1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression
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VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

  1. Chitra Mohan
  2. Lisa M Kim
  3. Nicole Hollar
  4. Tailai Li
  5. Eric Paulissen
  6. Cheuk T Leung  Is a corresponding author
  7. Ed Luk  Is a corresponding author
  1. Stony Brook University, United States
  2. University of Minnesota Medical School, United States
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Cite this article as: eLife 2018;7:e36654 doi: 10.7554/eLife.36654

Abstract

VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate VivosX. Disulfide crosslinks between cysteine-modified H2A.Z and/or H2A histones within nucleosomes were induced using a membrane-permeable oxidant. VivosX detected different combinations of H2A.Z and H2A within nucleosomes in yeast cells. This assay correctly reported the change in global H2A.Z occupancy previously observed when the deposition and eviction pathways of H2A.Z were perturbed. Homotypic H2A.Z/H2A.Z (ZZ) nucleosomes accumulated when assembly of the transcription preinitiation complex was blocked, revealing that the transcription machinery preferentially disassembles ZZ nucleosomes. VivosX works in human cells and distinguishes ZZ nucleosomes with one or two ubiquitin moieties, demonstrating that it can be used to detect protein-protein interactions inside cells from different species.

Data availability

Source data files of western blotting quantification are included as Figure Supplements

Article and author information

Author details

  1. Chitra Mohan

    Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Lisa M Kim

    Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Nicole Hollar

    Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Tailai Li

    Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Eric Paulissen

    Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Cheuk T Leung

    Department of Pharmacology, University of Minnesota Medical School, Minneapolis, United States
    For correspondence
    ctleung@umn.edu
    Competing interests
    The authors declare that no competing interests exist.
  7. Ed Luk

    Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, United States
    For correspondence
    ed.luk@stonybrook.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6619-2258

Funding

National Institute of General Medical Sciences (RO1 GM104111)

  • Ed Luk

National Cancer Institute (RO1 CA200652)

  • Cheuk T Leung

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Geeta J Narlikar, University of California, San Francisco, United States

Publication history

  1. Received: March 14, 2018
  2. Accepted: August 8, 2018
  3. Accepted Manuscript published: August 9, 2018 (version 1)
  4. Version of Record published: August 23, 2018 (version 2)

Copyright

© 2018, Mohan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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