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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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A Method for H2A.Z/H2B Dimer Exchange Within Nucleosomes In Vitro

  • 实验方案

  • 发表日期：

    2025年

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  • Benoit Guillemette,
  • Liette Laflamme,
  • Luc Gaudreau

摘要

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.