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Here, we determine the positions of fully wrapped nucleosomes across the yeast genome by chemical mapping through the histone H2A-A122C residue, which cleaves near the DNA entry\/exit sites. This approach reveals the most refined sequence-dependent profile of nucleosomes reported to date. Comparisons of the H2A-A122C approach with the H3-Q85C and H4-S47C methods clearly show a sequence preference for the chemical cleavage site. Notably, depletion of CC and GG dinucleotides at nucleotide positions \u221211 to \u22129 and +9 to +11 bp, respectively, from the nucleosome dyad position (zero) is consistently accompanied by enrichment of AA\/AT\/TA\/TT dinucleotides in both yeast and mouse genomes. Introducing consecutive C\u2022G base pairs to the corresponding sites in the Widom 601 sequence makes reconstituted nucleosomes more likely to shift into alternative positions without affecting the thermal stability of the nucleosome particle, implying a structural constraint imposed by the DNA sequence. Thus, CC and GG dinucleotides in the major groove blocks, in which the minor groove faces out from the histone octamer, at superhelix locations (SHL) \u22121.0 and +1.0, respectively, destabilize histone\u2013DNA interactions, serving as intrinsic determinants of nucleosome positioning in eukaryotic genomes.<\/jats:p>","DOI":"10.1101\/gr.281372.125","type":"journal-article","created":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T19:15:18Z","timestamp":1782414918000},"source":"Crossref","is-referenced-by-count":0,"title":["Locations of consecutive G\u2022C base pairs direct genomic nucleosome positioning"],"prefix":"10.1101","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9591-1875","authenticated-orcid":false,"given":"Hiroaki","family":"Kato","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Shimane University School of Medicine, Izumo, Shimane 693-8501, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomohiro","family":"Fuse","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Graduate School of Science and Engineering, Meisei University, Hino, Tokyo 191-8506, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9926-2961","authenticated-orcid":false,"given":"Shoko","family":"Sato","sequence":"additional","affiliation":[{"name":"Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yohei","family":"Kurihara","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Graduate School of Science and Engineering, Meisei University, Hino, Tokyo 191-8506, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8801-6589","authenticated-orcid":false,"given":"Wataru","family":"Kagawa","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Graduate School of Science and Engineering, Meisei University, Hino, Tokyo 191-8506, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3383-3554","authenticated-orcid":false,"given":"Takeshi","family":"Urano","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Shimane University School of Medicine, Izumo, Shimane 693-8501, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6440-9954","authenticated-orcid":false,"given":"Yasuyuki","family":"Ohkawa","sequence":"additional","affiliation":[{"name":"Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Higashi, Fukuoka 812-0054, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7412-3722","authenticated-orcid":false,"given":"Hitoshi","family":"Kurumizaka","sequence":"additional","affiliation":[{"name":"Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6939-2546","authenticated-orcid":false,"given":"Mitsuhiro","family":"Shimizu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Graduate School of Science and Engineering, Meisei University, Hino, Tokyo 191-8506, Japan;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"246","published-online":{"date-parts":[[2026,6,25]]},"container-title":["Genome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/gr.281372.125","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T19:15:20Z","timestamp":1782414920000},"score":1,"resource":{"primary":{"URL":"http:\/\/genome.cshlp.org\/lookup\/doi\/10.1101\/gr.281372.125"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,25]]},"references-count":0,"alternative-id":["10.1101\/gr.281372.125"],"URL":"https:\/\/doi.org\/10.1101\/gr.281372.125","relation":{},"ISSN":["1088-9051","1549-5469"],"issn-type":[{"value":"1088-9051","type":"print"},{"value":"1549-5469","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,25]]}}}