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Publication . Article . 2018

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation

Rullan Marc; Benzinger Dirk; Gregor W. Schmidt; Milias-Argeitis, Andreas; Mustafa Khammash;
Open Access   English  

doi: 10.3929/ethz-b-000267237 , 10.1016/j.molcel.2018.04.012

handle: 20.500.11850/267237 , 11370/19164865-28b8-4cff-94ab-735627838358

pmc: PMC5971206

pmid: 29775585

Published: 17 May 2018
Abstract
Summary Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells.
Rullan et al. develop an optogenetic framework for elucidating stochastic transcription at the single-cell level. Combining live-cell nascent RNA quantification with optogenetic transcription in an automated setup for spatiotemporal light delivery, the authors establish real-time light-based feedback control in single cells. The method is used to study transcriptional burst dynamics.
Highlights • Live single-cell quantification of light-activated transcriptional bursts in yeast • A platform for precise light targeting enables single-cell dynamic feedback control • Single-cell regulation markedly reduces cell-to-cell variability • Transcription factor activity modulates burst timing and duration
Graphical Abstract
Subjects by Vocabulary

Microsoft Academic Graph classification: Transcriptional regulation Single-cell analysis Optogenetics Saccharomyces cerevisiae biology.organism_classification biology Transcription factor Transcription (biology) Cell biology Transcriptional bursting Regulation of gene expression

Subjects

Optogenetics; Cybergenetics; Feedback regulation; Single cell; Transcription; Stochasticity; Transcriptional bursting; PP7; EL222, Optogenetics, Cybergenetics, Feedback regulation, Single cell, Transcription, Stochasticity, Transcriptional bursting, PP7, EL222, Article, Cell Biology, Molecular Biology

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Funded by
EC| CyberGenetics
Project
CyberGenetics
Cybergenetics: Theory and Design Tools for Biomolecular Control Systems
  • Funder: European Commission (EC)
  • Project Code: 743269
  • Funding stream: H2020 | ERC | ERC-ADG
Validated by funder
, EC| COSY-BIO
Project
COSY-BIO
Control Engineering of Biological Systems for Reliable Synthetic Biology Applications
  • Funder: European Commission (EC)
  • Project Code: 766840
  • Funding stream: H2020 | RIA
Validated by funder