{"title":"A H2A的方法。核小体内Z/H2B二聚体的体外交换。","authors":"Benoit Guillemette, Liette Laflamme, Luc Gaudreau","doi":"10.1007/978-1-0716-4486-7_3","DOIUrl":null,"url":null,"abstract":"<p><p>H2A.Z is a highly conserved variant of H2A that is incorporated in genomic regulatory regions and contributes to control gene expression and genomic stability. H2A.Z variant exchange involves the removal of H2A-H2B dimers from a pre-assembled nucleosome and their replacement with H2A.Z-H2B dimers. A specific family of chromatin remodeling complexes, homologous to the yeast Swr1 complex, has been shown to be capable of this histone exchange activity both in vivo and in vitro. Here, we describe an assay to measure the histone exchange activity of recombinant human p400 expressed in insect Sf9 cells on immobilized mononucleosomes in vitro, using Flag-tagged H2A.Z/H2B dimers. Histone exchange is then measured with a simple Western blot assay. The assay can be adapted to other histone exchange complexes/catalytic subunits purified from any species.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2919 ","pages":"47-66"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A Method for H2A.Z/H2B Dimer Exchange Within Nucleosomes In Vitro.\",\"authors\":\"Benoit Guillemette, Liette Laflamme, Luc Gaudreau\",\"doi\":\"10.1007/978-1-0716-4486-7_3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>H2A.Z is a highly conserved variant of H2A that is incorporated in genomic regulatory regions and contributes to control gene expression and genomic stability. H2A.Z variant exchange involves the removal of H2A-H2B dimers from a pre-assembled nucleosome and their replacement with H2A.Z-H2B dimers. A specific family of chromatin remodeling complexes, homologous to the yeast Swr1 complex, has been shown to be capable of this histone exchange activity both in vivo and in vitro. Here, we describe an assay to measure the histone exchange activity of recombinant human p400 expressed in insect Sf9 cells on immobilized mononucleosomes in vitro, using Flag-tagged H2A.Z/H2B dimers. Histone exchange is then measured with a simple Western blot assay. The assay can be adapted to other histone exchange complexes/catalytic subunits purified from any species.</p>\",\"PeriodicalId\":18490,\"journal\":{\"name\":\"Methods in molecular biology\",\"volume\":\"2919 \",\"pages\":\"47-66\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2025-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Methods in molecular biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1007/978-1-0716-4486-7_3\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in molecular biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/978-1-0716-4486-7_3","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
A Method for H2A.Z/H2B Dimer Exchange Within Nucleosomes In Vitro.
H2A.Z is a highly conserved variant of H2A that is incorporated in genomic regulatory regions and contributes to control gene expression and genomic stability. H2A.Z variant exchange involves the removal of H2A-H2B dimers from a pre-assembled nucleosome and their replacement with H2A.Z-H2B dimers. A specific family of chromatin remodeling complexes, homologous to the yeast Swr1 complex, has been shown to be capable of this histone exchange activity both in vivo and in vitro. Here, we describe an assay to measure the histone exchange activity of recombinant human p400 expressed in insect Sf9 cells on immobilized mononucleosomes in vitro, using Flag-tagged H2A.Z/H2B dimers. Histone exchange is then measured with a simple Western blot assay. The assay can be adapted to other histone exchange complexes/catalytic subunits purified from any species.
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For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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