Engineering and visualizing genome folding at high spatiotemporal resolution

 描述（由申请人提供）：在基因组生物学领域的关键未回答的问题是染色质折叠的动力学如何形成基因表达模式。我们对染色质高阶3-D折叠动力学的了解非常有限，这主要是由于缺乏以精确的时空方式在细胞群中精确成像、工程和监测循环的技术。在这里，我们建议通过开发工具来解决这些限制，以同步的方式动态改变染色质折叠的细胞群体以及单个细胞，并测量染色质循环及其在单细胞中以高空间分辨率转录的关系。具体目标1： 设计工具来控制循环动力学。我们将修改的因素，折叠染色质在不同的水平，如Ldb 1和CTCF融合到一个部分，其稳定性可以控制扩散配体。结合高分辨率5C和单分子成像，这些工具有望产生对核结构和基因表达机制关系的基本见解。在《特定目标2》中，我们计划设计光诱导系统来精确控制循环动力学。使用可以与设计的DNA结合蛋白结合使用的光激活二聚化结构域，我们试图设计用于快速促进或破坏各种尺度的染色质循环的因子。这项技术不仅可以在群体中进行研究，还可以在单细胞水平上进行研究。在具体目标3中：我们将开发试剂来研究在单细胞水平上与成环相关的转录动力学。我们将联合收割机RNA FISH与超分辨率成像相结合，开发一种高分辨率探索新生转录的时空结构的方法。结合高通量图像采集，我们将通过测量转录本不同部分的相对强度来区分转录的时间动态。我们将采用超分辨率成像（STORM）来测量转录位点的空间结构。这些实验有望揭示强迫染色质循环对转录周期不同阶段的影响，并阐明转录爆发动力学和物理基因结构之间的关系。

 DESCRIPTION (provided by applicant): 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 develp 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.