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

Información del proyecto

BURSTREG

Identificador del acuerdo de subvención: 755695

DOI

10.3030/755695

Proyecto cerrado

Fecha de la firma de la CE 6 Septiembre 2017

Fecha de inicio 1 Enero 2018

Fecha de finalización 31 Diciembre 2022

Financiado con arreglo a

• EXCELLENT SCIENCE - European Research Council (ERC)

Coste total

€ 1 950 775,00

Aportación de la UE

€ 1 950 775,00

Coordinado por

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)

Período documentado: 2022-07-01 hasta 2022-12-31

Resumen del contexto y de los objetivos generales del proyecto

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.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

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.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

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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