1. Chromosomes and Gene Expression
Download icon

RNA Polymerase II cluster dynamics predict mRNA output in living cells

  1. Won-Ki Cho
  2. Namrata Jayanth
  3. Brian P English
  4. Takuma Inoue
  5. J Owen Andrews
  6. William Conway
  7. Jonathan B Grimm
  8. Jan-Hendrik Spille
  9. Luke D Lavis
  10. Timothée Lionnet
  11. Ibrahim I Cisse  Is a corresponding author
  1. Massachusetts Institute of Technology, United States
Research Article
  • Cited 116
  • Views 9,088
  • Annotations
Cite this article as: eLife 2016;5:e13617 doi: 10.7554/eLife.13617

Abstract

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

Article and author information

Author details

  1. Won-Ki Cho

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Namrata Jayanth

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Brian P English

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Takuma Inoue

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. J Owen Andrews

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. William Conway

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Jonathan B Grimm

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Jan-Hendrik Spille

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Luke D Lavis

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Timothée Lionnet

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. Ibrahim I Cisse

    Department of Physics, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    icisse@mit.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Xiaowei Zhuang, Howard Hughes Medical Institute, Harvard University, United States

Publication history

  1. Received: December 8, 2015
  2. Accepted: May 2, 2016
  3. Accepted Manuscript published: May 3, 2016 (version 1)
  4. Version of Record published: June 30, 2016 (version 2)

Copyright

© 2016, Cho 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

  • 9,088
    Page views
  • 2,656
    Downloads
  • 116
    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)

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

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

Categories and tags

Research organism

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    The pioneer factor hypothesis is not necessary to explain ectopic liver gene activation

    Jeffrey L Hansen et al.
    Research Article

    The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and nonPF HNF4A in K562 lymphoblast cells. While co-expression of FOXA1 and HNF4A activated a burst of endoderm-specific gene expression, we found no evidence for a functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and 'pioneered' for each other, although FOXA1 required fewer copies of its motif for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we hypothesize an alternative to the PFH where 'pioneer activity' depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA.

    1. Chromosomes and Gene Expression
    2. Developmental Biology

    Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos

    Graham JM Hickey et al.
    Research Article

    Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal the order of chromatin states in establishing developmental gene poising in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with 'Placeholder' nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of Polycomb Repressive Complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.

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

    Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation

    Dorota Rousova et al.
    Research Article

    In meiosis, DNA double strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific “axis-tethered loop” chromosome organization. Through in vitro reconstitution and budding yeast genetics we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates to nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.