New Proximity Labeling Tools for Studying 3D Chromatin Structure and Function

项目概述将真核遗传物质作为染色质纤维在三个方面进行高度组织的包装 维度空间对于正确的基因表达和基因组维护至关重要。值得注意的是在拓扑上- 相关结构域(TADS)，包含由聚合体结合的绝缘染色质环区的邻域 粘附素和CCCTC结合因子(CTCF)同源二聚体。这些绝缘材料的完整性和维护 邻域很重要，因为环结构的破坏可能会导致基因错误调控和原生生物- 癌基因激活。然而，目前的生化工具还不够发达，无法研究问题。 关于邻里形成、调节和动态的机制。因此，我们需要分子工具 这使实时、高分辨率分析成为可能，而对细胞功能的干扰最小。 这项提议将结合分子生物学工具和光化学知识来开发 一种用于标记活细胞内分子相互作用组的光遗传学工具。一种可遗传编码的 光催化蛋白(SOPP)用于从添加了生物素的探针中产生活性中间体 转而，共价标记蛋白质定位径向距离内的生物分子相互作用。初步 实验核蛋白可以用较短的照射时间来标记，具有很高的时间特异性。我 预计这种方法将被应用于绘制其他细胞器的蛋白质相互作用图，使用 定量蛋白质组学数据作为对该方法的进一步验证。用于邻近标记的条件系统 利用Split-SOPP(SSOPP)将被开发用于邻近门控微环境测绘。最后，索普- 将合成并验证具有不同反应性和径向标记半径的可激活化学探针。 使用这种新的光遗传邻近标记工具，染色质的三维结构将是 利用核酸酶Dead Cas9(DCas9)-Sopp融合定位到基因组位置 RNA(SgRNAs)。SOPP-CTCF和SOPP-粘附素融合将用于绘制多位点相互作用图。这 方法应该提供对隔离区域内使基因参与的因素的动态的洞察。 此外，使用sSOPP方法进行邻近门控CTCF-粘附素标记将提供更准确的 环状区域互动图。最后，将使用dCas9-SOPP技术来研究 原癌基因的完整和破坏的相互作用组，包括环，尤其是与因子有关的环 参与基因调控。作为绘制分子间原子学图谱的一种更全面的方法，一个正交的 核酸标记方法将适用于这一工作流程，以创建一个能够映射的多路系统 在活细胞中，蛋白质和核酸染色质相邻的环相互作用。 拟议研究的成功完成将提供一种新的光遗传工具，将用于 通过更广泛的表观遗传学社区了解更高阶数的因果影响 基因调控中的染色体结构及其在癌症发生和发展中的作用。

PROJECT SUMMARY The highly-organized packing of eukaryotic genetic material as chromatin fibers in three- dimensional space is critical for proper gene expression and genome maintenance. Of note are topologically- associated domains (TADs) containing neighborhoods of insulated chromatin loop regions bound by clustered cohesin and CCCTC-binding factor (CTCF) homodimers. The integrity and maintenance of these insulated neighborhoods are important as disruptions to loop structure can result in gene misregulation and proto- oncogene activation. However, current biochemical tools are insufficiently developed to study questions concerning the mechanism of neighborhood formation, regulation and dynamics. Thus, we need molecular tools that enable real-time, high-resolution analysis with minimal pertubation on cellular function. This proposal will combine molecular biology tools with the knowledge of photochemistry to develop an optogenetic tool for labeling the molecular interactome within live cells. A genetically-encodable photocatalytic protein (SOPP) is used to generate reactive intermediates from a biotin-appended probe that in turn, covalently tag biomolecular interactions within radial distance of protein localization. Preliminary experiments nuclear proteins can be labeled with high temporal specificity using short irradiation times. I anticipate that this method will be applied towards mapping the protein interactomes of other organelles, using quantitative proteomic data as further confirmation of this method. Conditional systems for proximity labeling using split-SOPP (sSOPP) will be developed for proximity-gated microenvironment mapping. Finally, SOPP- activatable chemical probes with different reactivities and radial labeling radius will be synthesized and validated. Using this new optogenetic proximity labeling tool, the three-dimensional architecture of chromatin will be mapped using nuclease dead Cas9 (dCas9)-SOPP fusions guided to genomic sites using defined signal guide RNAs (sgRNAs). SOPP-CTCF and SOPP-cohesin fusions will be used to map multisite interactions. This approach should provide insight into the dynamics of factors that engage genes within insulated regions. Additionally, using the sSOPP approach for proximity-gated CTCF-cohesin labeling will provide a more accurate picture of loop region interactomics. Lastly, the dCas9-SOPP technology will be used to study the differences in the interactome of intact and disrupted proto-oncogene containing loops, especially with regard to the factors involved in gene regulation. As a more holistic approach to mapping molecular interatomics, an orthogonal nucleic acid labeling method will be adapted to this workflow to create a multiplexed system capable of mapping both protein and nucleic acid chromatin neighborhood loop interactomes in live cells. Successful completion of the proposed research will provide a new optogenetic tool that will be used by the broader epigenetics community to understand the causative impacts of higher-order chromosomal architecture on gene regulation, and their role in cancer development and progression.