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）配合物在调节染色体建筑和行动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家族的机械细节蛋白质很重要，因为每个重塑剂复合物都有助于独特的染色质结构维护。对这些蛋白质的机械理解需要解决它们的相互作用和影响染色体纤维。 CRS由ATPase活动亚基和许多辅助组成组成参与精心制作的蛋白质 - 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决它们的结构以及每个亚基如何与核小体单独相互作用。迄今为止，对于与核小体结合的重塑络合物复合物的高分辨率结构数据。一个对重塑研究的缺点是，它们通常依赖于溶液中核小体阵列的重构这不能概括真正的核环境。因此，增强对CR行为的理解需要一项技术来照亮核小体和之间发生的分子接触活核内的CR蛋白。该建议将定义一种解决分子的方法 Cr蛋白的动力学揭示了活酵母中核小体相互作用组。这将被实现使用一种方法，该方法允许通过采用使用组蛋白蛋白相互作用的共价捕获。位点特异性的，紫外线诱导的交联氨基酸，遗传掺入核小体中。空间详细信息将以两种方式实现。首先，交联链在组蛋白中的特定位置将提供有关CR亚基进行的核小体表面接触的见解。第二，当这种方法是与染色体免疫沉淀（CHIP）技术配对，可以确定交联事件的染色体定位，详细说明了沿染色质的相互作用的占用纤维。时间分辨率将借助同步细胞和交联的紫外线控制事件。与遗传编码的紫外线交联的体内交联的时间分辨非常适合解决染色体结构，组成和形成中的许多开放问题。最关键的是潜力揭示染色体途径中相互作用网络并识别序列这些机制所涉及的事件。特别是，这种方法将有助于定义组蛋白翻译后修饰事件会影响核小体表面的CR结合和动力学。这项工作将是第一个制定评估染色体结构和动态交联映射技术的技术重塑配合物，体内。这项工作将弥合广泛的差距，该差距跨越体内与体内 CRS的实验，并将与生物学相关的结构/功能动态分配给这些大错复合物。结果将大大推进染色质生物学领域。此外，CR是药物靶标对于疾病和发育障碍，强调了它们与公共卫生的相关性。 1