In vivo spatiotemporal mapping of genome-wide motions and gene-level transcriptional activity via integrated experimental platform and data-analytical pipeline

通过集成实验平台和数据分析管道对全基因组运动和基因水平转录活性进行体内时空绘图

• 批准号: 10663201
• 负责人: Alexandra Zidovska
• 金额: $ 32.4万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10663201
• 关键词: ATP phosphohydrolase; Address; Algorithms; Biochemical; Biochemistry; Biological; Biology; Cell Differentiation process; Cell Nucleus; Cells; Chromatin; Chromosome Mapping; Chromosome Territory; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Confocal Microscopy; Coupled; Coupling; Cytoskeleton; DNA Packaging; DNA Repair; DNA-Directed DNA Polymerase; Data Analytics; Data Collection; Data Set; Development; Diabetes Mellitus; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genome Mappings; Genomics; Goals; Heterogeneity; Histone H2B; Homeostasis; Human Genome; IL6 gene; Image; Individual; Interphase; Knowledge; Laws; Learning; Length; Link; Machine Learning; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Measures; Methods; Molecular Biology Techniques; Monitor; Motion; Movement; Mus; Nature; Neurons; Nuclear; Paint; Physics; Polymers; Process; Property; RNA Polymerase II; Research; Rheology; Role; Site; Spectrum Analysis; Structure; System; Technology; Time; Topoisomerase II; Transport Process; Visualization; developmental disease; embryonic stem cell; experimental study; genome-wide; genomic locus; in vivo; particle; physical science; segregation; self organization; spatiotemporal; stem cell differentiation; temporal measurement; tool; whole genome

Summary: The human genome is highly dynamic, yet the principles governing its movement are not known. Locally, chromatin undergoes constant remodeling and rearrangement associated with processes such as transcription, replication and DNA repair. At large length scales, chromatin dynamics is coherent over microns and seconds. How the local gene-level processes contribute to nucleus-wide chromatin motions remains an open question. To address this question, our overall approach is to map spatially and temporally resolved chromatin dynamics across the nucleus in mammalian cells in vivo, while connecting it with motion and transcriptional activity of specific genomic loci in real time. To do so, we will develop crosscutting tools integrating synergistically quantitative approaches derived from the physical sciences with the latest techniques from molecular biology and biochemistry. We will build an integrated experimental and analytical platform enabling simultaneous measurements of nucleus-wide and gene-specific motions in real time in vivo (Aim 1). Specifically, we will establish a data collection and analytical pipeline mapping chromatin motions across the nucleus in vivo using displacement correlation spectroscopy (DCS), while monitoring motions of genes visualized by CRISPR/dCas9 technology and tracked via new machine-learning assisted algorithms. In addition, our platform will monitor the spatiotemporal heterogeneity of chromatin across nucleus and toggle transcriptional activity of the tracked genes. Using this integrated platform, we will address the fundamental question of how gene-level transcription activity contributes to genome-wide motions (Aim 2). We will measure maps of chromatin motions and compaction across the whole genome, while simultaneously determining the local compaction and mobility of specific genes (MUC4, IL6) as a function of their transcriptional activity. Our findings will paint a new picture of the complexity and interconnectedness of gene- and genome-level dynamics and spatial heterogeneity. Finally, we will extend this approach to study interphase chromatin dynamics and compaction before and after cell differentiation of mouse embryonic stem cells (Aim 3). By linking gene-level activity to genome-wide compaction and motions, these results will have important implications for elucidating the role of chromatin dynamics in gene regulation and expression. Moreover, such knowledge will provide a framework for a mechanistic picture of chromatin dynamics in mammalian cells.

概括： 人类基因组是高度动态的，但控制其运动的原理尚不清楚。在当地， 染色质经历与以下过程相关的不断重塑和重排： 转录、复制和 DNA 修复。在大长度尺度上，染色质动力学在微米范围内是一致的 和秒。局部基因水平过程如何促进核范围染色质运动仍然是一个问题 开放式问题。为了解决这个问题，我们的总体方法是绘制空间和时间解析的地图 体内哺乳动物细胞核内的染色质动力学，同时将其与运动和 特定基因组位点的实时转录活性。为此，我们将开发横切工具 将源自物理科学的定量方法与最新的协同定量方法相结合 分子生物学和生物化学技术。我们将建立一个综合的实验和分析 能够在体内实时同时测量核范围和基因特异性运动的平台 （目标 1）。具体来说，我们将建立一个映射染色质运动的数据收集和分析管道 使用位移相关光谱（DCS）在体内穿过细胞核，同时监测 通过 CRISPR/dCas9 技术可视化基因，并通过新的机器学习辅助算法进行跟踪。在 此外，我们的平台将监测跨核和切换染色质的时空异质性 被追踪基因的转录活性。使用这个集成平台，我们将解决基本问题 基因水平转录活性如何促进全基因组运动的问题（目标 2）。我们将测量 整个基因组的染色质运动和压缩图，同时确定 特定基因（MUC4、IL6）的局部压缩和移动性作为其转录活性的函数。我们的 研究结果将描绘出基因和基因组水平的复杂性和相互关联性的新图景 动态和空间异质性。最后，我们将扩展这种方法来研究间期染色质 小鼠胚胎干细胞分化前后的动力学和压实（目标 3）。经过 将基因级活动与全基因组压缩和运动联系起来，这些结果将具有重要意义 阐明染色质动力学在基因调控和表达中的作用的意义。而且，这样的 这些知识将为哺乳动物细胞染色质动力学的机制图提供一个框架。