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

项目摘要 染色质结构和转录调控是细胞功能所必需的，它们的动态变化很大。 在发育和疾病方面都是相关的。然而，尽管几十年来令人惊叹的工作确定了 参与这些过程的分子玩家，并在全基因组范围内绘制它们的相互作用图，我们目前 无法描述连接3D染色质结构和转录动力学的功能。这一限制 源于染色质结构和基因表达来自内在的随机转变 在单细胞水平上，我们缺少与这些转变相关的关键时间参数。 因此，测量单个细胞染色质结构和随时间变化的转录的新工具至关重要。 以了解人类基因组是如何被读取的，并预测性地控制表观基因组。 在这里，我们建议开发一套新的活体单细胞成像技术来同时测量 大范围基因表达的3D染色质结构及其相关动力学的变化 时间尺度：来自单个拓扑相关结构域和增强子-启动子相互作用的动力学， 到与细胞周期中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 周期)，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和 基于染色质结构光学重建(ORCA)的基因座识别的端点分离 跟踪和追踪功能染色质单位内的3-12个点。这项新技术，我们称之为LIVE-ORCA， 将允许我们第一次测量整个拓扑相关区域的时间动力学 单细胞。我们将使用Live-ORCA结合转录爆发的延时成像来研究 启动子-增强子活性在细胞分化过程中的动态以及在 染色质网络。对于更长的时间尺度(跨多个细胞周期)，我们的方法将结合时间推移 基因表达显微镜，监测两个标记基因组座位之间的距离，作为活的报告 染色质结构，并使用ORCA对整个基因邻域进行末端染色质追踪。我们会 在两个系统中进行这些测量：在高度受控的合成报告器上，在那里我们可以诱导 要么是短期沉默，要么是长期的表观遗传记忆，以及在基因分化的时间点 表观遗传地致力于一种新的转录状态。此外，为了进一步研究其作用机制，本文还对其作用机制进行了研究。 表观遗传，我们将开发一种新的微流控设备，使我们能够跟踪染色质的变化 跨越单个细胞谱系的3D结构。最后，为了测试我们对数量的理解，我们将回顾并 第四，在这些单细胞数据和染色质动力学的理论模型之间。这项研究计划将 极大地促进了我们对染色质动力学及其在转录调控中的功能作用的理解，同时 同时，贡献了一套全新的新型成像技术和工程细胞系，将 作为4D核基因组和更广泛的科学界的跳板。

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.