Single-molecule visualization of transcription dynamics to understand regulatory mechanisms of cofactors

Transcription is a multi-step process involving many regulatory factors, including cofactors such as Mediator, chromatin remodelers and chromatin modifiers.

Cofactors are thought to enhance transcription efficiency, but a detailed mechanistic understanding of cofactor action is still lacking.

Inside single cells, the transcriptional process is highly dynamic, with regulatory factors transiently associating to DNA, and transcription occurring in stochastic bursts of high transcriptional activity, interspersed by periods of inactivity. How cofactors regulate these dynamics is so far largely unexplored.

In this proposal, we aim to elucidate the molecular mechanisms of cofactor action, by combining perturbation experiments with cutting-edge single-molecule imaging techniques to measure the DNA binding dynamics of regulatory factors (input) as well as the stochastic behavior of transcription (output) in living yeast cells.

First, bursting properties will be quantified at endogenous genes upon single and double cofactor perturbations to identify rate-limiting steps that are regulated by cofactors and to determine cooperativity between cofactors.

Second, the DNA binding dynamics of cofactors, transcription factors and the preinitiation complex will be determined in wildtype and upon perturbations of cooperating cofactors, allowing us to construct a kinetic model of transcription.

Third, building on our recently developed imaging technique, we will develop a new technology to simultaneously visualize the DNA binding of single cofactor molecules and transcriptional output at a single target locus to dissect the functional effect of cofactor DNA binding events on transcription activation.

Overall, our results will provide unique insight into the composition of and transitions between transcription regulatory steps, uncovering mechanisms how cofactors increase transcription efficiency.