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

Molecular Cell
Marc RullanMustafa Khammash

Abstract

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.

Citations

May 19, 2019·The EMBO Journal·Benjamin T DonovanTineke L Lenstra
Dec 4, 2019·Biotechnology and Bioengineering·Jidapas My An-AdirekkunMegan N McClean
Apr 14, 2020·PLoS Computational Biology·Jean-Baptiste LugagneMary J Dunlop
Oct 4, 2019·Nature Communications·Elisa PedoneLucia Marucci
Apr 1, 2019·Advanced Biosystems·Javier Santos-Moreno, Yolanda Schaerli
Oct 24, 2019·Development·Daniel KruegerStefano De Renzis
Mar 15, 2020·Nature Communications·Melinda Liu PerkinsMustafa Khammash
Oct 10, 2020·PLoS Computational Biology·Jan Mikelson, Mustafa Khammash
Oct 30, 2020·Yeast·David FigueroaFrancisco Salinas
Sep 16, 2020·Current Opinion in Biotechnology·César Carrasco-LópezJosé L Avalos
Dec 9, 2020·International Journal of Molecular Sciences·Alvaro BanderasPascal Hersen
Jan 26, 2021·ACS Synthetic Biology·Robert J LovelettJosé L Avalos
Oct 4, 2019·Current Opinion in Systems Biology·Jean-Baptiste Lugagne, Mary J Dunlop
Apr 28, 2021·ACS Synthetic Biology·Elisa PedoneLucia Marucci
Apr 29, 2021·Nature Communications·Giansimone PerrinoDiego di Bernardo
Jun 2, 2021·Nucleic Acids Research·Achim P PoppJ Christof M Gebhardt
Jun 3, 2021·ACS Synthetic Biology·Alina BurmeisterAlexander Grünberger
Jun 3, 2021·Cells·Anja J EngelGerhard Thiel
Jun 2, 2021·Current Opinion in Microbiology·Giansimone PerrinoGuy-Bart Stan
Aug 5, 2021·Nature Reviews. Microbiology·Lisa Zillig, Harrison Steel
Jul 10, 2018·Chemical Reviews·Aba LosiAndreas Möglich
Oct 27, 2018·Analytical Chemistry·Pieter E OomenAndrew G Ewing
Oct 7, 2021·Nature Communications·Chetan AdityaJakob Ruess
Sep 26, 2021·Nature Communications·Sant KumarMustafa Khammash
Jan 8, 2022·ACS Synthetic Biology·Zhiqian WangHoujin Zhang

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Methods Mentioned

BETA
chip
smFISH
fluorescence recovery after photobleaching
restriction-ligation
PCRs
PCR
restriction ligation

Software Mentioned

MATLAB MathWorks
CellX
smFISH
MATLAB
YouScope

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