Single molecule analysis of nucleosomes by high-speed atomic force microscopy

D.P. Melters, K.C. Neuman, Y. Dalal
National Institutes of Health,
United States

Keywords: high-speed AFM, chromatin, nucleosome, genes


Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for regulating gene regulation. At the single molecule level, how accessibility is controlled on the chromatin fiber is a fundamental question in the field. Here, we developed a single-molecule tracking method where we analyzed thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for nucleosomes. Furthermore, we found that an essential nucleosome binding protein reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how this nucleosome binding protein modulates centromeric chromatin dynamics in vivo. Overexpressing the nucleosome binding protein resulted in reduced centromeric transcription and impaired loading of new centromeric nucleosomes. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility, resulting in fine-tuning of regulation of chromatin accessibility.