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A synthetic biology approach to probing nucleosome symmetry

  1. Yuichi Ichikawa
  2. Caitlin F Connelly
  3. Alon Appleboim
  4. Thomas CR Miller
  5. Hadas Jacobi
  6. Nebiyu A Abshiru
  7. Hsin-Jung Chou
  8. Yuanyuan Chen
  9. Upasna Sharma
  10. Yupeng Zheng
  11. Paul M Thomas
  12. Hsuiyi V Chen
  13. Vineeta Bajaj
  14. Christoph W Müller
  15. Neil L Kelleher
  16. Nir Friedman
  17. Daniel NA Bolon
  18. Oliver J Rando Is a corresponding author
  19. Paul D Kaufman Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. The Hebrew University, Israel
  3. The Francis Crick Institute, United Kingdom
  4. Northwestern University, United States
  5. European Molecular Biology Laboratory (EMBL), Germany
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Cite as: eLife 2017;6:e28836 doi: 10.7554/eLife.28836

Abstract

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read , we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read by effector proteins in the cell.

Article and author information

Author details

  1. Yuichi Ichikawa

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Caitlin F Connelly

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Alon Appleboim

    School of Computer Science and Engineering, The Hebrew University, Jerusalem, Israel
    Competing interests
    The authors declare that no competing interests exist.
  4. Thomas CR Miller

    Molecular Machines Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Hadas Jacobi

    School of Computer Science and Engineering, The Hebrew University, Jerusalem, Israel
    Competing interests
    The authors declare that no competing interests exist.
  6. Nebiyu A Abshiru

    National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Hsin-Jung Chou

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Yuanyuan Chen

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Upasna Sharma

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Yupeng Zheng

    National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Paul M Thomas

    National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Hsuiyi V Chen

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Vineeta Bajaj

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Christoph W Müller

    Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  15. Neil L Kelleher

    National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-8815-3372
  16. Nir Friedman

    School of Computer Science and Engineering, The Hebrew University, Jerusalem, Israel
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-9678-3550
  17. Daniel NA Bolon

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Oliver J Rando

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    Oliver.Rando@umassmed.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0003-1516-9397
  19. Paul D Kaufman

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    paul.kaufman1@umassmed.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0003-3089-313X

Funding

National Institute of General Medical Sciences (R01GM100164)

  • Yuichi Ichikawa
  • Caitlin F Connelly
  • Hsin-Jung Chou
  • Hsuiyi V Chen
  • Oliver J Rando
  • Paul D Kaufman

European Commission (340712)

  • Alon Appleboim
  • Hadas Jacobi
  • Nir Friedman

National Institute of General Medical Sciences (P41GM108569)

  • Nebiyu A Abshiru
  • Yupeng Zheng
  • Paul M Thomas
  • Neil L Kelleher

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

Reviewing Editor

  1. Jerry L Workman, Reviewing Editor, Stowers Institute for Medical Research, United States

Publication history

  1. Received: May 19, 2017
  2. Accepted: September 12, 2017
  3. Accepted Manuscript published: September 12, 2017 (version 1)
  4. Accepted Manuscript updated: September 13, 2017 (version 2)

Copyright

© 2017, Ichikawa 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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