1. Chromosomes and Gene Expression

The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry

  1. Rupam Choudhury
  2. Anuroop Venkateswaran Venkatasubramani
  3. Jie Hua
  4. Marco Borsò
  5. Celeste Franconi
  6. Sarah Kinkley
  7. Ignasi Forné
  8. Axel Imhof  Is a corresponding author
  1. Ludwig-Maximilians-Universität München, Germany
  2. Max Planck Institute for Molecular Genetics, Germany
https://doi.org/10.7554/eLife.95718
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Cite this article
  1. Rupam Choudhury
  2. Anuroop Venkateswaran Venkatasubramani
  3. Jie Hua
  4. Marco Borsò
  5. Celeste Franconi
  6. Sarah Kinkley
  7. Ignasi Forné
  8. Axel Imhof
(2024)
The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry
eLife 13:e95718.
https://doi.org/10.7554/eLife.95718
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Abstract

Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity-biotinylation method targeting the RNA and proteins constituents. The method that we termed Antibody-Mediated-Proximity-Labelling-coupled to Mass Spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. This analysis identified a number of known RNA binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

Data availability

The datasets produced in this study are available in the ProteomeXchange Consortium via the PRIDE 64 partner repository with the identifiers: PXD044295 (Proteomics) and PXD044296 (Histone PTMs).

The following data sets were generated

Article and author information

Author details

  1. Rupam Choudhury

    Department of Molecular Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.

  2. Anuroop Venkateswaran Venkatasubramani

    Department of Molecular Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2119-8741

  3. Jie Hua

    Department of Molecular Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4910-0945

  4. Marco Borsò

    Protein Analysis Unit, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7467-7960

  5. Celeste Franconi

    Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.

  6. Sarah Kinkley

    Department of Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4997-4749

  7. Ignasi Forné

    Protein Analysis Unit, Ludwig-Maximilians-Universität München, Martinsried, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0309-907X

  8. Axel Imhof

    Department of Molecular Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
    For correspondence
    Imhof@lmu.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2993-8249

Funding

Deutsche Forschungsgemeinschaft (419067076)

  • Rupam Choudhury

Deutsche Forschungsgemeinschaft (213249687)

  • Marco Borsò

Deutsche Forschungsgemeinschaft (325871075)

  • Rupam Choudhury

Deutsche Forschungsgemeinschaft (QBM)

  • Anuroop Venkateswaran Venkatasubramani

Volkswagen Foundation (97131)

  • Celeste Franconi
  • Sarah Kinkley

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

Copyright

© 2024, Choudhury 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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  1. Rupam Choudhury
  2. Anuroop Venkateswaran Venkatasubramani
  3. Jie Hua
  4. Marco Borsò
  5. Celeste Franconi
  6. Sarah Kinkley
  7. Ignasi Forné
  8. Axel Imhof
(2024)
The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry
eLife 13:e95718.
https://doi.org/10.7554/eLife.95718

Share this article

https://doi.org/10.7554/eLife.95718

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