Abstract

Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of S. cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Antisense transcription varies highly across the regulatory landscapes analyzed, but antisense transcription in itself does not affect sense transcription at the same locus. Our findings collectively show that a diverse array of factors regulate transcription elongation by precisely balancing Pol II activity.

Data availability

The accession number for the Illumina sequencing reported in this paper is Gene Expression Omnibus (GEO): GSE159603.

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

Article and author information

Author details

  1. Mary Couvillion

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  2. Kevin M Harlen

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  3. Kate C Lachance

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  4. Kristine L Trotta

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8166-7696
  5. Erin Smith

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  6. Christian Brion

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  7. Brendan M Smalec

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  8. L Stirling Churchman

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    churchman@genetics.med.harvard.edu
    Competing interests
    L Stirling Churchman, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3888-2574

Funding

National Institutes of Health (R01-HG007173)

  • L Stirling Churchman

National Institutes of Health (F31 HG010570)

  • Kate C Lachance

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

Copyright

© 2022, Couvillion 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

  • 3,427
    views
  • 611
    downloads
  • 11
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Mary Couvillion
  2. Kevin M Harlen
  3. Kate C Lachance
  4. Kristine L Trotta
  5. Erin Smith
  6. Christian Brion
  7. Brendan M Smalec
  8. L Stirling Churchman
(2022)
Transcription elongation is finely tuned by dozens of regulatory factors
eLife 11:e78944.
https://doi.org/10.7554/eLife.78944

Share this article

https://doi.org/10.7554/eLife.78944

Further reading

    1. Chromosomes and Gene Expression
    Daphne R Knudsen-Palmer, Pravrutha Raman ... Antony M Jose
    Research Article

    Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode Caenorhabditis elegans can selectively impair the silencing of some genes. Here, we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells, and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, are enriched within ‘pUG zones’ matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

    1. Chromosomes and Gene Expression
    Shuvra Shekhar Roy, Sulochana Bagri ... Shantanu Chowdhury
    Research Article

    Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained.