The Yeast Minichromosome System Consisting of Highly Positioned Nucleosomes in Vivo.

In eukaryotic genomes, the nucleosome is the structural and functional unit, and its position and dynamics are important for gene expression control and epigenetic regulation. Epigenetics is an important mechanism in development and homeostasis, and aberrant epigenetics is a common feature in cancer. Although understanding the mechanistic basis that determines nucleosome positioning in vivo is important for elucidating chromatin function and epigenetic regulation, a suitable experimental system to examine such mechanisms is still being developed. Herein, we examined nucleosome organization in yeast minichromosomes, using a parallel mapping method we previously developed that involve site-directed chemical cleavage and micrococcal nuclease digestion. This parallel mapping is capable of revealing the differences in the occupancy and the stability of individual nucleosomes in the minichromosome. Based on the previously characterized minichromosome, we engineered a set of new minichromosomes, aimed at strengthening the positioning of the nucleosomes. The site-directed chemical mapping method demonstrated that the nucleosome positioning in the newly designed yeast minichromosome system was significantly more stable. This system will be useful for elucidating the determinants of nucleosome organization, such as DNA sequences and/or nucleosome binding proteins, and for determining the relationships between nucleosome dynamics and epigenetic regulation, which are targets for therapeutic agents.