Light-Induced Transcription Activation for Time-Lapse Microscopy Experiments in Living Cells
Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.
Key wordsOptogenetics Transcription dynamics Automated microscopy Image quantification
We thank Pranas Grigaitis for help and the DKFZ light microscopy core facility for technical support. This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant RI 1283/14-1 to K.R.) and the project ENHANCE within the NCT 3.0 program of the National Center for Tumor Diseases (NCT), Heidelberg.
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