Robert K. McGinty

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.