Combined SPT and FCS methods highlight a mechanism of RNAP II oversampling in cell nuclei

The orchestration of gene expression is a key question in both fundamental and applied research. Different models for transcription regulation were proposed, yet the dynamic regulation of RNA polymerase II (RNAP II) activity remains a matter of debate. To improve our knowledge of this topic, we investigated RNAP II motility in eukaryotic cells by combining Single Particle Tracking (SPT) and Fluorescence Correlation Spectroscopy (FCS) techniques, to take advantage of their different sensitivities. Thanks to calibrated samples, we first developed a benchmark for measurements and analysis to eliminate the main potential instrumental biases. We then applied this workflow to study the diffusion of RPB1, the catalytic subunit of RNA pol II. By a cross-analysis of FCS and SPT, we were able to highlight different RPB1 motility states and characterize them. We could distinguish between a very slow Brownian diffusion state, a stationary state, and two different modes of confined diffusion. Interestingly, our analysis also highlighted the oversampling by RPB1 of nuclear subdomains. We propose a novel model of highly dynamic spatio-temporal transcription regulation based on these results. Altogether, our results highlight the importance of combining microscopy approaches at different time scales to get a full insight into the real complexity of molecular diffusion distribution kinetics.