#	Pagina
attuale pagina	/open-h2020/projects/211819/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - BURSTREG (Single-molecule visualization of transcription dynamics to understand regulatory mechanisms of transcriptional bursting and its effects on cellular fitness)

Teaser

Summary

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

In this project, we aim to understand how regulatory DNA sequences and regulatory factors control bursting. We have identified several factors that affect bursting and are now quantitatively characterizing their role in bursting regulation using a cutting-edge single-molecule RNA imaging techniques to directly measure transcriptional bursting in living yeast cells. 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.

Final results

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 are using these cutting-edge techniques to quantitively understand how regulatory factors and their binding dynamics control bursting. Overall, we expect that these approaches will reveal how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.