1. Chromosomes and Gene Expression
  2. Computational and Systems Biology

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

  1. Adrian L Sanborn  Is a corresponding author
  2. Benjamin T Yeh
  3. Jordan T Feigerle
  4. Cynthia V Hao
  5. Raphael J L Townshend
  6. Erez Lieberman-Aiden
  7. Ron O Dror
  8. Roger D Kornberg  Is a corresponding author
  1. Stanford University, United States
  2. Baylor College of Medicine, United States
  3. Stanford University School of Medicine, United States
https://doi.org/10.7554/eLife.68068
Share this article
Cite this article
  1. Adrian L Sanborn
  2. Benjamin T Yeh
  3. Jordan T Feigerle
  4. Cynthia V Hao
  5. Raphael J L Townshend
  6. Erez Lieberman-Aiden
  7. Ron O Dror
  8. Roger D Kornberg
(2021)
Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator
eLife 10:e68068.
https://doi.org/10.7554/eLife.68068
  2. Download BibTeX
  3. Download .RIS

Abstract

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Data availability

All data from in vivo activation and in vitro screens are included in tables as source data files. PDB files of structural models of Med15-AD interactions are included in Figure 6-source data 2. All sequencing data have been deposited in GEO, under the accession code GSE173156.

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

Article and author information

Author details

  1. Adrian L Sanborn

    Department of Structural Biology, Department of Computer Science, Stanford University, Stanford, United States
    For correspondence
    a@adriansanborn.com
    Competing interests
    The authors declare that no competing interests exist.

  2. Benjamin T Yeh

    David Geffen School of Medicine, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9397-6392

  3. Jordan T Feigerle

    Department of Molecular Physiology and Biophysics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Cynthia V Hao

    Department of Structural Biology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2183-0698

  5. Raphael J L Townshend

    Department of Computer Science, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Erez Lieberman-Aiden

    Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Ron O Dror

    Department of Computer Science, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Roger D Kornberg

    Department of Structural Biology, Stanford University School of Medicine, Stanford, United States
    For correspondence
    kornberg@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2425-7519

Funding

National Institutes of Health (R01-DK121366 and R01-AI021144)

  • Roger D Kornberg

U.S. Department of Energy (Office of Science Graduate Student Research (SCGSR) program (DE-SC0014664))

  • Raphael J L Townshend

National Institutes of Health (F32-GM126704)

  • Jordan T Feigerle

National Institutes of Health (R01-GM127359)

  • Ron O Dror

U.S. Department of Energy (Scientific Discovery through Advanced Computing (SciDAC) program)

  • Ron O Dror

National Science Foundation (Physics Frontiers Center Award (PHY1427654))

  • Erez Lieberman-Aiden

Welch Foundation (Q-1866)

  • Erez Lieberman-Aiden

U.S. Department of Agriculture (Agriculture and Food Research Initiative Grant (2017-05741))

  • Erez Lieberman-Aiden

National Institutes of Health (4D Nucleome Grant (U01HL130010))

  • Erez Lieberman-Aiden

National Institutes of Health (Encyclopedia of DNA Elements Mapping Center Award (UM1HG009375))

  • Erez Lieberman-Aiden

U.S. Department of Defense (National Defense Science & Engineering Graduate (NDSEG) Fellowship)

  • Adrian L Sanborn

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

Copyright

© 2021, Sanborn 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

  • 8,601
    views
  • 1,023
    downloads
  • 139
    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. Adrian L Sanborn
  2. Benjamin T Yeh
  3. Jordan T Feigerle
  4. Cynthia V Hao
  5. Raphael J L Townshend
  6. Erez Lieberman-Aiden
  7. Ron O Dror
  8. Roger D Kornberg
(2021)
Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator
eLife 10:e68068.
https://doi.org/10.7554/eLife.68068

Share this article

https://doi.org/10.7554/eLife.68068

Categories and tags

Research organisms

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

    Hans Tobias Gustafsson, Lucas Ferguson ... Oliver J Rando
    Research Article

    Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Major nuclear locales define nuclear genome organization and function beyond A and B compartments

    Omid Gholamalamdari, Tom van Schaik ... Andrew S Belmont
    Research Article

    Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here, we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Although gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.

    1. Chromosomes and Gene Expression

    SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing

    Ashwin Govindan, Nicholas K Conrad
    Research Article

    O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.