Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo
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 be...
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| creator | Fuse, Tomohiro Katsumata, Koji Morohoshi, Koya Mukai, Yukio Ichikawa, Yuichi Kurumizaka, Hitoshi Yanagida, Akio Urano, Takeshi Kato, Hiroaki Shimizu, Mitsuhiro |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0186974 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0186974</identifier><identifier>PMID: 29073207</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Aldose-Ketose Isomerases - genetics ; Analysis ; Bias ; Binding sites ; Biochemistry ; Biology and Life Sciences ; Chemistry ; Chromatin ; Chromosome Mapping - methods ; Deoxyribonucleic acid ; DNA ; DNA sequencing ; DNA, Fungal - genetics ; Engineering ; Free radicals ; Fungi ; Gene expression ; Gene mapping ; Genetic aspects ; Genetic Loci - genetics ; Genomes ; Histone H4 ; Hydroxyl Radical - metabolism ; Hydroxyl radicals ; Laboratories ; Mapping ; Methods ; Micrococcal Nuclease - metabolism ; Mutation ; Nuclease ; Nucleases ; Nuclei ; Nucleosomes ; Nucleosomes - genetics ; Nucleosomes - metabolism ; Pharmacy ; Physical Sciences ; Physiological aspects ; Research and Analysis Methods ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - cytology ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae Proteins - genetics ; Science education</subject><ispartof>PloS one, 2017-10, Vol.12 (10), p.e0186974-e0186974</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Fuse et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Fuse et al 2017 Fuse et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c736t-5ecf5dda6c93ba46af173a53b0b43accc69b34d35cf6db93c4052e6e766f8bcf3</citedby><cites>FETCH-LOGICAL-c736t-5ecf5dda6c93ba46af173a53b0b43accc69b34d35cf6db93c4052e6e766f8bcf3</cites><orcidid>0000-0002-6939-2546</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658119/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658119/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,861,882,2096,2915,23847,27905,27906,53772,53774,79349,79350</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29073207$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Dalal, Yamini</contributor><creatorcontrib>Fuse, Tomohiro</creatorcontrib><creatorcontrib>Katsumata, Koji</creatorcontrib><creatorcontrib>Morohoshi, Koya</creatorcontrib><creatorcontrib>Mukai, Yukio</creatorcontrib><creatorcontrib>Ichikawa, Yuichi</creatorcontrib><creatorcontrib>Kurumizaka, Hitoshi</creatorcontrib><creatorcontrib>Yanagida, Akio</creatorcontrib><creatorcontrib>Urano, Takeshi</creatorcontrib><creatorcontrib>Kato, Hiroaki</creatorcontrib><creatorcontrib>Shimizu, Mitsuhiro</creatorcontrib><title>Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. 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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. 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>29073207</pmid><doi>10.1371/journal.pone.0186974</doi><tpages>e0186974</tpages><orcidid>https://orcid.org/0000-0002-6939-2546</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Aldose-Ketose Isomerases - genetics Analysis Bias Binding sites Biochemistry Biology and Life Sciences Chemistry Chromatin Chromosome Mapping - methods Deoxyribonucleic acid DNA DNA sequencing DNA, Fungal - genetics Engineering Free radicals Fungi Gene expression Gene mapping Genetic aspects Genetic Loci - genetics Genomes Histone H4 Hydroxyl Radical - metabolism Hydroxyl radicals Laboratories Mapping Methods Micrococcal Nuclease - metabolism Mutation Nuclease Nucleases Nuclei Nucleosomes Nucleosomes - genetics Nucleosomes - metabolism Pharmacy Physical Sciences Physiological aspects Research and Analysis Methods Saccharomyces cerevisiae Saccharomyces cerevisiae - cytology Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - genetics Science education |
| title | Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-18T04%3A13%3A26IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Parallel%20mapping%20with%20site-directed%20hydroxyl%20radicals%20and%20micrococcal%20nuclease%20reveals%20structural%20features%20of%20positioned%20nucleosomes%20in%20vivo&rft.jtitle=PloS%20one&rft.au=Fuse,%20Tomohiro&rft.date=2017-10-26&rft.volume=12&rft.issue=10&rft.spage=e0186974&rft.epage=e0186974&rft.pages=e0186974-e0186974&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0186974&rft_dat=%3Cgale_plos_%3EA511535391%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1956442056&rft_id=info:pmid/29073207&rft_galeid=A511535391&rft_doaj_id=oai_doaj_org_article_f5de810afb8b4edf90c387f40f733d1a&rfr_iscdi=true |