skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Hierarchy in regulator interactions with distant transcriptional activation domains empowers rheostatic regulation
Abstract Transcription factors carry long intrinsically disordered regions often containing multiple activation domains. Despite numerous recent high‐throughput identifications and characterizations of activation domains, the interplay between sequence motifs, activation domains, and regulator binding in intrinsically disordered transcription factor regions remains unresolved. Here, we map sequence motifs and activation domains in anArabidopsis thalianaNAC transcription factor clade, revealing that although sequence motifs and activation domains often coincide, no systematic overlap exists. Biophysical analyses using NMR spectroscopy show that the long intrinsically disordered region of senescence‐associated transcription factor ANAC046 is devoid of residual structure. We identify two activation domain/sequence motif regions, one at each end that both bind a panel of six positive and negative regulator domains from biologically relevant regulators promiscuously. Binding affinities measured using isothermal titration calorimetry reveal a hierarchy for regulator binding of the two ANAC046 activation domain/sequence motif regions defining these as regulatory hotspots. Despite extensive dynamic intramolecular contacts along the disordered chain revealed using paramagnetic relaxation enhancement experiments and simulations, the regions remain uncoupled in binding. Together, the results imply rheostatic regulation by ANAC046 through concentration‐dependent regulator competition, a mechanism likely mirrored in other transcription factors with distantly located activation domains.  more » « less
Award ID(s):
2112056
PAR ID:
10598691
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
Protein Science
Date Published:
Journal Name:
Protein Science
Volume:
34
Issue:
6
ISSN:
0961-8368
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; (, Molecular Biology and Evolution)
    O'Connell, Mary (Ed.)
    Abstract The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of “functional affinity” in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein–protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; (, bioRxiv)
    Abstract Sequence-specific activation by transcription factors is essential for gene regulation1,2. Key to this are activation domains, which often fall within disordered regions of transcription factors3,4and recruit co-activators to initiate transcription5. These interactions are difficult to characterize via most experimental techniques because they are typically weak and transient6,7. Consequently, we know very little about whether these interactions are promiscuous or specific, the mechanisms of binding, and how these interactions tune the strength of gene activation. To address these questions, we developed a microfluidic platform for expression and purification of hundreds of activation domains in parallel followed by direct measurement of co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). By applying STAMMPPING to quantify direct interactions between eight co-activators and 204 human activation domains (>1,500Kds), we provide the first quantitative map of these interactions and reveal 334 novel binding pairs. We find that the metazoan-specific co-activator P300 directly binds >100 activation domains, potentially explaining its widespread recruitment across the genome to influence transcriptional activation. Despite sharing similar molecular properties (e.g.enrichment of negative and hydrophobic residues), activation domains utilize distinct biophysical properties to recruit certain co-activator domains. Co-activator domain affinity and occupancy are well-predicted by analytical models that account for multivalency, andin vitroaffinities quantitatively predict activation in cells with an ultrasensitive response. Not only do our results demonstrate the ability to measure affinities between even weak protein-protein interactions in high throughput, but they also provide a necessary resource of over 1,500 activation domain/co-activator affinities which lays the foundation for understanding the molecular basis of transcriptional activation. 
    more » « less
  3. ; ; ; ; ; ; ; ; (, Research Square)
    Abstract Plants tailor their architecture to warm ambient temperatures through the central thermosensory transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), yet the sequence features that confer this activity remain poorly defined. Here, we combine targeted mutagenesis, phase-separation assays, and transgenic complementation to dissect how structured and disordered regions of PIF4 contribute to its function in thermomorphogenesis. A long N-terminal intrinsically disordered region (IDR) enables PIF4 to form gel-like condensates in vitro andin planta. Within this IDR, we identify an acidic transactivation domain (TAD) and an extended basic segment that carries a nuclear-localization signal and the canonical basic motif of the basic helix-loop-helix (bHLH) domain. The basic segment is both necessary and sufficient to drive PIF4 condensate formation, while the TAD merely tunes condensate properties. Strikingly, alanine substitutions that abolish TAD-mediated transactivation, disrupt DNA binding, or greatly reduce phase-separation propensity have no significant effect on thermomorphogenetic hypocotyl elongation. By contrast, replacing three conserved basic residues in the first helix of the HLH domain disrupts PIF4 oligomerization and abolishes thermo-induced hypocotyl growth. We conclude that the ability of PIF4 to oligomerize, rather than to bind DNA or recruit the transcriptional machinery, is the primary determinant of its thermomorphogenetic activity. 
    more » « less
  4. ; (, GENETICS)
    Kaplan, C (Ed.)
    Abstract Transcription factors activate gene expression in development, homeostasis, and stress with DNA binding domains and activation domains. Although there exist excellent computational models for predicting DNA binding domains from protein sequence, models for predicting activation domains from protein sequence have lagged, particularly in metazoans. We recently developed a simple and accurate predictor of acidic activation domains on human transcription factors. Here, we show how the accuracy of this human predictor arises from the clustering of aromatic, leucine, and acidic residues, which together are necessary for acidic activation domain function. When we combine our predictor with the predictions of convolutional neural network (CNN) models trained in yeast, the intersection is more accurate than individual models, emphasizing that each approach carries orthogonal information. We synthesize these findings into a new set of activation domain predictions on human transcription factors. 
    more » « less
  5. ; ; ; ; (, Molecular Microbiology)
    Abstract Bacteria contain conserved mechanisms to control the intracellular levels of metal ions. Metalloregulatory transcription factors bind metal cations and play a central role in regulating gene expression of metal transporters. Often, these transcription factors regulate transcription by binding to a specific DNA sequence in the promoter region of target genes. Understanding the preferred DNA‐binding sequence for transcriptional regulators can help uncover novel gene targets and provide insight into the biological role of the transcription factor in the host organism. Here, we identify consensus DNA‐binding sequences and subsequent transcription regulatory networks for two metalloregulators from the ferric uptake regulator (FUR) and diphtheria toxin repressor (DtxR) superfamilies inThermus thermophilusHB8. By homology search, we classify the DtxR homolog as a manganese‐specific, MntR (TtMntR), and the FUR homolog as a peroxide‐sensing, PerR (TtPerR). Both transcription factors repress separate ZIP transporter genes in vivo, andTtPerR acts as a bifunctional transcription regulator by activating the expression of ferric and hemin transport systems. We showTtPerR andTtMntR bind DNA in the presence of manganese in vitro and in vivo; however,TtPerR is unable to bind DNA in the presence of iron, likely due to iron‐mediated histidine oxidation. Unlike canonical PerR homologs,TtPerR does not appear to contribute to peroxide detoxification. Instead, theTtPerR regulon and DNA binding sequence are more reminiscent of Fur or Mur homologs. Collectively, these results highlight the similarities and differences between two metalloregulatory superfamilies and underscore the interplay of manganese and iron in transcription factor regulation. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Text Encoded Conversion
  • XML
  • Save / Share this Record
  • Send to Email