Genome-wide mechanisms and dynamics of ATP-dependent chromatin remodeling complexes

ATP依赖性染色质重塑复合物的全基因组机制和动力学

• 批准号: 10799754
• 负责人: Sandipan Brahma
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799754
• 关键词: ARID1A gene; Acute; Address; Affect; Automobile Driving; Binding; Biochemical; Bioinformatics; Biological Process; British Columbia; Catalytic Domain; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Complex; DNA; DNA Packaging; Data; Defect; Development; Disease; Enhancers; Environment; Family; Foundations; Fred Hutchinson Cancer Research Center; Frequencies; Future; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Heterogeneity; Histones; Human Genome; Individual; Kinetics; Knowledge; Label; Lead; Link; Malignant Neoplasms; Maps; Mentors; Modeling; Molecular; Molecular Machines; Mus; Mutation; Nucleosomes; Phase; Polycomb; Positioning Attribute; Process; Protein Complex Subunit; Protein Subunits; Proteins; Regulation; Regulator Genes; Regulatory Element; Repression; Research; Research Personnel; Residual state; Resolution; Role; SMARCA4 gene; SMARCB1 gene; SWI/SNF Family Complex; Saccharomycetales; Scientist; Site; Specificity; Structure; Supervision; Techniques; Testing; Time; Tissues; Training; Transcriptional Regulation; Universities; Variant; cell type; chromatin remodeling; chromosomal location; design; developmental disease; embryonic stem cell; epigenomics; exome sequencing; genome sequencing; genome-wide; in vivo; insight; molecular imaging; nervous system disorder; novel; novel strategies; polybromo; preference; promoter; recruit; single molecule; stem cell differentiation; stem cells; superresolution microscopy; targeted treatment; transcription factor

PROJECT SUMMARY/ABSTRACT The SWI/SNF family ATP-dependent chromatin remodelers are multi-subunit protein complexes that dynamically position chromosomal nucleosomes to modulate DNA accessibility, transcription-factor binding, and cell-type-specific gene expression. SWI/SNF function is crucial at several stages of mammalian development, and recent human whole-genome and exome sequencing studies revealed striking mutational frequencies in genes encoding SWI/SNF subunits across a range of diseases - from neurologic disorders to over 20% of all cancers. Disease-associated SWI/SNF mutations often cause the loss of a protein subunit that further destabilizes the complex and results in altered subunit composition and functions. Of note, heterogeneity in SWI/SNF subunit composition is also observed naturally within and across cell types, hypothesized to result in complex- and tissue-specific SWI/SNF functions. Despite their importance, how these subunits determine the substrate preference and mechanistic functions of SWI/SNF complexes are not well understood, largely due to the lack of robust approaches to characterize where they bind within the genome and how they interact with chromatin genome-wide. Thus, the main goal of this proposal is to address this key knowledge gap. Since chromatin remodelers directly interact with nucleosomes, it is important to clarify remodeler-bound nucleosome compositions and structures. Standard biochemical characterization of soluble components purified from cellular extracts cannot determine the nucleosome structures associated with chromatin-bound complexes. It is also important to understand the kinetic parameters of remodeler-chromatin interactions, such as how fast a remodeler is recruited to its target sites, and how long does it remain bound at its genomic sites. The candidate proposes to address these questions by using structural and functional epigenomics approaches and live-cell single-molecule imaging, to characterize remodeler-chromatin interactions in the context of the complex and dynamic chromatin environment inside cells. Aim 1 is to determine the genome-wide occupancy of SWI/SNF complexes with distinct subunit compositions. Aim 2 is to determine a) the structures and histone composition of remodeler-bound nucleosomes genome-wide, and b) the kinetic parameters of remodeler-chromatin interactions in live cells. Aim 3 is to study the interaction of SWI/SNF with repressive chromatin. The mentored phase of this project will be completed under the sponsorship of Dr. Steven Henikoff at the Fred Hutchinson Cancer Research Center. The candidate will acquire training in live-cell single-molecule imaging under the supervision of Dr. Sheila Teves at the University of British Columbia. The proposed research and training will provide a strong foundation for the candidate to develop as an independent investigator, studying chromatin remodeling mechanisms and dynamics in the regulation of fundamental cellular processes.

项目摘要/摘要 SWI/SNF家族ATP依赖性染色质重塑剂是多生产蛋白复合物， 动态定位染色体核小体以调节DNA可及性，转录因子结合， 和细胞型特异性基因表达。 SWI/SNF功能在哺乳动物的几个阶段至关重要 开发以及最近的人类全基因组和外显子组测序研究揭示了惊人的突变 从神经系统疾病到跨多种疾病的SWI/SNF亚基的基因频率 超过20％的癌症。与疾病相关的SWI/SNF突变通常会导致蛋白质亚基的流失 进一步破坏了复合物的稳定性，并导致亚基组成和功能改变。值得注意的是， 还观察到SWI/SNF亚基组合物中的异质性在细胞类型内和跨细胞类型中自然观察到 假设可以产生复杂的和组织特异性的SWI/SNF功能。尽管它们的重要性，但如何 亚基确定SWI/SNF复合物的底物偏好和机械功能不好 理解，很大程度上是由于缺乏强大的方法来表征它们在基因组中结合的位置 以及它们如何与染色质基因组相互作用。因此，该提议的主要目标是解决此键 知识差距。由于染色质重塑与核小体直接相互作用，因此澄清很重要 结合重塑的核小体组成和结构。可溶性的标准生化表征 从细胞提取物中纯化的成分无法确定与 染色质结合的复合物。了解重塑剂 - 染色质的动力学参数也很重要 互动，例如将重塑器招募到其目标站点的速度，以及它保持多长时间的约束 它的基因组部位。候选人建议通过使用结构和功能来解决这些问题 表观基因组学方法和活细胞单分子成像，以表征重塑剂 - 染色质 在细胞内部复杂而动态的染色质环境的背景下的相互作用。目标1是 确定具有不同亚基组成的SWI/SNF复合物的全基因组占用率。目标2是 确定a）重塑核小体基因组的结构和组蛋白组成，b） 活细胞中重塑 - 染色质相互作用的动力学参数。目标3是研究 swi/snf带有抑制性染色质。该项目的指导阶段将在 弗雷德·哈钦森癌症研究中心的史蒂文·亨科夫（Steven Henikoff）博士的赞助。候选人会 在大学的Sheila Teves博士的监督下，获得活细胞单分子成像的培训 不列颠哥伦比亚省。拟议的研究和培训将为候选人提供坚实的基础 发展为独立研究者，研究染色质重塑机制和动力学 基本细胞过程的调节。