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bioRxiv · 10.1101/132050

Real-Time Observation Of Light-Controlled Transcription In Living Cells

Rademacher, A.·Erdel, F.·Trojanowski, J.·Rippe, K.
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Abstract

Gene expression is a tightly controlled process that is coordinated in space and time. To dissect its dynamic regulation with high temporal resolution, we introduce an optogenetic tool termed BLInCR (Blue Light-Induced Chromatin Recruitment) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a reporter gene cluster in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable [~]2 minutes after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models for transcription activation suggests that the gene cluster undergoes a maturation process when activated. BLInCR will facilitate the study of transcription dynamics in living cells.

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Rademacher, A., Erdel, F., Trojanowski, J., Rippe, K.. 2017-04-28. Real-Time Observation Of Light-Controlled Transcription In Living Cells. https://doi.org/10.1101/132050

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