Dataset Information

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

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.

SUBMITTER: Fuse T

PROVIDER: S-EPMC5658119 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Fuse Tomohiro T Katsumata Koji K Morohoshi Koya K Mukai Yukio Y Ichikawa Yuichi Y Kurumizaka Hitoshi H Yanagida Akio A Urano Takeshi T Kato Hiroaki H Shimizu Mitsuhiro M

PloS one 20171026 10

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 c ...[more]

PMID: 29073207

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Project description:BackgroundTranscription is affected by nucleosomal resistance against polymerase passage. In turn, nucleosomal resistance is determined by DNA sequence, histone chaperones and remodeling enzymes. The contributions of these factors are widely debated: one recent title claims "… DNA-encoded nucleosome organization…" while another title states that "histone-DNA interactions are not the major determinant of nucleosome positions." These opposing conclusions were drawn from similar experiments analyzed by idealized methods. We attempt to resolve this controversy to reveal nucleosomal competency for transcription.Methodology/principal findingsTo this end, we analyzed 26 in vivo, nonlinked, and in vitro genome-wide nucleosome maps/replicates by new, rigorous methods. Individual H2A nucleosomes are reconstituted inaccurately by transcription, chaperones and remodeling enzymes. At gene centers, weakly positioned nucleosome arrays facilitate rapid histone eviction and remodeling, easing polymerase passage. Fuzzy positioning is not due to artefacts. At the regional level, transcriptional competency is strongly influenced by intrinsic histone-DNA affinities. This is confirmed by reproducing the high in vivo occupancy of translated regions and the low occupancy of intergenic regions in reconstitutions from purified DNA and histones. Regional level occupancy patterns are protected from invading histones by nucleosome excluding sequences and barrier nucleosomes at gene boundaries and within genes.Conclusions/significanceDense arrays of weakly positioned nucleosomes appear to be necessary for transcription. Weak positioning at exons facilitates temporary remodeling, polymerase passage and hence the competency for transcription. At regional levels, the DNA sequence plays a major role in determining these features but positions of individual nucleosomes are typically modified by transcription, chaperones and enzymes. This competency is reduced at intergenic regions by sequence features, barrier nucleosomes, and proteins, preventing accessibility regulation of untargeted genes. This combination of DNA- and protein-influenced positioning regulates DNA accessibility and competence for regulatory protein binding and transcription. Interactive nucleosome displays are offered at http://chromatin.unl.edu/cgi-bin/skyline.cgi.

Project description:Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells1-3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes3-6 and DNase I hypersensitive sites7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling8,9 that may be related to heterogeneity in chromatin accessibility10-12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.

Dataset Information

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

Publications

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

Similar Datasets

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