Biology subjects

Meeussen, J. V. W.

Publications and source records attributed to Meeussen, J. V. W..

4 recordsLinked to original sources

bioRxiv2026

A Cooperative Mechanism of Eukaryotic Transcription Factor Target Search

Rapid gene activation requires transcription factors (TFs) to locate their target motifs within vast genomes. In bacteria, TF target search is accelerated by combining 3D diffusion with 1D sliding, called facilitated diffusion, yet whether eukaryotic TFs rely on similar strategies has remained unresolved due to the lack of direct measurements in living cells. Here, we directly visualize eukaryotic TF target search in living cells by labeling a single TF per nucleus and visualizing its binding to its endogenous locus. Using the budding yeast TF Gal4, we find that efficient target localization occurs near the diffusion limit and does not require facilitated diffusion. Instead, rapid association requires cooperative self-interactions mediated by an intrinsically-disordered central region (IDR), independent of the activation domain. Replacing the Gal4 IDR with human self-interacting IDRs (EWS or FUS) restores efficient search, demonstrating that self-interactions are a general and portable feature for search. A second structured dimerization domain further cooperatives with the IDR to stabilize binding at neighboring motifs, revealing two mechanistically separable forms of cooperativity that together govern TF function. These findings highlight that facilitated diffusion is not strictly required and establish cooperative IDR-driven self-interactions as a key mechanism to enable rapid target recognition in eukaryotic cells.

molecular biology↗

bioRxiv2025

Recruitment of Fpt1 to tRNA genes requires TFIIIB and the N-terminal TPR array of TFIIIC subunit {tau}131

Transfer-RNA genes (tDNAs) in budding yeast recruit varying amounts of Fpt1, a regulator of RNA polymerase III (RNAPIII) occupancy. Fpt1 occupancy resembles that of the general transcription factor TFIIIC but how Fpt1 is recruited to tDNAs remains unclear. Here we show that both TFIIIB and TFIIIC are required for Fpt1 binding under active as well as repressive RNAPIII conditions. Depletion of TFIIIB reduced Fpt1 occupancy without affecting TFIIIC. In contrast, TFIIIC depletion led to reduced Fpt1 and a gene-specific reduction in TFIIIB occupancy. Moreover, upon depletion of TFIIIC, Fpt1 and TFIIIB were lost to different extents. We identified the C-terminal intrinsically disordered region of Fpt1 as critical for increased Fpt1 binding under repressive conditions. Within this region, a short -helix was predicted to interact with the N-terminal tetratricopeptide repeat array of the TFIIIC subunit {tau}131, a region also known to interact with TFIIIB. Deletion of this -helix abrogated stress-induced Fpt1 recruitment to tDNAs, as did mutations in the predicted interaction surface of {tau}131, while having a milder effect on TFIIIB occupancy. Together, these findings uncovered a dual and dynamic mechanism of Fpt1 recruitment to tDNAs with independent contributions of TFIIIB and TFIIIC.

molecular biology↗

bioRxiv2023

Transcription factor exchange enables prolonged transcriptional bursts

Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding is related to transcription activation. Here, we devised a microscopy method to simultaneously measure transient TF binding at a single locus and the effect of these binding events on transcription. We show that DNA binding of the yeast TF Gal4 activates transcription of a target gene within a few seconds, with at least [~]20% efficiency and with a high initiation rate of [~]1 RNA/s. Gal4 DNA dissociation decreases transcription rapidly. Moreover, at a gene with multiple binding sites, individual Gal4 molecules only rarely stay bound throughout the entire burst, but instead frequently exchange during a burst to increase the transcriptional burst duration. Our results suggest a mechanism for enhancer regulation in more complex eukaryotes, where TF cooperativity and exchange enable robust transcription activation.

molecular biology↗

bioRxiv2022

Transcription factor clusters enable target search but do not contribute to target gene activation

Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and how it influences gene expression is not well understood. Here, we use quantitative microscopy in living cells to study the regulation and function of clustering of the budding yeast TF Gal4 in its endogenous context. Our results show that Gal4 cluster formation is facilitated by, but does not completely depend on DNA binding and intrinsically disordered regions. Gal4 cluster properties are regulated by the Gal4-inhibitor Gal80 and Gal4 concentration. Moreover, we discover that clustering acts as a double-edged sword: self-interactions aid TF recruitment to target genes, but recruited Gal4 molecules that are not DNA-bound do not contribute to, and may even inhibit, transcription activation. We propose that cells need to balance the different effects of TF clustering on target search and transcription activation to facilitate proper gene expression.

molecular biology↗