Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation
- University of California, Berkeley, United States
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.
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Relative accessability of HSV1 genomic DNA compared with its host cell (ATAC-seq)NCBI Gene Expression Omnibus, GSE117335.
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Single Particle Tracking data for U2OS cells after infectionZenodo, 10.5281/zenodo.1313872.
Article and author information
Author details
David Trombley McSwiggen
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
- Jessica K Tyler, Weill Cornell Medicine, United States
Publication history
- Received: March 26, 2019
- Accepted: April 29, 2019
- Accepted Manuscript published: April 30, 2019 (version 1)
- Accepted Manuscript updated: May 7, 2019 (version 2)
- 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.
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Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation
eLife 8:e47098.
https://doi.org/10.7554/eLife.47098
Further reading
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- Cell Biology
- Chromosomes and Gene Expression
Dynamic regulation of transcription is crucial for the cellular responses to various environmental or developmental cues. Gdown1 is a ubiquitously expressed, RNA polymerase II (Pol II) interacting protein, essential for the embryonic development of metazoan. It tightly binds Pol II in vitro and competitively blocks the binding of TFIIF and possibly other transcriptional regulatory factors, yet its cellular functions and regulatory circuits remain unclear. Here, we show that human GDOWN1 strictly localizes in the cytoplasm of various types of somatic cells and exhibits a potent resistance to the imposed driving force for its nuclear localization. Combined with the genetic and microscope-based approaches, two types of the functionally coupled and evolutionally conserved localization regulatory motifs are identified, including the CRM1-dependent nucleus export signal (NES) and a novel Cytoplasmic Anchoring Signal (CAS) that mediates its retention outside of the nuclear pore complexes (NPC). Mutagenesis of CAS alleviates GDOWN1’s cytoplasmic retention, thus unlocks its nucleocytoplasmic shuttling properties, and the increased nuclear import and accumulation of GDOWN1 results in a drastic reduction of both Pol II and its associated global transcription levels. Importantly, the nuclear translocation of GDOWN1 occurs in response to the oxidative stresses, and the ablation of GDOWN1 significantly weakens the cellular tolerance. Collectively, our work uncovers the molecular basis of GDOWN1’s subcellular localization and a novel cellular strategy of modulating global transcription and stress-adaptation via controlling the nuclear translocation of GDOWN1.
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In a departure from previous findings, new results suggest that free-floating pieces of DNA which carry additional copies of cancer-driving genes do not tend to cluster or have increased transcription.
