Wednesday, April 2, 2025
4/2/2025
New Proximity Labeling Tools for Studying 3D Chromatin Structure and Function - 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.
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