Transcription factor residence time dominates over concentration in transcription activation

Abstract

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

Transcription Factor Dynamics

“…Some TFs exhibit broad residence time distributions, consistent with a continuum of affinities across diverse genomic targets (Normanno et al 2015;Stavreva et al 2019;Garcia et al 2021). A novel kinetic analysis suggests on the other hand the existence of 5-6 discrete dissociation rates ranging from subseconds to minutes (Popp et al 2020;Reisser et al 2020). Despite their differences, the different SMT analyses converge on the fact that only a minority of TF-binding events extend beyond the seconds regime, consistent with dozens of earlier studies by FRAP (Hemmerich et al 2011).…”

“…Several observations are consistent with the one-to-one model: (1) the transcription machinery assembles within seconds, well within typical TF residence times (Zhang et al 2016;Nguyen et al 2020) Stavreva et al 2019). While the simple two-state model might be valid in simple systems, it often breaks down in higher eukaryotes (Bartman et al 2016;Corrigan et al 2016;Rodriguez et al 2019;Lammers et al 2020;Popp et al 2020). In a striking example, pluripotency genes cease to transcribe early in differentiation, long before their enhancers lose TF occupancy (Hamilton et al 2019).…”

“…The linker histone H1 exchanges from chromatin with similarly fast kinetics (∼20 sec-3 min) (Lever et al 2000;Misteli et al 2000), while the architectural proteins cohesin and CTCF fall in between core histones and TFs (min) (Hansen et al 2017). The residence time depends on the affinity between a TF and its target (Clauß et al 2017;Callegari et al 2019;Donovan et al 2019;Popp et al 2020), but can be modulated across loci, cell types, and cell states. For example, the residence time of the gTF TBP (TATA-binding protein) ranges from seconds in the developing zebrafish embryo (Reisser et al 2018), interphase U2OS cells, and in vitro assays (Zhang et al 2016), to hours at the histone locus in Drosophila cells (Guglielmi et al 2013).…”

Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient

“…Because endogenous enhancers contain multiple binding sites for different transcription factors, accounting for these sites and their interactions leads to a combinatorial explosion of model parameters ( Garcia et al, 2016 , 2020 ); determin- ing the values of these parameters from simple experiments constitutes a computational—and conceptual—challenge ( Vincent et al, 2016 ; Garcia et al, 2016 , 2020 ). To render complex transcrip- tional regulatory systems tractable to theory, minimal synthetic enhancers have been engineered in bacteria ( Garcia and Phillips, 2011 ; Brewster et al, 2014 ; Razo-Mejia et al, 2018 ; Phillips et al, 2019 ), eukaryotic cells ( Popp et al, 2020 ), and developing organisms ( Fakhouri et al, 2010 ; Sayal et al, 2016 ). In such experiments, a short, synthetic DNA sequence with only one to a few binding sites for a single transcription factor drives the expression of a reporter gene.…”

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

“…Already from the seminal work of Jacob and Monod 1 , a central regulatory role was recognized for the equilibrium occupancy of response elements by TFs, which depends on their concentration, affinity, and, in eukaryotes, the chromatin accessibility of their binding sites 2,3 . However, recent studies have highlighted the importance of transient TF-DNA interactions, and indicate that the kinetics of TF binding and dissociation play an important role in regulating transcription in vivo [4][5][6][7][8][9][10][11] . In particular, the importance of the TF residence time in determining the transcriptional burst duration 6,10,11 and size 6,10 has been demonstrated.…”