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Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications

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Expressed Protein Ligation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2133))

Abstract

Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable “lnc-tag,” we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.

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Acknowledgments

We thank Dr. Carolin Lechner for having established some of the procedures described and Dr. Andreas Bachmann for the compounds used in the reactions. This work was supported by funding by the SystemsX program and EPFL.

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Authors and Affiliations

  1. École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Nora Guidotti & Beat Fierz

Authors
  1. Nora Guidotti
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  2. Beat Fierz
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Corresponding author

Correspondence to Beat Fierz .

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Editors and Affiliations

  1. ProteoDesign, Barcelona, Barcelona, Spain

    Miquel Vila-Perelló

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Guidotti, N., Fierz, B. (2020). Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications. In: Vila-Perelló, M. (eds) Expressed Protein Ligation. Methods in Molecular Biology, vol 2133. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0434-2_13

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  • DOI: https://doi.org/10.1007/978-1-0716-0434-2_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0433-5

  • Online ISBN: 978-1-0716-0434-2

  • eBook Packages: Springer Protocols

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