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Purification of Yeast Native Reagents for the Analysis of Chromatin Function-I: Nucleosomes for Reconstitution and Manipulation of Histone Marks

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Histones

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1528))

Abstract

Purification of native biological material provides powerful tools for the functional analysis of enzymes and proteins in chromatin. In particular, histone proteins harbor numerous post-translational modifications, which may differ between species, tissues, and growth conditions and are lacking on recombinant histones. Moreover, the physiological substrate of most enzymes that modify histones is chromatin and the majority of these enzymes need to be part of a multiprotein assembly to be able to act on chromatin. For the yeast Saccharomyces cerevisiae different chromatin purification protocols are available but often result in poor yields or rely on genetic manipulation. We present a simple purification protocol that can yield up to 150 μg of pure native chromatin per liter of yeast culture. The purified material can be obtained from mutant cells lacking specific histone modifications and can be used in in vitro chromatin assembly for biochemical studies. Based on the extremely high degree of conservation throughout eukaryotes, this modifiable native chromatin can be used in studies with factors from other organisms including humans.

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Acknowledgments

We are grateful to Rhea Utley for significant corrections to this manuscript. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR; MOP-14308). N.L. was supported by a Canadian Government Foreign Affairs/International Trade studentship. J.C. holds the Canada Research Chair in Chromatin Biology and Molecular Epigenetics.

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Authors and Affiliations

  1. St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, CHU de Québec Research Center- Oncology axis, Hôtel-Dieu de Québec, 9 McMahon Street, Quebec City, QC, Canada, G1R 3S3

    Nicolas Lacoste, Wajid Bhat & Jacques Côté

  2. Institut Curie/CNRS, UMR3664, Paris, France

    Nicolas Lacoste

Authors
  1. Nicolas Lacoste
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  2. Wajid Bhat
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  3. Jacques Côté
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Correspondence to Jacques Côté .

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Editors and Affiliations

  1. Université de Sherbrooke Department of Biology, Sherbrooke, Québec, Canada

    Benoit Guillemette

  2. Department of Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Luc R. Gaudreau

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Lacoste, N., Bhat, W., Côté, J. (2017). Purification of Yeast Native Reagents for the Analysis of Chromatin Function-I: Nucleosomes for Reconstitution and Manipulation of Histone Marks. In: Guillemette, B., Gaudreau, L. (eds) Histones. Methods in Molecular Biology, vol 1528. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6630-1_3

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  • DOI: https://doi.org/10.1007/978-1-4939-6630-1_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6628-8

  • Online ISBN: 978-1-4939-6630-1

  • eBook Packages: Springer Protocols

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