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

真核生物遗传物质作为染色质纤维的高度组织化包装在三个细胞中， 三维空间对于正确的基因表达和基因组维持是至关重要的。在拓扑学上- 相关结构域（TADs），包含由成簇的 粘附素和CCCTC结合因子（CTCF）同源二聚体。这些绝缘的完整性和维护 邻近区域是重要的，因为环结构的破坏可导致基因失调和原核表达。 癌基因激活然而，目前的生物化学工具还不足以研究问题， 关于邻里关系形成、调节和动态的机制。因此，我们需要分子工具 这使得能够进行实时、高分辨率分析，同时对细胞功能的干扰最小。 这项建议将联合收割机分子生物学工具与光化学知识相结合， 用于标记活细胞内的分子相互作用组的光遗传学工具。一种基因编码的 光催化蛋白（SOPP）用于从生物素附加的探针产生活性中间体， 转向，共价标记蛋白定位的径向距离内的生物分子相互作用。初步 实验核蛋白可以使用短的照射时间以高的时间特异性标记。我 预计这种方法将应用于绘制其他细胞器的蛋白质相互作用组，使用 定量蛋白质组学数据作为该方法的进一步确认。用于邻近标记的条件系统 使用分裂SOPP（sSOPP）将被开发用于邻近门控微环境映射。最后，SOPP- 将合成并验证具有不同反应性和径向标记半径的可活化化学探针。 使用这种新的光遗传学邻近标记工具，染色质的三维结构将被 使用核酸酶死亡Cas9（dCas 9）-SOPP融合物作图，所述融合物使用限定的信号引导物引导至基因组位点 RNA（sgRNA）。SOPP-CTCF和SOPP-粘附蛋白融合体将用于绘制多位点相互作用。这 这种方法应该提供深入了解的因素，从事基因在绝缘区域内的动态。 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CTCF-粘附素标记。 loop region interactomics的图片最后，dCas 9-SOPP技术将用于研究 完整和破坏的原癌基因环的相互作用组，特别是关于因子 参与基因调控。作为一种更全面的方法来映射分子原子间，正交 核酸标记方法将适合于该工作流程，以创建能够映射 活细胞中的蛋白质和核酸染色质邻近环相互作用组。 拟议研究的成功完成将提供一种新的光遗传学工具， 更广泛的表观遗传学社区，以了解更高层次的因果影响， 染色体结构对基因调控的影响，以及它们在癌症发展和进展中的作用。

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.