MINFLUX nanoscopy links chromatin nanostructure to transcriptional state in living cells.

A central question in gene regulation is the relationship between the nanoscale organization of chromatin and transcriptional activity. However, directly visualizing and quantifying this process in living cells at nanometer resolution remains challenging. Here, we integrate live-cell MINFLUX nanoscopy with the DNA probe 5-HMSiR-Hoechst and a HaloTag system to map chromatin architecture at loci colabeled with RNA polymerase II (Pol II) and heterochromatin protein 1α (HP1α)-markers of distinct transcriptional states. High-resolution snapshots, captured via spatiotemporal segmentation of single-molecule localizations, reveal that HP1α loci exhibit densely packed chromatin with high DNA localization density, indicating a stable, rigid state. In contrast, Pol II loci are predominantly open and extended and exhibit low DNA localization density, implying a highly flexible and dynamic conformation. Critically, we establish a direct positive correlation between the degree of HP1α aggregation and the level of local chromatin compaction. Our work thus provides nanoscale insight into how transcriptional activity is physically encoded in living cell chromatin.