Using single-molecule approach to visualize the nucleosome assembly in yeast nucleoplasmic extracts

In eukaryotic cells, the smallest subunit of chromatin is the nucleosome, which consists of a segment of DNA wound on histone protein cores. Despite many years of effort, the process of nucleosome assembly and disassembly is still not very clear. Here, we present a convenient method to investigate the process of nucleosome assembly at the single molecule level. We invented a novel system derived from the yeast nucleoplasmic extracts (YNPE), and demonstrated that the YNPE supports the nucleosome assembly under physiological condition. By combining the total internal reflection fluorescence microscopy with microfluidic flow-cell technique, the dynamic process of nucleosome assembly in YNPE was visualized at single-molecule level. Our system provides a novel in vitro single-molecule tool to investigate the dynamics of nucleosome assembly under physiological conditions.