Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 3 of 3 results

Transcription activation is enhanced by multivalent interactions independent of liquid-liquid phase separation.

blue CRY2/CIB1 HeLa U-2 OS Transgene expression
bioRxiv, 28 Jan 2021 DOI: 10.1101/2021.01.27.428421 Link to full text
Abstract: Transcription factors (TFs) consist of a DNA binding and an activation domain (AD) that are considered to be independent and exchangeable modules. However, recent studies conclude that also the physico-chemical properties of the AD can control TF assembly at chromatin via driving a phase separation into “transcriptional condensates”. Here, we dissected the mechanism of transcription activation at a reporter gene array with real-time single-cell fluorescence microscopy readouts. Our comparison of different synthetic TFs reveals that the phase separation propensity of the AD correlates with high transcription activation capacity by increasing binding site occupancy, residence time and the recruitment of co-activators. However, we find that the actual formation of phase separated TF liquid-like droplets has a neutral or inhibitory effect on transcription induction. Thus, our study suggests that the ability of a TF to phase separate reflects the functionally important property of the AD to establish multivalent interactions but does not by itself enhance transcription.

Light-Induced Transcription Activation for Time-Lapse Microscopy Experiments in Living Cells.

blue CRY2/CIB1 U-2 OS
Methods Mol Biol, 13 Aug 2019 DOI: 10.1007/978-1-4939-9674-2_17 Link to full text
Abstract: 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.

Real-time observation of light-controlled transcription in living cells.

blue CRY2/CIB1 U-2 OS
J Cell Sci, 9 Nov 2017 DOI: 10.1242/jcs.205534 Link to full text
Abstract: Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) 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 cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min 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 of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.
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