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Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications

  • Nora Guidotti
  • Beat FierzEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Protein semisynthesis Native chemical ligation Expressed protein ligation Asymmetric nucleosomes Chromatin Epigenetics Histone modifications Bivalent domains 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.École Polytechnique Fédérale de Lausanne (EPFL)LausanneSwitzerland

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