Engineering and visualizing genome folding at high spatiotemporal resolution

摘要 在基因组生物学领域，关键的未回答的问题是染色质折叠形状的动力学如何 基因表达模式我们对染色质高阶三维折叠动力学的了解严重不足， 有限的，主要是由于缺乏技术，以精确的成像，工程师和监测循环， 以时空方式跨越细胞群体。在这里，我们建议通过以下方式解决这些限制： 开发工具，以同步方式动态改变细胞群中的染色质折叠， 以及单个细胞，并测量染色质循环及其与高空间转录的关系 单细胞的分辨率。在具体目标1中，我们将设计工具来控制循环动力学。我们将 修饰在不同水平折叠染色质的因子，如Ldb1和CTCF，通过融合到一个部分， 稳定性可以通过可扩散配体来控制。结合高分辨率5C和单分子 对这些工具进行成像，有望使人们对核结构之间的关系有一个基本的了解 和基因表达机制。在《特定目标2》中，我们计划设计光诱导系统， 循环动力学的精确控制。使用光活化的二聚化结构域， 结合设计的DNA结合蛋白，我们试图设计用于快速促进或 在不同尺度上破坏染色质循环。这项技术不仅可以在人群中进行研究， 也是在单细胞水平上。在具体目标3：我们将开发试剂来研究转录 在单细胞水平上与循环有关的动力学。我们将联合收割机RNA FISH与超分辨率 成像，以开发一种探索新生转录的空间和时间结构的方法， 高分辨率。结合高通量图像采集，我们将区分时间动态 通过测量转录物不同部分的相对强度来确定转录的强度。我们将 采用超分辨率成像（STORM）来测量转录位点的空间结构。这些 实验预计将揭示强迫染色质循环对不同阶段的影响， 转录周期并阐明转录爆发动力学与物理基因之间的关系 结构

Abstract Critical unanswered questions in the field of genome biology are how the dynamics of chromatin folding shape gene expression patterns. Our knowledge of the dynamics of higher-order 3-D folding of chromatin is severely limited, largely due to the lack of technologies to precisely image, engineer and monitor looping in a precise spatiotemporal manner across a population of cells. Here we propose to address these limitations by developing tools to dynamically alter chromatin folding in a synchronous manner across populations of cells as well as individual cells, and measure chromatin looping and its relationship to transcription at high spatial resolution in single cells. In Specific Aim 1 we will design tools to control looping dynamics. We will modify factors that fold chromatin at various levels, such as Ldb1 and CTCF by fusion to a moiety whose stability can be controlled by diffusible ligands. In combination with hi resolution 5C and single molecule imaging these tools are expected to generate fundamental insights into the relationship of nuclear architecture and gene expression mechanisms. In Specific Aim 2 we plan to engineer light-inducible systems for the precise control of looping dynamics. Using light activated dimerization domains that can be used in conjunction with designer DNA binding proteins we attempt to engineer factors used to rapidly promote or disrupt chromatin looping at various scales. This technology should enable studies not only in populations but also at the single cell level. In Specific Aim 3: we will develop reagents to study the transcriptional dynamics in relation to looping at the single cell level. We will combine RNA FISH with super-resolution imaging to develop a methodology for exploring the spatial and temporal structure of nascent transcription at high resolution. Combined with high-throughput image acquisition, we will discriminate the temporal dynamics of transcription by measuring the relative intensities arising from the different parts of the transcript. We will employ super-resolution imaging (STORM) to measure the spatial structure of transcription sites. These experiments are expected to reveal the impact of forced chromatin looping on distinct stages of the transcription cycle and elucidate the relationship between transcriptional burst kinetics and physical gene structure.