Abstract

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase-separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization.

Data availability

The GEO accession number for the ATAC-seq data is: GSE117335. The SPT trajectory data are available via Zenodo at DOI:10.5281/zenodo.1313872. The software used to generate these data is available at https://gitlab.com/tjian-darzacq-lab.

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

Article and author information

Author details

  1. David Trombley McSwiggen

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3844-7433
  2. Anders S Hansen

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Sheila S Teves

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1220-2414
  4. Hervé Marie-Nelly

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  5. Yvonne Hao

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  6. Alec Basil Heckert

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  7. Kayla K Umemoto

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  8. Claire Dugast-Darzacq

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  9. Robert Tjian

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jmlim@berkeley.edu
    Competing interests
    Robert Tjian, is one of the three founding funders of eLife, and a member of eLife's Board of Directors.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0539-8217
  10. Xavier Darzacq

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    darzacq@berkeley.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2537-8395

Funding

National Institutes of Health (UO1- 497 EB021236)

  • David Trombley McSwiggen
  • Anders S Hansen
  • Yvonne Hao
  • Alec Basil Heckert
  • Kayla K Umemoto
  • Claire Dugast-Darzacq
  • Xavier Darzacq

National Institutes of Health (U54-DK107980)

  • David Trombley McSwiggen
  • Anders S Hansen
  • Yvonne Hao
  • Alec Basil Heckert
  • Kayla K Umemoto
  • Claire Dugast-Darzacq
  • Xavier Darzacq

California Institute for Regenerative Medicine (LA1-08013)

  • Anders S Hansen
  • Alec Basil Heckert
  • Xavier Darzacq

Howard Hughes Medical Institute (003061)

  • David Trombley McSwiggen
  • Anders S Hansen
  • Sheila S Teves
  • Yvonne Hao
  • Alec Basil Heckert
  • Kayla K Umemoto
  • Robert Tjian

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

Reviewing Editor

  1. Jessica K Tyler, Weill Cornell Medicine, United States

Version history

  1. Received: March 26, 2019
  2. Accepted: April 29, 2019
  3. Accepted Manuscript published: April 30, 2019 (version 1)
  4. Accepted Manuscript updated: May 7, 2019 (version 2)
  5. Version of Record published: May 16, 2019 (version 3)

Copyright

© 2019, McSwiggen 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

  • 14,297
    Page views
  • 2,365
    Downloads
  • 180
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, 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. David Trombley McSwiggen
  2. Anders S Hansen
  3. Sheila S Teves
  4. Hervé Marie-Nelly
  5. Yvonne Hao
  6. Alec Basil Heckert
  7. Kayla K Umemoto
  8. Claire Dugast-Darzacq
  9. Robert Tjian
  10. Xavier Darzacq
(2019)
Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation
eLife 8:e47098.
https://doi.org/10.7554/eLife.47098

Share this article

https://doi.org/10.7554/eLife.47098

Further reading

    1. Chromosomes and Gene Expression
    Allison Coté, Aoife O'Farrell ... Arjun Raj
    Research Article

    Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Maria L Adelus, Jiacheng Ding ... Casey E Romanoski
    Research Article

    Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.