Journal article Open Access

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


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    <dct:title>An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation</dct:title>
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    <dct:description>&lt;p&gt;&lt;strong&gt;Highlights&lt;/strong&gt;&lt;/p&gt; &lt;ul&gt; &lt;li&gt;Live single-cell quantification of light-activated transcriptional bursts in yeast&lt;/li&gt; &lt;li&gt;A platform for precise light targeting enables single-cell dynamic feedback control&lt;/li&gt; &lt;li&gt;Single-cell regulation markedly reduces cell-to-cell variability&lt;/li&gt; &lt;li&gt;Transcription factor activity modulates burst timing and duration&lt;/li&gt; &lt;/ul&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Summary&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to&amp;nbsp;analyze how the dynamic activity of transcriptional&amp;nbsp;regulators affects stochastic transcription. By&amp;nbsp;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&amp;nbsp;&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;. 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.&lt;/p&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt;</dct:description>
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