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

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Imaging Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2038))

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.

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References

  1. Darzacq X, Yao J, Larson DR, Causse SZ, Bosanac L, de Turris V, Ruda VM, Lionnet T, Zenklusen D, Guglielmi B, Tjian R, Singer RH (2009) Imaging transcription in living cells. Annu Rev Biophys 38:173–196. https://doi.org/10.1146/annurev.biophys.050708.133728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Martin RM, Rino J, Carvalho C, Kirchhausen T, Carmo-Fonseca M (2013) Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity. Cell Rep 4(6):1144–1155. https://doi.org/10.1016/j.celrep.2013.08.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cho WK, Jayanth N, English BP, Inoue T, Andrews JO, Conway W, Grimm JB, Spille JH, Lavis LD, Lionnet T, Cisse II (2016) RNA Polymerase II cluster dynamics predict mRNA output in living cells. elife 5:e13617. https://doi.org/10.7554/eLife.13617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Larson DR, Fritzsch C, Sun L, Meng X, Lawrence DS, Singer RH (2013) Direct observation of frequency modulated transcription in single cells using light activation. elife 2:e00750. https://doi.org/10.7554/eLife.00750

    Article  PubMed  PubMed Central  Google Scholar 

  5. Chubb J, Trcek T, Shenoy S, Singer R (2006) Transcriptional pulsing of a developmental gene. Curr Biol 16(10):1018–1025. https://doi.org/10.1016/j.cub.2006.03.092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445

    Article  CAS  Google Scholar 

  7. Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy SM, Phair RD, Singer RH (2007) In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol 14(9):796–806. https://doi.org/10.1038/nsmb1280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Normanno D, Boudarene L, Dugast-Darzacq C, Chen J, Richter C, Proux F, Benichou O, Voituriez R, Darzacq X, Dahan M (2015) Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher. Nat Commun 6:7357. https://doi.org/10.1038/ncomms8357

    Article  PubMed  PubMed Central  Google Scholar 

  9. Janicki SM, Tsukamoto T, Salghetti SE, Tansey WP, Sachidanandam R, Prasanth KV, Ried T, Shav-Tal Y, Bertrand E, Singer RH, Spector DL (2004) From silencing to gene expression: real-time analysis in single cells. Cell 116(5):683–698

    Article  CAS  Google Scholar 

  10. Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, Tucker CL (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods 7(12):973–975. https://doi.org/10.1038/nmeth.1524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rademacher A, Erdel F, Trojanowski J, Schumacher S, Rippe K (2017) Real-time observation of light-controlled transcription in living cells. J Cell Sci 130(24):4213–4224. https://doi.org/10.1242/jcs.205534

    Article  CAS  PubMed  Google Scholar 

  12. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church GM, Zhang F (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500(7463):472–476. https://doi.org/10.1038/nature12466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Motta-Mena LB, Reade A, Mallory MJ, Glantz S, Weiner OD, Lynch KW, Gardner KH (2014) An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol 10(3):196–202. https://doi.org/10.1038/nchembio.1430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rullan M, Benzinger D, Schmidt GW, Milias-Argeitis A, Khammash M (2018) An Optogenetic platform for real-time, single-cell interrogation of stochastic transcriptional regulation. Mol Cell 70(4):745–756 e746. https://doi.org/10.1016/j.molcel.2018.04.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol 11(3):198–200. https://doi.org/10.1038/nchembio.1753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. R Core Team (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  17. Soutoglou E, Misteli T (2008) Activation of the cellular DNA damage response in the absence of DNA lesions. Science 320(5882):1507–1510. https://doi.org/10.1126/science.1159051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bintu L, Yong J, Antebi YE, McCue K, Kazuki Y, Uno N, Oshimura M, Elowitz MB (2016) Dynamics of epigenetic regulation at the single-cell level. Science 351(6274):720–724. https://doi.org/10.1126/science.aab2956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. RStudio Team (2015) RStudio: integrated development for R. RStudio, Inc, Boston, MA

    Google Scholar 

  20. Pau G, Fuchs F, Sklyar O, Boutros M, Huber W (2010) EBImage—an R package for image processing with applications to cellular phenotypes. Bioinformatics 26(7):979–981. https://doi.org/10.1093/bioinformatics/btq046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wickham H (2011) The split-apply-combine strategy for data analysis. J Stat Softw 40(1):1–29

    Article  Google Scholar 

  22. Wickham H (2016) ggplot2: elegant graphics for data analysis. Springer, New York

    Book  Google Scholar 

  23. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  24. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675

    Article  CAS  Google Scholar 

  25. Eck S, Worz S, Muller-Ott K, Hahn M, Biesdorf A, Schotta G, Rippe K, Rohr K (2016) A spherical harmonics intensity model for 3D segmentation and 3D shape analysis of heterochromatin foci. Med Image Anal 32:18–31. https://doi.org/10.1016/j.media.2016.03.001

    Article  PubMed  Google Scholar 

  26. Thevenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7(1):27–41. https://doi.org/10.1109/83.650848

    Article  CAS  PubMed  Google Scholar 

  27. Wu B, Chao JA, Singer RH (2012) Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys J 102(12):2936–2944. https://doi.org/10.1016/j.bpj.2012.05.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Shin Y, Berry J, Pannucci N, Haataja MP, Toettcher JE, Brangwynne CP (2017) Spatiotemporal control of intracellular phase transitions using light-activated optodroplets. Cell 168(1–2):159–171 e114. https://doi.org/10.1016/j.cell.2016.11.054

    Article  CAS  PubMed  Google Scholar 

  29. Modat M, Cash DM, Daga P, Winston GP, Duncan JS, Ourselin S (2014) Global image registration using a symmetric block-matching approach. J Med Imaging (Bellingham) 1(2):024003. https://doi.org/10.1117/1.JMI.1.2.024003

    Article  Google Scholar 

  30. Ferguson ML, Larson DR (2013) Measuring transcription dynamics in living cells using fluctuation analysis. In: Shav-Tal Y (ed) imaging gene expression: methods and protocols. Humana Press, New York, pp 47–60

    Chapter  Google Scholar 

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Acknowledgments

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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Authors and Affiliations

  1. Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany

    Jorge Trojanowski, Anne Rademacher, Fabian Erdel & Karsten Rippe

  2. Centre de Biologie Intégrative (CBI), CNRS, UPS, Toulouse, France

    Fabian Erdel

Authors
  1. Jorge Trojanowski
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  2. Anne Rademacher
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  3. Fabian Erdel
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  4. Karsten Rippe
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Corresponding author

Correspondence to Karsten Rippe .

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Editors and Affiliations

  1. The Mina & Everard Goodman, Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, Israel

    Yaron Shav-Tal

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Trojanowski, J., Rademacher, A., Erdel, F., Rippe, K. (2019). Light-Induced Transcription Activation for Time-Lapse Microscopy Experiments in Living Cells. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_17

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  • DOI: https://doi.org/10.1007/978-1-4939-9674-2_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9673-5

  • Online ISBN: 978-1-4939-9674-2

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