Single Molecule Analysis of CENP-A Chromatin by High-Speed Atomic Force Microscopy

Abstract

AbstractChromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated in the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze 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 dynamicsin vitroinform us about chromatin accessibilityin vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the MSD and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamicsin vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. Thus, changes which alter chromatin accessibilityin vitro, also correspondingly alter transcriptionin vivo. These data suggest a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance mobility.