1. Chromosomes and Gene Expression

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo

  1. Natalia Petrenko
  2. Yi Jin
  3. Koon Ho Wong
  4. Kevin Struhl  Is a corresponding author
  1. Harvard Medical School, United States
  2. University of Macau, China
https://doi.org/10.7554/eLife.28447
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  1. Natalia Petrenko
  2. Yi Jin
  3. Koon Ho Wong
  4. Kevin Struhl
(2017)
Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo
eLife 6:e28447.
https://doi.org/10.7554/eLife.28447
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Abstract

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

Data availability

The following data sets were generated
The following previously published data sets were used
    1. Paul et al.
    (2015) Saccharomyces cerevisiae S288c Genome sequencing
    Publicly available via DNA Data Bank of Japan (accession no. SRP047524).
    https://trace.ddbj.nig.ac.jp/DRASearch/study?acc=SRP047524

Article and author information

Author details

  1. Natalia Petrenko

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.

  2. Yi Jin

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.

  3. Koon Ho Wong

    Faculty of Health Sciences, University of Macau, Zhuhai Shi, China
    Competing interests
    No competing interests declared.

  4. Kevin Struhl

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    kevin@hms.harvard.edu
    Competing interests
    Kevin Struhl, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4181-7856

Funding

National Institutes of Health (GM 30186)

  • Kevin Struhl

Croucher Foundation

  • Koon Ho Wong

University of Macau (MYRG2015-00186-FHS)

  • Koon Ho Wong

University of Macau (MYRG2016-0-0211-FHS)

  • Koon Ho Wong

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

Reviewing Editor

  1. Alan G Hinnebusch, National Institutes of Health, United States

Version history

  1. Received: December 24, 2016
  2. Accepted: July 9, 2017
  3. Accepted Manuscript published: July 12, 2017 (version 1)
  4. Version of Record published: July 26, 2017 (version 2)
  5. Version of Record updated: September 18, 2017 (version 3)

Copyright

© 2017, Petrenko 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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  1. Natalia Petrenko
  2. Yi Jin
  3. Koon Ho Wong
  4. Kevin Struhl
(2017)
Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo
eLife 6:e28447.
https://doi.org/10.7554/eLife.28447

Share this article

https://doi.org/10.7554/eLife.28447

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Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species

    Natalia Petrenko, Kevin Struhl
    Research Article Updated

    The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity.

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    Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

    F Javier DeHaro-Arbona, Charalambos Roussos ... Sarah Bray
    Research Article

    Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.