Nucleosomes influence multiple steps during replication initiation

  1. Ishara F Azmi
  2. Shinya Watanabe
  3. Michael F Maloney
  4. Sukhyun Kang
  5. Jason A Belsky
  6. David M MacAlpine
  7. Craig L Peterson
  8. Stephen P Bell  Is a corresponding author
  1. Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
  2. University of Massachusetts Medical School, United States
  3. Duke University Medical Center, United States

Abstract

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.

Article and author information

Author details

  1. Ishara F Azmi

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Shinya Watanabe

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael F Maloney

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Sukhyun Kang

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Jason A Belsky

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2945-6282
  6. David M MacAlpine

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Craig L Peterson

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Stephen P Bell

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    spbell@mit.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2876-610X

Funding

Howard Hughes Medical Institute (Investigator)

  • Stephen P Bell

National Institute of General Medical Sciences (GM54096 and GM104097)

  • David M MacAlpine
  • Craig L Peterson

American Cancer Society (123700-PF-13-071-01-DM)

  • Ishara F Azmi

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

Reviewing Editor

  1. Robert Sclafani, University of Colorado School of Medicine, United States

Version history

  1. Received: October 19, 2016
  2. Accepted: March 20, 2017
  3. Accepted Manuscript published: March 21, 2017 (version 1)
  4. Version of Record published: April 21, 2017 (version 2)

Copyright

© 2017, Azmi 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

  • 3,089
    Page views
  • 780
    Downloads
  • 40
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

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. Ishara F Azmi
  2. Shinya Watanabe
  3. Michael F Maloney
  4. Sukhyun Kang
  5. Jason A Belsky
  6. David M MacAlpine
  7. Craig L Peterson
  8. Stephen P Bell
(2017)
Nucleosomes influence multiple steps during replication initiation
eLife 6:e22512.
https://doi.org/10.7554/eLife.22512

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Sevim Kahraman, Kimitaka Shibue ... Rohit N Kulkarni
    Tools and Resources

    Pancreatic a-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human a-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality a-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live a-cells from dissociated human islet cells with ~ 95% purity. The a-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form a-pseudoislets. The a-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key a-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in a-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary a-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Valentin Chabert, Geun-Don Kim ... Andreas Mayer
    Research Article

    Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.