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Characterization of Posttranslational Modifications on Histone Variants

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Book cover Histone Variants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1832))

Abstract

The study of histone variants and histone posttranslational modifications (PTMs) is a trending topic in different fields including developmental biology, neurobiology, and immunology; as well as in the understanding of molecular mechanisms leading to diverse diseases, such as cancer. Since the establishment of histone PTMs starts immediately after their synthesis and it continues once they are assembled into chromatin, here we describe a classic protocol of subcellular fractionation aiming to study histones at different stages of maturation. This includes newly synthesized histones enriched in cytosolic fractions; a pool of newly synthesized, evicted, and stored histones enriched in nuclear soluble fractions; and chromatin-associated histones enriched in chromatin pellet. To study specific histone variants and the establishment of their PTMs, we describe a protocol for obtaining histone variants expressed in bacteria. In addition, we describe a Triton-Acetic acid-Urea (TAU) gel electrophoresis protocol adapted to work on mini-gels, which can be coupled to Western blot to analyze PTMs on histone variants. Finally, we describe a Chromatin immunoprecipitation (ChIP) assay for studying histone PTMs, or tagged histone variants, on specific DNA sequences.

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Acknowledgments

We thank Francisca Alvarez for valuable advice on the TAU gel electrophoresis and ChIP protocols, Karla Veas for providing purified H3.1 and H3.3. F.S. is supported by a grant from Comisión Nacional de Ciencia y Tecnología (PCHA/Doctorado Nacional/2014-21140346). AL’s team is supported by grants from Comisión Nacional de Ciencia y Tecnología (FONDECYT 1160480 and Basal Project AFB 170004).

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Author notes

  1. Francisco Saavedra and Sebastián Marty-Lombardi contributed equally to this work.

Authors and Affiliations

  1. Fundación Ciencia & Vida, Santiago, Chile

    Francisco Saavedra, Sebastián Marty-Lombardi & Alejandra Loyola

Authors
  1. Francisco Saavedra
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  2. Sebastián Marty-Lombardi
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  3. Alejandra Loyola
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Corresponding author

Correspondence to Alejandra Loyola .

Editor information

Editors and Affiliations

  1. Institut Curie, PSL Research University CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France

    Guillermo A. Orsi

  2. Institut Curie, PSL Research University CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France

    Geneviève Almouzni

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Saavedra, F., Marty-Lombardi, S., Loyola, A. (2018). Characterization of Posttranslational Modifications on Histone Variants. In: Orsi, G., Almouzni, G. (eds) Histone Variants. Methods in Molecular Biology, vol 1832. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8663-7_2

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  • DOI: https://doi.org/10.1007/978-1-4939-8663-7_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8662-0

  • Online ISBN: 978-1-4939-8663-7

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