Department of Biochemistry and Biophysics, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Protein Sci. 2022 Jun;31(6):e4339. doi: 10.1002/pro.4339.
Quantitative analysis of chromatin protein-nucleosome interactions is essential to understand regulation of genome-templated processes. However, current methods to measure nucleosome interactions are limited by low throughput, low signal-to-noise, and/or the requirement for specialized instrumentation. Here, we report a Lanthanide Chelate Excite Time-Resolved Fluorescence Resonance Energy Transfer (LANCE TR-FRET) assay to efficiently quantify chromatin protein-nucleosome interactions. The system makes use of commercially available reagents, offers robust signal-to-noise with minimal sample requirements, uses a conventional fluorescence microplate reader, and can be adapted for high-throughput workflows. We determined the nucleosome-binding affinities of several chromatin proteins and complexes, which are consistent with measurements obtained through orthogonal biophysical methods. We also developed a TR-FRET competition assay for high-resolution footprinting of chromatin protein-nucleosome interactions. Finally, we set up a TR-FRET competition assay using the LANA peptide to quantitate nucleosome acidic patch binding. We applied this assay to establish a proof-of-principle for regulation of nucleosome acidic patch binding by methylation of chromatin protein arginine anchors. Overall, our TR-FRET assays allow facile, high-throughput quantification of chromatin interactions and are poised to complement mechanistic chromatin biochemistry, structural biology, and drug discovery programs.
定量分析染色质蛋白-核小体相互作用对于理解基因组模板过程的调控至关重要。然而，目前测量核小体相互作用的方法受到低通量、低信噪比和/或对专用仪器的要求的限制。在这里，我们报告了一种镧系螯合物激发时间分辨荧光共振能量转移（LANCE TR-FRET）测定法，用于高效定量染色质蛋白-核小体相互作用。该系统利用市售试剂，具有稳健的信噪比，最小的样品需求，使用常规荧光微孔板读数器，并可适应高通量工作流程。我们确定了几种染色质蛋白和复合物的核小体结合亲和力，这些亲和力与通过正交生物物理方法获得的测量结果一致。我们还开发了一种用于染色质蛋白-核小体相互作用的高分辨率足迹分析的 TR-FRET 竞争测定法。最后，我们使用 LANA 肽建立了一种用于定量核小体酸性斑结合的 TR-FRET 竞争测定法。我们应用该测定法建立了由染色质蛋白精氨酸锚定的甲基化调节核小体酸性斑结合的原理验证。总的来说，我们的 TR-FRET 测定法允许轻松、高通量地定量染色质相互作用，并为机制染色质生物化学、结构生物学和药物发现计划提供补充。
