Live-cell multiplex super-resolution imaging of chromatin state transitions

染色质状态转变的活细胞多重超分辨率成像

• 批准号: 10264073
• 负责人: Lacramioara Bintu
• 金额: $ 102.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10264073
• 关键词: 3-Dimensional; Adopted; Algorithms; Amaze; Animals; Architecture; Back; Behavior; Biological Assay; Cell Cycle; Cell Differentiation process; Cell Fraction; Cell Line; Cell Lineage; Cell physiology; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; Color; Communities; Complex; DNA; Dancing; Development; Disease; Dose; ES Cell Line; Engineering; Enhancers; Epigenetic Process; Equilibrium; Evolution; Excision; Gene Activation; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Genome; Genomics; Health; Hi-C; Human; Human Genome; Image; Imaging technology; Individual; Knock-in; Label; Ligation; Link; MYC gene; Measurement; Measures; Mediating; Memory; Methods; Microfluidic Microchips; Microscopy; Modeling; Modification; Molecular; Mus; Nature; Neighborhoods; Neural Crest; Nuclear; Optics; Phase; Polycomb; Polymers; Population; Positioning Attribute; Process; Proteins; RNA; Reporter; Reporter Genes; Research; Resolution; Role; Shapes; Signal Transduction; Sister; Structure; System; Techniques; Technology; Testing; Theoretical model; Time; Transcript; Transcriptional Regulation; Work; cellular imaging; chromatin remodeling; cohesin; data modeling; embryonic stem cell; epigenetic memory; epigenome; experimental study; forgetting; genome-wide; genomic locus; human embryonic stem cell; imaging approach; innovation; long term memory; novel; novel imaging technology; particle; promoter; reconstruction; recruit; stem; three dimensional structure; tool

Project Summary Chromatin structure and transcription regulation are essential for cellular function, and their dynamics are highly correlated both in development and in disease. However, despite decades of amazing work identifying the molecular players involved in these processes, and mapping their interactions genome-wide, we are currently unable to describe the function connecting 3D chromatin structure and transcription dynamics. This limitation stems from the fact that chromatin structure and gene expression emerge from intrinsically stochastic transitions at the single-cell level, and we are missing the critical temporal parameters associated with these transitions. Therefore, new tools to measure both chromatin structure and transcription over time in single cells are critical for understanding how the human genome is read and for predictively controlling the epigenome. Here, we propose to develop a new set of live single-cell imaging technologies to simultaneously measure changes in 3D chromatin structures and their associated dynamics of gene expression across a large range of timescales: from dynamics of individual topologically associated domains and enhancer-promoter interactions, to changes associated with stable epigenetic memory across cell cycles. For the shorter timescales (under a cell cycle), our new imaging approach combines live super-resolution microscopy of fluorescently labeled loci with end-point demultiplexing of loci identity using Optical Reconstruction of Chromatin Architecture (ORCA), in order to track and trace 3-12 points within a functional chromatin unit. This new technique, which we call live-ORCA, will allow us to measure for the first time the temporal dynamics of an entire topologically associated domain in single cells. We will use live-ORCA in conjunction with time-lapse imaging of transcriptional bursting to study the dynamics of promoter-enhancer activity throughout cell differentiation and under perturbations of the chromatin network. For the longer timescale (across multiple cell cycles), our approach will combine time-lapse microscopy of gene expression, monitoring the distance between two tagged genomic loci as a live reporter of chromatin structure, and end-point chromatin tracing of the entire gene neighborhood using ORCA. We will perform these measurements in two systems: at a highly controlled synthetic reporter where we can induce either short-term silencing or long-term epigenetic memory, and at time points in differentiation when genes commit epigenetically to a new transcriptional state. Moreover, in order to further investigate the mechanism of epigenetic inheritance, we will develop a novel microfluidic device that allows us to track changes in chromatin 3D structures across individual cell lineages. Finally, to test our quantitative understanding, we will go back and forth between these single-cell data and theoretical modelling of chromatin dynamics. This research plan will greatly advance our understanding of chromatin dynamics and its functional role in transcription regulation, while at the same time contributing a whole new set of novel imaging technologies and engineered cell lines that will serve as a jumping board for the 4D Nucleome and broader scientific community.

项目摘要 染色质结构和转录调节对于细胞功能至关重要，它们的动力学高度 在发育和疾病中都相关。但是，尽管几十年来， 参与这些过程的分子参与者，并绘制其整个基因组的相互作用，我们目前是 无法描述连接3D染色质结构和转录动力学的功能。这个限制 源于以下事实：染色质结构和基因表达来自内在的随机转变 在单细胞级别，我们缺少与这些过渡相关的关键时间参数。 因此，在单个单元格中测量染色质结构和转录的新工具至关重要 为了了解人类基因组的读取和预测控制表观基因组。 在这里，我们建议开发一套新的实时单细胞成像技术，以同时测量 3D染色质结构的变化及其基因表达的相关动力学在大量范围内 时间表：从单个拓扑相关的域和增强子促销相互作用的动力学， 与跨细胞周期稳定的表观遗传记忆相关的变化。对于较短的时间尺度（在单元格下 循环），我们的新成像方法结合了荧光标记基因座的实时超分辨率显微镜与 使用染色质体系结构（ORCA）的光学重建的基因座身份的终点消除终点 在功能性染色质单元中跟踪和追踪3-12点。我们称之为Live-Orca的这项新技术 将允许我们首次测量整个拓扑相关域的时间动力学 单细胞。我们将使用Live-Orca与转录爆发的延时成像一起研究 整个细胞分化和在扰动下的启动子增强剂活性的动力学 染色质网络。对于更长的时间尺度（跨多个细胞周期），我们的方法将结合延时 基因表达的显微镜检查，监测两个标记的基因组基因座之间的距离 使用ORCA对整个基因邻域的染色质结构和终点染色质追踪。我们将 在两个系统中执行这些测量：在高度控制的合成报告基因中，我们可以在其中诱导 短期沉默或长期表观遗传记忆，以及在基因分化时的时间点 表观遗传在新的转录状态下提交。此外，为了进一步研究 表观遗传遗传，我们将开发一种新型的微流体设备，使我们能够跟踪染色质的变化 跨个别细胞谱系的3D结构。最后，为了测试我们的定量理解，我们将回去 在这些单细胞数据与染色质动力学的理论建模之间进行第四。该研究计划将 大大提高了我们对染色质动力学及其在转录调节中的功能作用的理解，而 同时，贡献了一套全新的新型成像技术和工程细胞系 作为4D核心和更广泛的科学界的跳跃板。