More Like this

1. Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

   https://doi.org/10.1371/journal.pone.0286193  

   Suwita, Johannes P.; Voong, Calvin K.; Ly, Elina; Goodrich, James A.; Kugel, Jennifer F. (August 2023, PLOS ONE)

   D’Auria, Sabato (Ed.)

   The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs. 

   more » « less
2. Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

   Yizhuo Yang, Keith J. (January 2023, bioRxiv)

   na (Ed.)

   T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long “daisy chained” structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various “half” binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution. 

   more » « less
3. Structural and mechanistic diversity in p53-mediated regulation of organismal longevity across taxonomical orders

   https://doi.org/10.1371/journal.pcbi.1012382  

   Osbourne, Romani; Thayer, Kelly M (May 2025, PLOS Computational Biology)

   Skolnick, Jeffrey (Ed.)

   The link between p53 tumor suppressive functions and organismal lifespan is multifaceted. Its DNA-repair mechanism is longevity-enhancing while its role in cellular senescence pathways induces pro-aging phenotypes. To understand how p53 may regulate organismal lifespan, cross-species genotype-phenotype (GP) studies of the p53 DNA-binding domain (DBD) have been used to assess the correlation of amino acid changes to lifespan. Amino acid changes in non-DNA-binding regions such as the transactivation (TAD), proline-rich (PRD), regulatory (REG), and tetramerization (TET) are largely unexplored. In addition, existing GP correlation tools such as SigniSite do not account for phylogenetic relationships between aligned sequences in correlating genotypic differences to phenotypes such as lifespan. To identify phylogenetically significant, longevity-correlated residues in full-length p53 alignments, we developed a Python- and R-based workflow, Relative Evolutionary Scoring (RES). While RES-predicted longevity-associated residues (RPLARs) are concentrated primarily in the DBD, the PRD, TET, and REG domains also house RPLARs. While yeast functional assay enrichment reveals that RPLARs may be dispensable for p53-mediated transactivation, PEPPI and Rosetta-based protein-protein interaction prediction suggests a role for RPLARs in p53 stability and interaction interfaces of tumor suppressive protein-protein complexes. With experimental validation of the RPLARs’ roles in p53 stability, transactivation, and involvement in senescence-regulatory pathways, we can gain crucial insights into mechanisms underlying dysregulated tumor suppression and accelerated aging. 

   more » « less
4. DNA controls the dimerization of the human FoxP1 forkhead domain

   https://doi.org/10.1016/j.xcrp.2024.101854  

   Kolimi, Narendar; Ballard, Jake; Peulen, Thomas; Goutam, Rajen; Duffy, Francis X.; Ramírez-Sarmiento, César A.; Babul, Jorge; Medina, Exequiel; Sanabria, Hugo (March 2024, Cell Reports Physical Science)

   Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences and gating access to genes. Even when the binding of TFs and their cofactors to DNA is reversible, indicating a reversible control of gene expression, there is little knowledge about the molecular effect DNA has on TFs. Using single-molecule multiparameter fluorescence spectroscopy, molecular dynamics simulations, and biochemical assays, we find that the monomeric form of the forkhead (FKH) domain of the human FoxP1 behaves as a disordered protein and increases its folded population when it dimerizes. Notably, DNA binding promotes a disordered FKH dimer bound to DNA, negatively controlling the stability of the dimeric FoxP1:DNA complex. The DNA-mediated reversible regulation on FKH dimers suggests that FoxP1-dependent gene suppression is unstable, and it must require the presence of other dimerization domains or cofactors to revert the negative impact exerted by the DNA. 

   more » « less
5. Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

   https://doi.org/10.1371/journal.pgen.1009039  

   Kuang, Yi; Pyo, Anna; Eafergan, Natanel; Cain, Brittany; Gutzwiller, Lisa M.; Axelrod, Ofri; Gagliani, Ellen K.; Weirauch, Matthew T.; Kopan, Raphael; Kovall, Rhett A.; et al (September 2021, PLOS Genetics)

   Aster, Jon Christopher (Ed.)

   Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the C bf/ S u(H)/ L ag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called S u(H) p aired s ites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo . Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation. 

   more » « less