1. Chromosomes and Gene Expression
  2. Structural Biology and Molecular Biophysics

Mechanisms governing target search and binding dynamics of hypoxia-inducible factors

  1. Yu Chen
  2. Claudia Cattoglio
  3. Gina M Dailey
  4. Qiulin Zhu
  5. Robert Tjian  Is a corresponding author
  6. Xavier Darzacq  Is a corresponding author
  1. University of California, Berkeley, United States
https://doi.org/10.7554/eLife.75064
Share this article
Cite this article
  1. Yu Chen
  2. Claudia Cattoglio
  3. Gina M Dailey
  4. Qiulin Zhu
  5. Robert Tjian
  6. Xavier Darzacq
(2022)
Mechanisms governing target search and binding dynamics of hypoxia-inducible factors
eLife 11:e75064.
https://doi.org/10.7554/eLife.75064
  2. Download BibTeX
  3. Download .RIS

Abstract

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, another main determinant of chromatin binding and diffusion behavior is the AD-containing intrinsically disordered region (IDR). Using Cut&Run and RNA-seq as orthogonal genomic approaches we also confirmed IDR-dependent binding and activation of a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search and binding process that contribute to functional target site selectivity on chromatin.

Data availability

NGS data have been deposited in NCBI's GEO under accession number GSE207575.SPT raw data are accessible through DOI: 10.5281/zenodo.5559234.

The following data sets were generated

Article and author information

Author details

  1. Yu Chen

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7856-4648

  2. Claudia Cattoglio

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6100-0491

  3. Gina M Dailey

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8988-963X

  4. Qiulin Zhu

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  5. Robert Tjian

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jmlim@berkeley.edu
    Competing interests
    Robert Tjian, Robert Tjian is one of the three founding funders of eLife, and a member of eLife's Board of Directors. Is a co-founder of Eikon Therapeutics.

  6. Xavier Darzacq

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    darzacq@berkeley.edu
    Competing interests
    Xavier Darzacq, is a co-founder of Eikon Therapeutics.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2537-8395

Funding

National Institutes of Health (U54-CA231641-01)

  • Xavier Darzacq

Howard Hughes Medical Institute

  • Robert Tjian

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

Copyright

© 2022, Chen 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

  • 2,224
    views
  • 344
    downloads
  • 32
    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. Yu Chen
  2. Claudia Cattoglio
  3. Gina M Dailey
  4. Qiulin Zhu
  5. Robert Tjian
  6. Xavier Darzacq
(2022)
Mechanisms governing target search and binding dynamics of hypoxia-inducible factors
eLife 11:e75064.
https://doi.org/10.7554/eLife.75064

Share this article

https://doi.org/10.7554/eLife.75064

Categories and tags

Research organism

Further reading

    1. Chromosomes and Gene Expression

    Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

    Carmina Lichauco, Eric J Foss ... Antonio Bedalov
    Research Article

    The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

    1. Chromosomes and Gene Expression

    Cryogenic Electron Microscopy: MagIC beads for scarce macromolecules

    Carlos Moreno-Yruela, Beat Fierz
    Insight

    Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics

    Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging

    Liza Dahal, Thomas GW Graham ... Xavier Darzacq
    Research Article

    Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.