Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo

Tomohiro Fuse, Koji Katsumata, Koya Morohoshi, Yukio Mukai, Yuichi Ichikawa, Hitoshi Kurumizaka, Akio Yanagida, Takeshi Urano, Hiroaki Kato, Mitsuhiro Shimizu

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    Abstract

    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.

    Original languageEnglish
    Article numbere0186974
    JournalPLoS One
    Volume12
    Issue number10
    DOIs
    Publication statusPublished - 2017 Oct 1

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    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)

    Cite this

    Fuse, T., Katsumata, K., Morohoshi, K., Mukai, Y., Ichikawa, Y., Kurumizaka, H., Yanagida, A., Urano, T., Kato, H., & Shimizu, M. (2017). Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo. PLoS One, 12(10), [e0186974]. https://doi.org/10.1371/journal.pone.0186974