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Using RNA Tags for Multicolor Live Imaging of Chromatin Loci and Transcription in Drosophila Embryos

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Book cover RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

Elucidating the biological implications of higher order chromatin architectures in animal development requires simultaneous, quantitative measurements of chromatin dynamics and transcriptional activity in living specimen. Here we describe a multicolor labeling and live imaging approach in embryos of the fruit fly Drosophila melanogaster. The method allows simultaneous measurement of movements of specific loci and their transcriptional activity for developmental genes, enabling new approaches to probe the interaction between 3D chromatin architecture and regulation of gene expression in development.

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References

  1. Furlong EEM, Levine M (2018) Developmental enhancers and chromosome topology. Science 361(6409):1341–1345. https://doi.org/10.1126/science.aau0320

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dekker J, Belmont AS, Guttman M, Leshyk VO, Lis JT, Lomvardas S, Mirny LA, O’Shea CC, Park PJ, Ren B, Politz JCR, Shendure J, Zhong S, Network DN (2017) The 4D nucleome project. Nature 549(7671):219–226. https://doi.org/10.1038/nature23884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Vermunt MW, Zhang D, Blobel GA (2019) The interdependence of gene-regulatory elements and the 3D genome. J Cell Biol 218(1):12–26. https://doi.org/10.1083/jcb.201809040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Beagrie RA, Scialdone A, Schueler M, Kraemer DC, Chotalia M, Xie SQ, Barbieri M, de Santiago I, Lavitas LM, Branco MR, Fraser J, Dostie J, Game L, Dillon N, Edwards PA, Nicodemi M, Pombo A (2017) Complex multi-enhancer contacts captured by genome architecture mapping. Nature 543(7646):519–524. https://doi.org/10.1038/nature21411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gibcus JH, Samejima K, Goloborodko A, Samejima I, Naumova N, Nuebler J, Kanemaki MT, Xie L, Paulson JR, Earnshaw WC, Mirny LA, Dekker J (2018) A pathway for mitotic chromosome formation. Science 359(6376):eaaao6135. https://doi.org/10.1126/science.aao6135

    Article  CAS  Google Scholar 

  6. Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332(6028):475–478. https://doi.org/10.1126/science.1202142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hocine S, Raymond P, Zenklusen D, Chao JA, Singer RH (2013) Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat Methods 10(2):119–121. https://doi.org/10.1038/nmeth.2305

    Article  CAS  PubMed  Google Scholar 

  8. Golding I, Paulsson J, Zawilski SM, Cox EC (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123(6):1025–1036. https://doi.org/10.1016/j.cell.2005.09.031

    Article  CAS  PubMed  Google Scholar 

  9. Garcia HG, Tikhonov M, Lin A, Gregor T (2013) Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Curr Biol 23(21):2140–2145. https://doi.org/10.1016/j.cub.2013.08.054

    Article  CAS  PubMed  Google Scholar 

  10. Fukaya T, Lim B, Levine M (2016) Enhancer control of transcriptional bursting. Cell 166(2):358–368. https://doi.org/10.1016/j.cell.2016.05.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lucas T, Ferraro T, Roelens B, De Las Heras Chanes J, Walczak AM, Coppey M, Dostatni N (2013) Live imaging of bicoid-dependent transcription in Drosophila embryos. Curr Biol 23(21):2135–2139. https://doi.org/10.1016/j.cub.2013.08.053

    Article  CAS  PubMed  Google Scholar 

  12. Saad H, Gallardo F, Dalvai M, Tanguy-le-Gac N, Lane D, Bystricky K (2014) DNA dynamics during early double-strand break processing revealed by non-intrusive imaging of living cells. PLoS Genet 10(3):e1004187. https://doi.org/10.1371/journal.pgen.1004187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mariame B, Kappler-Gratias S, Kappler M, Balor S, Gallardo F, Bystricky K (2018) Real-time visualization and quantification of human cytomegalovirus replication in living cells using the ANCHOR DNA labeling technology. J Virol 92(18):e00571–e00518. https://doi.org/10.1128/JVI.00571-18

    Article  PubMed  PubMed Central  Google Scholar 

  14. Dubarry N, Pasta F, Lane D (2006) ParABS systems of the four replicons of Burkholderia cenocepacia: new chromosome centromeres confer partition specificity. J Bacteriol 188(4):1489–1496. https://doi.org/10.1128/JB.188.4.1489-1496.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gratz SJ, Harrison MM, Wildonger J, O’Connor-Giles KM (2015) Precise genome editing of Drosophila with CRISPR RNA-guided Cas9. Methods Mol Biol 1311:335–348. https://doi.org/10.1007/978-1-4939-2687-9_22

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kondo S, Ueda R (2013) Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195(3):715–721. https://doi.org/10.1534/genetics.113.156737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104(9):3312–3317. https://doi.org/10.1073/pnas.0611511104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chen H, Levo M, Barinov L, Fujioka M, Jaynes JB, Gregor T (2018) Dynamic interplay between enhancer-promoter topology and gene activity. Nat Genet 50(9):1296–1303. https://doi.org/10.1038/s41588-018-0175-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Chen H, Xu Z, Mei C, Yu D, Small S (2012) A system of repressor gradients spatially organizes the boundaries of Bicoid-dependent target genes. Cell 149(3):618–629. https://doi.org/10.1016/j.cell.2012.03.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bateman JR, Lee AM, Wu CT (2006) Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. Genetics 173(2):769–777. https://doi.org/10.1534/genetics.106.056945

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Garcia HG, Gregor T (2018) Live imaging of mRNA synthesis in Drosophila. Methods Mol Biol 1649:349–357. https://doi.org/10.1007/978-1-4939-7213-5_23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194(4):1029–1035. https://doi.org/10.1534/genetics.113.152710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Hongtao Chen & Thomas Gregor

  2. Joseph Henry Laboratories of Physics, Princeton University, Princeton, NJ, USA

    Thomas Gregor

  3. Department of Developmental and Stem Cell Biology, UMR3738, Institut Pasteur, Paris, France

    Thomas Gregor

Authors
  1. Hongtao Chen
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  2. Thomas Gregor
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Corresponding author

Correspondence to Thomas Gregor .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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Chen, H., Gregor, T. (2020). Using RNA Tags for Multicolor Live Imaging of Chromatin Loci and Transcription in Drosophila Embryos. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_22

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

  • eBook Packages: Springer Protocols

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