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Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging

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Chromatin Immunoprecipitation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1689))

Abstract

Epigenetic complexes regulate chromatin dynamics via binding to and assembling on chromatin. However, the mechanisms of chromatin binding and assembly of epigenetic complexes within cells remain incompletely understood, partly due to technical challenges. Here, we present a new approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) that enables to assess the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was developed based on chromatin immunoprecipitation followed by single-molecule fluorescence microscopy imaging. In this method, an epigenetic complex subunit fused with a gene coding for a fluorescent protein is stably expressed in its corresponding knockout cells. Nucleosomes associated with epigenetic complexes are isolated from cells at native conditions and incubated with biotinylated antibodies. The resulting complexes are immobilized on a quartz slide that had been passivated and functionalized with NeutrAvidin. Image stacks are then acquired by using single-molecule TIRF microscopy. The individual spots imaged by TIRF microscopy represent single protein–nucleosome complexes. The number of copies of the protein complexes on a nucleosome is inferred from the fluorescence photobleaching measurements. Sm-ChIPi is a sensitive and direct method that can quantify the cellular assembly stoichiometry of epigenetic complexes on chromatin.

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Acknowledgment

This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to X.R.).

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Authors and Affiliations

  1. Department of Chemistry, University of Colorado Denver, Denver, CO, 80217-3364, USA

    Roubina Tatavosian

  2. Department of Chemistry, University of Colorado Denver, Renovated Science Building, Rm 3071, 1151 Arapahoe Street, Denver, CO, 80204, USA

    Xiaojun Ren

Authors
  1. Roubina Tatavosian
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  2. Xiaojun Ren
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Corresponding author

Correspondence to Xiaojun Ren .

Editor information

Editors and Affiliations

  1. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

    Neus Visa

  2. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

    Antonio Jordán-Pla

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Tatavosian, R., Ren, X. (2018). Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging. In: Visa, N., Jordán-Pla, A. (eds) Chromatin Immunoprecipitation. Methods in Molecular Biology, vol 1689. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7380-4_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7380-4_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7379-8

  • Online ISBN: 978-1-4939-7380-4

  • eBook Packages: Springer Protocols

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