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Automated 3D Gene Position Analysis Using a Customized Imaris Plugin: XTFISHInsideNucleus

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Plant Chromatin Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1675))

Abstract

Fluorescence in situ hybridization (FISH) is commonly used to visualize chromosomal regions or genomic loci within the nucleus, and can largely contribute to unraveling the link between structure and function in the nucleus. Three-dimensional (3D) analyses are required to best capture the nucleus’ organizing principles, but the experimental setup and computational analyses are far from trivial. Here, we present a robust workflow for 3D FISH against repeats and single copy loci in embedded intact nuclei from Arabidopsis leaves. We then describe in detail the image acquisition, subsequent image deconvolution before 3D image processing, and the image reconstruction. We developed an automated batch image processing pipeline using a customized, open source plugin implemented in the Imaris environment.

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References

  1. Zorn C, Cremer T, Cremer C, Zimmer J (1976) Laser UV microirradiation of interphase nuclei and post-treatment with caffeine. A new approach to establish the arrangement of interphase chromosomes. Hum Genet 35(1):83–89

    Article  CAS  PubMed  Google Scholar 

  2. Cremer T, Cremer C, Schneider T, Baumann H, Hens L, Kirsch-Volders M (1982) Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments. Hum Genet 62(3):201–209

    Article  CAS  PubMed  Google Scholar 

  3. Shaw PJ, Abranches R, Paula Santos A, Beven AF, Stoger E, Wegel E, Gonzalez-Melendi P (2002) The architecture of interphase chromosomes and nucleolar transcription sites in plants. J Struct Biol 140(1–3):31–38. doi:10.1016/S1047-8477(02)00537-3

    Article  CAS  PubMed  Google Scholar 

  4. Pecinka A, Schubert V, Meister A, Kreth G, Klatte M, Lysak MA, Fuchs J, Schubert I (2004) Chromosome territory arrangement and homologous pairing in nuclei of Arabidopsis thaliana are predominantly random except for NOR-bearing chromosomes. Chromosoma 113(5):258–269. doi:10.1007/s00412-004-0316-2

    Article  CAS  PubMed  Google Scholar 

  5. Berr A, Pecinka A, Meister A, Kreth G, Fuchs J, Blattner FR, Lysak MA, Schubert I (2006) Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata. Plant J 48(5):771–783. doi:10.1111/j.1365-313X.2006.02912.x

    Article  CAS  PubMed  Google Scholar 

  6. Cremer T, Cremer C (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2(4):292–301

    Article  CAS  PubMed  Google Scholar 

  7. Ruault M, Dubarry M, Taddei A (2008) Re-positioning genes to the nuclear envelope in mammalian cells: impact on transcription. Trends Genet 24(11):574–581. doi:10.1016/j.tig.2008.08.008

    Article  CAS  PubMed  Google Scholar 

  8. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394(6693):592–595

    Article  CAS  PubMed  Google Scholar 

  9. Feng C-M, Qiu Y, Van Buskirk EK, Yang EJ, Chen M (2014) Light-regulated gene repositioning in Arabidopsis. Nat Commun 5:3027. doi:10.1038/ncomms4027

    PubMed  PubMed Central  Google Scholar 

  10. Bauwens S, Katsanis K, Van Montagu M, Van Oostveldt P, Engler G (1994) Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana. Plant J 6(1):123–131. doi:10.1046/j.1365-313X.1994.6010123.x

    Article  Google Scholar 

  11. Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu C-t (2015) Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 6:7147. doi:10.1038/ncomms8147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bourbousse C, Mestiri I, Zabulon G, Bourge M, Formiggini F, Koini MA, Brown SC, Fransz P, Bowler C, Barneche F (2015) Light signaling controls nuclear architecture reorganization during seedling establishment. Proc Natl Acad Sci U S A 112(21):E2836–E2844. doi:10.1073/pnas.1503512112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Howe ES, Murphy SP, Bass HW (2013) Three-dimensional acrylamide fluorescence in situ hybridization for plant cells. Methods Mol Biol (Clifton, NJ) 990:53–66. doi:10.1007/978-1-62703-333-6_6

    Article  CAS  Google Scholar 

  14. She W, Grimanelli D, Baroux C (2014) An efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in Arabidopsis. J Vis Exp 88:51530. doi:10.3791/51530

    Google Scholar 

  15. Lysak M, Fransz P, Schubert I (2006) Cytogenetic analyses of Arabidopsis. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols. Humana Press, Totowa, NJ, pp 173–186. doi:10.1385/1-59745-003-0:173

    Chapter  Google Scholar 

  16. Bey TD, Koini M, Fransz PF (2017) Fluorescence in situ hybridization (FISH) and immunolabeling on 3D preserved nuclei. In: Bemer M, Baroux C (eds) Plant chromatin dynamics: methods and protocols. Springer, New York, NY. doi:10.1007/978-1-4939-7318-7_27

    Google Scholar 

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Acknowledgments

The authors thank Dr. Jana Döhner (Microscopy Imaging Facility, University of Zürich) for technical assistance with image acquisition and deconvolution; Dr. Peter Majer, Sacha Guyer, (Bitplane CH) for advice on the customized plugin; Drs. Wenjing She, Kostas Kritsas (University of Zürich), Imen Mestiri, and Fredy Barneche (IBENS Paris) for sharing experimental protocols as described here. This work was supported by a SystemsX iPhD grant (2014/235), the University of Zürich and the Baugarten Stiftung.

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Authors and Affiliations

  1. Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland

    Mariamawit S. Ashenafi & Célia Baroux

Authors
  1. Mariamawit S. Ashenafi
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  2. Célia Baroux
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Correspondence to Célia Baroux .

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Editors and Affiliations

  1. Department of Molecular Biology, Wageningen University & Research, Wageningen, The Netherlands

    Marian Bemer

  2. Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland

    Célia Baroux

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Ashenafi, M.S., Baroux, C. (2018). Automated 3D Gene Position Analysis Using a Customized Imaris Plugin: XTFISHInsideNucleus. In: Bemer, M., Baroux, C. (eds) Plant Chromatin Dynamics. Methods in Molecular Biology, vol 1675. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7318-7_32

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  • DOI: https://doi.org/10.1007/978-1-4939-7318-7_32

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7317-0

  • Online ISBN: 978-1-4939-7318-7

  • eBook Packages: Springer Protocols

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