂的蛋白质-染色质稳定相互作用。这使其成为一项具有挑战性的任务，完全解析它们的结构以及每个亚基如何单独与核小体相互作用。到目前为止，仍然没有结合到核小体的重塑体复合物的高分辨率结构数据。一重塑研究的一个缺点是，它们通常依赖于在溶液中重建核小体阵列无法再现真实的核环境。因此，要提高对CR行为的认识，需要一种技术来阐明发生在核小体之间的分子接触，活细胞核内的CR蛋白。该提案将确定一种解决分子 CR蛋白的动力学揭示了活酵母中的核小体-CR相互作用组。完成这项工作的方法是使用一种方法，该方法允许通过使用共价捕获细胞核中的组蛋白-蛋白相互作用，位点特异性的、UV诱导的交联剂氨基酸，其被遗传地掺入核小体中。空间细节将通过两种方式实现。首先，交联剂在组蛋白内的特定位置将提供了深入了解由CR亚基的核小体表面接触。其次，当这种方法与染色体免疫沉淀（CHiP）技术配对，将有可能确定交联事件的染色体定位，详细说明沿着染色质的相互作用的占据情况光纤借助于同步电池和交联的UV控制，将获得时间分辨率活动使用遗传编码的UV交联剂的时间分辨体内交联理想地适合于解决许多关于染色体结构、组成和形成的悬而未决的问题。最关键的是，有可能揭示染色体通路内的相互作用网络并确定序列与这些机制有关的事件。特别是，这种方法将有助于确定组蛋白翻译后修饰事件影响CR在核小体表面的结合和动力学。这项工作将是第一个研究该技术，以评估染色体的结构和动态交联映射，重塑复合物，在体内。这项工作将弥合一个广泛的差距，跨越在体外与体内 CRs的实验，并将生物学相关的结构/功能动力学分配给这些复杂的配合物这一结果将极大地推动染色质生物学领域的发展。此外，CR是医学靶点强调其与公共卫生的相关性。 1