Single-molecule visualization of transcription dynamics to understand regulatory mechanisms of transcriptional bursting and its effects on cellular fitness

Project Information

BURSTREG

Grant agreement ID: 755695

DOI

10.3030/755695

Project closed

EC signature date 6 September 2017

Start date 1 January 2018

End date 31 December 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 950 775,00

EU contribution

€ 1 950 775,00

Coordinated by

STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS
Netherlands

Periodic Reporting for period 4 - BURSTREG (Single-molecule visualization of transcription dynamics to understand regulatory mechanisms of transcriptional bursting and its effects on cellular fitness)

Reporting period: 2022-07-01 to 2022-12-31

Summary of the context and overall objectives of the project

Transcription, the process where DNA is copied to RNA to eventually form proteins, is often not continuous in time, but happens in bursts of high activity, followed by periods of inactivity. These bursts result in variability in expression between cells, which can influence cell fate decisions and disease progression. In addition, tumor cell populations often show increased gene expression heterogeneity compared to normal tissues, which forms a major barrier in the efficient diagnosis and treatment. Although the source of this heterogeneity was originally thought to be genetic mutations that accumulated over time, it is becoming clear that tumor heterogeneity can also be caused by non-genetic variations, such as epigenetic or gene expression variations. It is unclear why tumors display increased variability, and how this may be beneficial for tumor progression. The overarching goal of the current study is to test the mechanistic basis and functional effect of stochastic gene expression heterogeneity.
Expression variation of a gene is affected by bursting, as changing the duration and the rate of switching between periods of activity and inactivity directly changes its cell-to-cell variation. However, even though transcriptional bursting is conserved from bacteria to yeast to human cells, the origin and regulators of bursting remain largely unknown. In this project, we aim to understand how regulatory DNA sequences and regulatory factors control bursting. In addition, we examine the effect of different bursting patterns on fitness. Overall our project will give unprecedented insight into the mechanism and function of gene expression heterogeneity, which may reveal important insight into how tumors may utilize this to select for heterogeneity.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In this project, we aim to understand how regulatory DNA sequences and regulatory factors control bursting. Using a cutting-edge single-molecule RNA imaging techniques to directly measure transcriptional bursting in living yeast cells, we showed how bursting is regulated by DNA supercoiling, spatial clustering of transcription factors and by chromatin remodeling of promoter nucleosomes. For example, we found that remodeling of different promoter nucleosomes are controlling different aspects of bursting. In addition, as proposed in this project, we have developed a novel platform to visualize the binding of upstream regulatory factors and the RNA output in the same cell. This novel imaging assay allowed us to uncovered that the dwell time transcription factors to their regulatory sequence directly determines the number of RNAs produced in a burst. We also found evidence that the transcription factor dwell time inside cells is reduced by several orders of magnitude by promoter nucleosomes, which represents a novel mechanism for transcription regulation by chromatin.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

To understand how bursting is controlled, we have developed a novel single-molecule imaging techniques to simultaneously observe transcription factor binding and transcription kinetics at a single endogenous locus in real time. As far as we are aware, this technique showed for the first time the correlation and timing of transcription factors regulation on the stochastic production of transcriptional bursts in living cells. Currently, we are continuing to develop similar novel methods to improve transcription measurements at single-molecule resolution. We have used these cutting-edge techniques to quantatively show how regulatory factors and their binding dynamics control bursting. Overall, these approaches have revealed how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.

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