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.
Highlights
We show that the positions of the nucleosomes were mapped more accurately by this gelbased parallel mapping, as compared to Micrococcal nuclease (MNase) mapping alone
Since MNase has endonuclease and exonuclease activities with sequence preferences [26,27,28,29,30], and the sensitivity to MNase is different among nucleosomes [7,8,9,10,46,47], mapping the positions of nucleosomes by MNase digestion is often difficult, depending on their locations in the genomes
The chemical mapping method requires the H4 S47C strain, in which the Ser47 residue in histone H4 is substituted with cysteine in Saccharomyces cerevisiae
Summary
We established a conventional gel-based method for nucleosome mapping in vivo, by indirect end-labeling and primer extension mapping using the H4 S47C-dependent sitedirected chemical cleavage, combined in parallel with MNase digestion. Using the isolated nuclei as the substrate for the chemical cleavage as well as the MNase digestion, followed by indirect end-labeling or primer extension mapping, we were able to map the linker DNA regions and the centers of nucleosomes.
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