Tracing Reversible Light-Induced Chromatin Binding with Near-infrared Fluorescent Proteins.

Blue light-induced chromatin recruitment (BLInCR) is a versatile optogenetic tool to enrich effector proteins at specific loci within the nucleus using illumination in the 400-500 nm range. The resulting chromatin binding reaction is reversible on the time scale of minutes. BLInCR is advantageous over ligand-binding induced methods since it does not require a change of growth medium for the relatively slow depletion of the inducer from the nucleus. However, applying BLInCR for reversibility experiments is challenging because of the need to spectrally separate light-induced activation from visualization of the chromatin locus and effector and/or reader proteins by light microscopy. Here, we describe an improved BLInCR protocol for light-dependent association and dissociation of effectors using the near-infrared fluorescent protein iRFP713. Due to its spectral properties, iRFP713 can be detected separately from the red fluorescent protein mCherry. Thus, it becomes possible to trace two proteins labeled with iRFP713 and mCherry independently of the light activation reaction. This approach largely facilitates applications of the BLInCR system for experiments that test the reversibility, persistence, and memory of chromatin states.