1. Chromosomes and Gene Expression
  2. Genetics and Genomics

Massively multiplex single-molecule oligonucleosome footprinting

  1. Nour J Abdulhay
  2. Colin P McNally
  3. Laura J Hsieh
  4. Sivakanthan Kasinathan
  5. Aidan Keith
  6. Laurel S Estes
  7. Mehran Karimzadeh
  8. Jason G Underwood
  9. Hani Goodarzi
  10. Geeta J Narlikar
  11. Vijay Ramani  Is a corresponding author
  1. University of California San Francisco, United States
  2. Stanford University, United States
  3. Vector Institute, Canada
  4. Pacific Biosciences of California, Inc, United States
  5. University of California, San Francisco, United States
https://doi.org/10.7554/eLife.59404
Share this article
Cite this article
  1. Nour J Abdulhay
  2. Colin P McNally
  3. Laura J Hsieh
  4. Sivakanthan Kasinathan
  5. Aidan Keith
  6. Laurel S Estes
  7. Mehran Karimzadeh
  8. Jason G Underwood
  9. Hani Goodarzi
  10. Geeta J Narlikar
  11. Vijay Ramani
(2020)
Massively multiplex single-molecule oligonucleosome footprinting
eLife 9:e59404.
https://doi.org/10.7554/eLife.59404
  2. Download BibTeX
  3. Download .RIS

Abstract

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure.

Data availability

All raw data will be made available at GEO Accession GSE162410; processed data is available at Zenodo (https://doi.org/10.5281/zenodo.3834705). All scripts and notebooks for reproducing analyses in the paper are available at https://github.com/RamaniLab/SAMOSA.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Nour J Abdulhay

    Biochemistry & Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  2. Colin P McNally

    Biochemistry & Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  3. Laura J Hsieh

    Biochemistry & Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  4. Sivakanthan Kasinathan

    Pediatrics, Stanford University, Palo Alto, United States
    Competing interests
    No competing interests declared.

  5. Aidan Keith

    Biochemistry & Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  6. Laurel S Estes

    Biochemistry & Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  7. Mehran Karimzadeh

    Vector Institute, Toronto, Canada
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7324-6074

  8. Jason G Underwood

    Pacific Biosciences of California, Inc, Menlo Park, United States
    Competing interests
    Jason G Underwood, J.G.U. is an employee of Pacific Biosciences, Inc. and holds stock in this company..

  9. Hani Goodarzi

    Biochemistry & Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  10. Geeta J Narlikar

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    Geeta J Narlikar, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1920-0147

  11. Vijay Ramani

    Biochemistry & Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    vijay.ramani@ucsf.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3345-5960

Funding

Sandler Foundation

  • Vijay Ramani

American Cancer Society

  • Laura J Hsieh

National Institutes of Health (R01GM123977)

  • Hani Goodarzi

National Institutes of Health (R35GM127020)

  • Geeta J Narlikar

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

Reviewing Editor

  1. Job Dekker, University of Massachusetts Medical School, United States

Version history

  1. Received: May 28, 2020
  2. Accepted: November 24, 2020
  3. Accepted Manuscript published: December 2, 2020 (version 1)
  4. Version of Record published: December 14, 2020 (version 2)

Copyright

© 2020, Abdulhay 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.

Metrics

  • 5,588
    views
  • 547
    downloads
  • 61
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nour J Abdulhay
  2. Colin P McNally
  3. Laura J Hsieh
  4. Sivakanthan Kasinathan
  5. Aidan Keith
  6. Laurel S Estes
  7. Mehran Karimzadeh
  8. Jason G Underwood
  9. Hani Goodarzi
  10. Geeta J Narlikar
  11. Vijay Ramani
(2020)
Massively multiplex single-molecule oligonucleosome footprinting
eLife 9:e59404.
https://doi.org/10.7554/eLife.59404

Share this article

https://doi.org/10.7554/eLife.59404

Categories and tags

Research organism

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals

    Lisa Baumgartner, Jonathan J Ipsaro ... Julius Brennecke
    Research Advance

    Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino’s chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino’s chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression

    Human DDX6 regulates translation and decay of inefficiently translated mRNAs

    Ramona Weber, Chung-Te Chang
    Research Article

    Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

    1. Chromosomes and Gene Expression

    Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation

    Marwan Anoud, Emmanuelle Delagoutte ... Jean-Paul Concordet
    Research Article

    Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades’ radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.