Live‐cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting

Abstract

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single‐molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Normative models of enhancer function

“…In contrast, live imaging of the nascent mRNA could put constraints on T E [1]. In that case, the filtering time scale is the elongation time, typically on the order of a few minutes, while the reported transcriptional response times—and thus estimates of T E —would range from minutes to 1 − 2 hours [9, 26].…”

“…Likewise, short TF residence times (ii) and the importance of TF arrival ordering (iii) contradict the conceptual picture where stable enhanceosomes are assembled in equilibrium [4]. Kinetic schemes may be required to match the reported characteristics of bursty gene expression (vi) [26], or realize high cooperativity (vii) [27]. Thermodynamic models undisputedly have statistical power to predict expression from regulatory sequence even in eukaryotes [28], yet this does not resolve their biophysical inconsistencies or rule out non-equilibrium models.…”

Short residence times of DNA-bound transcription factors can reduce gene expression noise and increase the transmission of information in a gene regulation system

“…First, in the last decade methods to quantify the ability of a regulated gene to acquire information about the concentration of a TF have become standardized (10,44). Also, the effect of residence time on noise can be quantified with techniques that simultaneously quantify TF-DNA binding events and the production of mRNA molecules, which are being developed (16). It is especially difficult to obtain multiple alleles of a TF with different residence times without also altering the TF's affinity to DNA.…”

“…The residence time is equal to the inverse of the dissociation rate kd between a TF and DNA. Theoretical and experimental work has shown that the dissociation rate can affect gene expression, affect the size of gene expression bursts (16)(17)(18)(19), and modulate gene expression noise (20)(21)(22)(23). However, it is difficult to discern whether the dissociation rate affects gene expression by altering the residence time or the affinity between a TF and DNA, because both depend on the dissociation constant kd (affinity is given by the ratio Keq=kd/ka [M], where ka is association rate between a TF and DNA).…”

“…For example, it is difficult to measure residence time and simultaneously quantify the rate of gene expression. It is also hard to quantify the number of TF binding events at a specific binding site in a given time (16,24,31). Moreover, it is challenging to experimentally modify only residence time without also affecting affinity.…”