Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. However, due to its sequence preference, the interpretations of the positions and occupancies of nucleosomes using MNase have remained controversial. Next-generation sequencing (NGS) has also been utilized for analyses of MNase-digests, but some technical biases are commonly present in the NGS experiments. Here, we established a gel-based method to map nucleosome positions in Saccharomyces cerevisiae, using isolated nuclei as the substrate for the histone H4 S47C-site-directed chemical cleavage in parallel with MNase digestion. The parallel mapping allowed us to compare the chemically and enzymatically cleaved sites by indirect end-labeling and primer extension mapping, and thus we could determine the nucleosome positions and the sizes of the nucleosome-free regions (or nucleosome-depleted regions) more accurately, as compared to nucleosome mapping by MNase alone. The analysis also revealed that the structural features of the nucleosomes flanked by the nucleosome-free region were different from those within regularly arrayed nucleosomes, showing that the structures and dynamics of individual nucleosomes strongly depend on their locations. Moreover, we demonstrated that the parallel mapping results were generally consistent with the previous genome-wide chemical mapping and MNase-Seq results. Thus, the gel-based parallel mapping will be useful for the analysis of a specific locus under various conditions.

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.

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