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： 人类的基因组是高度动态的，但尚不清楚管理其运动的原则。在本地， 染色质经历与过程相关的恒定重塑和重排 转录，复制和DNA修复。在大长度上，染色质动力学在微米上是连贯的 和几秒钟。局部基因水平过程如何促进整个核染色质运动的促进 公开问题。为了解决这个问题，我们的整体方法是在空间和时间上绘制 在体内的哺乳动物细胞中跨核的染色质动力学，同时将其与运动连接起来 特定基因组基因局的转录活性实时。为此，我们将开发横切工具 整合从物理科学得出的协同定量方法与最新的 分子生物学和生物化学的技术。我们将建立一个集成的实验和分析 平台可以实时在体内实时同时测量整个核和基因特异性运动 （目标1）。具体而言，我们将建立数据收集和分析管道映射染色质运动 使用位移相关光谱法（DC）跨体内核心核，同时监测 由CRISPR/DCAS9技术可视化并通过新的机器学习辅助算法进行跟踪的基因。在 此外，我们的平台将监视染色质跨核和切换的染色质的时空异质性 追踪基因的转录活性。使用此集成平台，我们将解决基本 基因级转录活性如何促进全基因组运动的问题（AIM 2）。我们将衡量 整个基因组染色质运动和压实的地图，同时确定 特定基因（MUC4，IL6）的局部压实和迁移率是其转录活性的函数。我们的 调查结果将描绘出基因和基因组水平的复杂性和相互联系的新图片 动力学和空间异质性。最后，我们将扩展这种方法来研究间相染色质 小鼠胚胎干细胞的细胞分化之前和之后的动力学和压实（AIM 3）。经过 将基因级活性与全基因组压实和运动联系起来，这些结果将具有重要的 阐明染色质动力学在基因调节和表达中的作用的影响。而且，这样的 知识将为哺乳动物细胞中染色质动力学的机理图像提供一个框架。