Abstract
Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell’s nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.
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References
van Steensel B, Dekker J (2010) Genomics tools for unraveling chromosome architecture. Nat Biotechnol 28(10):1089–1095
Ay F, Noble WS (2015) Analysis methods for studying the 3D architecture of the genome. Genome Biol 16:183
Williamson I, Berlivet S, Eskeland R, Boyle S, Illingworth RS, Paquette D, Dostie J, Bickmore WA (2014) Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization. Genes Dev 28(24):2778–2791
Dion V, Gasser SM (2013) Chromatin movement in the maintenance of genome stability. Cell 152(6):1355–1364
Huet S, Lavelle C, Ranchon H, Carrivain P, Victor JM, Bancaud A (2014) Relevance and limitations of crowding, fractal, and polymer models to describe nuclear architecture. Int Rev Cell Mol Biol 307:443–479
Hajjoul H, Mathon J, Ranchon H, Goiffon I, Mozziconacci J, Albert B, Carrivain P, Victor JM, Gadal O, Bystricky K, Bancaud A (2013) High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome. Genome Res 23(11):1829–1838
Wang R, Mozziconacci J, Bancaud A, Gadal O (2015) Principles of chromatin organization in yeast: relevance of polymer models to describe nuclear organization and dynamics. Curr Opin Cell Biol 34:54–60
Albert B, Mathon J, Shukla A, Saad H, Normand C, Leger-Silvestre I, Villa D, Kamgoue A, Mozziconacci J, Wong H, Zimmer C, Bhargava P, Bancaud A, Gadal O (2013) Systematic characterization of the conformation and dynamics of budding yeast chromosome XII. J Cell Biol 202(2):201–210
Meister P, Gehlen LR, Varela E, Kalck V, Gasser SM (2010) Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol 470:535–567
Berger AB, Cabal GG, Fabre E, Duong T, Buc H, Nehrbass U, Olivo-Marin JC, Gadal O, Zimmer C (2008) High-resolution statistical mapping reveals gene territories in live yeast. Nat Methods 5(12):1031–1037
Serge A, Bertaux N, Rigneault H, Marguet D (2008) Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods 5(8):687–694
Hajjoul HM, Mathon J, Viero Y, Bancaud A (2011) Optimized micromirrors for three-dimensional single-particle tracking in living cells. Appl Phys Lett 98:243701
Therizols P, Duong T, Dujon B, Zimmer C, Fabre E (2010) Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres. Proc Natl Acad Sci U S A 107(5):2025–2030
Weber SC, Thompson MA, Moerner WE, Spakowitz AJ, Theriot JA (2012) Analytical tools to distinguish the effects of localization error, confinement, and medium elasticity on the velocity autocorrelation function. Biophys J 102(11):2443–2450
Backlund MP, Joyner R, Weis K, Moerner WE (2014) Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope. Mol Biol Cell 25(22):3619–3629
Michaelis C, Ciosk R, Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91(1):35–45
Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135(6 Pt 2):1685–1700
Lassadi I, Bystricky K (2011) Tracking of single and multiple genomic loci in living yeast cells. Methods Mol Biol 745:499–522
Loiodice I, Dubarry M, Taddei A (2014) Scoring and manipulating gene position and dynamics using FROS in budding yeast. Curr Protoc Cell Biol 62: Unit 22 17 21–14
Dubarry M, Loiodice I, Chen CL, Thermes C, Taddei A (2011) Tight protein-DNA interactions favor gene silencing. Genes Dev 25(13):1365–1370
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
Shaham S (2006) WormBook: methods in cell biology. The C. elegans Research Community (ed), WormBook
Winey M, Yarar D, Giddings TH Jr, Mastronarde DN (1997) Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes. Mol Biol Cell 8(11):2119–2132
Zhang B, Zerubia J, Olivo-Marin J (2007) Gaussian approximations of fluorescence microscope point-spread function models. Appl Opt 46(10):1819
Backlund MP, Joyner R, Moerner WE (2015) Chromosomal locus tracking with proper accounting of static and dynamic errors. Phys Rev E Stat Nonlin Soft Matter Phys 91(6):062716
Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont AS (2006) Long-range directional movement of an interphase chromosome site. Curr Biol 16(8):825–831
Saner N, Karschau J, Natsume T, Gierlinski M, Retkute R, Hawkins M, Nieduszynski CA, Blow JJ, de Moura AP, Tanaka TU (2013) Stochastic association of neighboring replicons creates replication factories in budding yeast. J Cell Biol 202(7):1001–1012
Acknowledgement
This work was supported by ATS-Nudgene and Emergence-CLEMgene of the Toulouse-IDEX. O.G. and C.N. are supported by Agence Nationale de la Recherche (ANDY). We thank I. Léger-Silvestre for thoughtful discussions and technical advices. Julien Mathon wrote the MTT-based executable files.
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Wang, R., Normand, C., Gadal, O. (2016). High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae . In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_4
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