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，它们的动态是高度相关的。 在发育和疾病中都有关联。然而，尽管几十年来的惊人工作确定了 参与这些过程的分子参与者，并在全基因组范围内绘制它们的相互作用，我们目前正在 无法描述连接3D染色质结构和转录动力学的功能。这种限制 源于这样一个事实，即染色质结构和基因表达是从本质上随机的转变中出现的， 在单细胞水平，我们错过了与这些转变相关的关键时间参数。 因此，新的工具来衡量染色质结构和转录随着时间的推移在单细胞是至关重要的 用于理解人类基因组是如何被读取的以及用于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单细胞成像技术， 3D染色质结构的变化及其相关的基因表达动态在很大范围内， 时间尺度：从单个拓扑相关结构域和增强子-启动子相互作用的动力学， 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 循环），我们的新成像方法结合了荧光标记位点的实时超分辨率显微镜， 使用染色质结构的光学重建（ORCA）对基因座身份进行终点解复用， 跟踪和追踪功能染色质单位内的3-12个点。这项新技术，我们称之为活体ORCA 将使我们能够第一次测量整个拓扑相关域的时间动态， 单细胞我们将使用实时ORCA结合转录爆发的延时成像来研究 在整个细胞分化过程中以及在微扰下，启动子-增强子活性的动态 染色质网络对于较长的时间尺度（跨多个细胞周期），我们的方法将结合联合收割机的时间推移 基因表达的显微镜，监测两个标记的基因组位点之间的距离，作为一个活的报告者， 染色质结构，以及使用ORCA对整个基因邻域的终点染色质追踪。我们将 在两个系统中进行这些测量：在一个高度受控的合成报告，我们可以诱导 无论是短期沉默或长期表观遗传记忆，并在分化的时间点，当基因 在表观遗传学上形成一种新的转录状态此外，为了进一步研究 表观遗传，我们将开发一种新的微流体装置，使我们能够跟踪染色质的变化， 单个细胞谱系的3D结构。最后，为了测试我们的定量理解，我们将回过头来， 在这些单细胞数据和染色质动力学的理论模型之间。该研究计划将 大大推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， 与此同时，我们还提供了一整套新的成像技术和工程细胞系， 作为4D Nucleome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.