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Imaging Chromosome Territory and Gene Loci Positions in Cells Grown on Soft Matrices

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Imaging Gene Expression

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

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Abstract

It is well established that the genome is non-randomly organized in the interphase nucleus with gene rich chromosome territories toward the nuclear interior, while gene poor chromosome territories are proximal to the nuclear periphery. In vivo tissue stiffness and architecture modulates cell type-specific genome organization and gene expression programs. However, the impact of external mechanical forces on the non-random organization of the genome is not completely understood. Here we describe a modified protocol for visualizing chromosome territories and gene loci positions in cells exposed to reduced matrix stiffness by employing soft polyacrylamide matrices. 3-Dimensional Fluorescence In Situ Hybridization (3D-FISH) protocol followed by image analyses performed on cells exposed to extracellular matrices of varying stiffness properties, enables the determination of the dynamics of chromosome territories as well as gene loci in the interphase nucleus. This will be useful in understanding how chromosome territories respond to changes in substrate stiffness and the potential correlation between the repositioning of chromosome territories and their respective transcriptional profiles.

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References

  1. Bolzer A, Kreth G, Solovei I et al (2005) Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS Biol 3:e157

    Article  Google Scholar 

  2. Cremer M, Küpper K, Wagler B, Wizelman L, von Hase J, Weiland Y, Kreja L, Diebold J, Speicher MR, Cremer T (2003) Inheritance of gene density-related higher order chromatin arrangements in normal and tumor cell nuclei. J Cell Biol 162:809–820

    Article  CAS  Google Scholar 

  3. Sun HB, Shen J, Yokota H (2000) Size-dependent positioning of human chromosomes in interphase nuclei. Biophys J 79:184–190

    Article  CAS  Google Scholar 

  4. Mayer R, Brero A, von Hase J, Schroeder T, Cremer T, Dietzel S (2005) Common themes and cell type specific variations of higher order chromatin arrangements in the mouse. BMC Cell Biol 6:44

    Article  Google Scholar 

  5. Lieberman-Aiden E, van Berkum NL, Williams L et al (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326:289–293

    Article  CAS  Google Scholar 

  6. van Berkum NL, Lieberman-Aiden E, Williams L, Imakaev M, Gnirke A, Mirny LA, Dekker J, Lander ES (2010) Hi-C: a method to study the three-dimensional architecture of genomes. J Vis Exp https://doi.org/10.3791/1869

  7. Kalhor R, Tjong H, Jayathilaka N, Alber F, Chen L (2011) Genome architectures revealed by tethered chromosome conformation capture and population-based modeling. Nat Biotechnol 30:90–98

    Article  Google Scholar 

  8. Acevedo-Acevedo S, Crone WC (2015) Substrate stiffness effect and chromosome missegregation in hIPS cells. J Negat Results Biomed 14:22

    Article  Google Scholar 

  9. Cremer M, von Hase J, Volm T, Brero A, Kreth G, Walter J, Fischer C, Solovei I, Cremer C, Cremer T (2001) Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells. Chromosom Res 9:541–567

    Article  CAS  Google Scholar 

  10. Croft JA, Bridger JM, Boyle S, Perry P, Teague P, Bickmore WA (1999) Differences in the localization and morphology of chromosomes in the human nucleus. J Cell Biol 145:1119–1131

    Article  CAS  Google Scholar 

  11. Georges PC, Janmey PA (2005) Cell type-specific response to growth on soft materials. J Appl Physiol 98:1547–1553

    Article  Google Scholar 

  12. Miller RT, Janmey PA (2015) Relationship of and cross-talk between physical and biologic properties of the glomerulus. Curr Opin Nephrol Hypertens 24:393–400

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Tanabe H, Müller S, Neusser M, von Hase J, Calcagno E, Cremer M, Solovei I, Cremer C, Cremer T (2002) Evolutionary conservation of chromosome territory arrangements in cell nuclei from higher primates. Proc Natl Acad Sci U S A 99:4424–4429

    Article  CAS  Google Scholar 

  14. Parada LA, McQueen PG, Misteli T (2004) Tissue-specific spatial organization of genomes. Genome Biol 5:R44

    Article  Google Scholar 

  15. Zink D, Amaral MD, Englmann A et al (2004) Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei. J Cell Biol 166:815–825

    Article  CAS  Google Scholar 

  16. Meaburn KJ, Gudla PR, Khan S, Lockett SJ, Misteli T (2009) Disease-specific gene repositioning in breast cancer. J Cell Biol 187:801–812

    Article  CAS  Google Scholar 

  17. Meaburn KJ, Agunloye O, Devine M, Leshner M, Roloff GW, True LD, Misteli T (2016) Tissue-of-origin-specific gene repositioning in breast and prostate cancer. Histochem Cell Biol 145:433–446

    Article  CAS  Google Scholar 

  18. Roskelley CD, Desprez PY, Bissell MJ (1994) Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction. Proc Natl Acad Sci U S A 91:12378–12382

    Article  CAS  Google Scholar 

  19. Caron JM (1990) Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol 10:1239–1243

    Article  CAS  Google Scholar 

  20. Assoian RK, Klein EA (2008) Growth control by intracellular tension and extracellular stiffness. Trends Cell Biol 18:347–352

    Article  CAS  Google Scholar 

  21. Chen H, Comment N, Chen J, Ronquist S, Hero A, Ried T, Rajapakse I (2015) Chromosome conformation of human fibroblasts grown in 3-dimensional spheroids. Nucleus 6:55–65

    Article  CAS  Google Scholar 

  22. Chen H, Seaman L, Liu S, Ried T, Rajapakse I (2017) Chromosome conformation and gene expression patterns differ profoundly in human fibroblasts grown in spheroids versus monolayers. Nucleus 8:383–391

    Article  CAS  Google Scholar 

  23. Wang Y, Nagarajan M, Uhler C, Shivashankar GV (2017) Orientation and repositioning of chromosomes correlate with cell geometry-dependent gene expression. Mol Biol Cell 28:1997–2009

    Article  CAS  Google Scholar 

  24. Pradhan R, Ranade D, Sengupta K (2018) Emerin modulates spatial organization of chromosome territories in cells on softer matrices. Nucleic Acids Res 46:5561–5586

    Article  CAS  Google Scholar 

  25. Fischer RS, Myers KA, Gardel ML, Waterman CM (2012) Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior. Nat Protoc 7:2056–2066

    Article  CAS  Google Scholar 

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Acknowledgments

This work is supported by IISER Pune and Wellcome Trust–Department of Biotechnology India Alliance (Grant number: 500164/Z/09/Z) by funding through an intermediate fellowship to K.S. Council of Scientific and Industrial Research, New Delhi supported RP by Senior Research Fellowship. We gratefully acknowledge facilities and equipment of Indian Institute of Science Education and Research (IISER), Pune. We thank the IISER Pune Microscopy Facility and Chromosome Biology Lab (CBL) members for their comments and suggestions.

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Authors and Affiliations

  1. Biology, Main Building, Indian Institute of Science Education and Research (IISER), Pune, Maharashtra, India

    Roopali Pradhan & Kundan Sengupta

Authors
  1. Roopali Pradhan
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  2. Kundan Sengupta
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Correspondence to Kundan Sengupta .

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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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Pradhan, R., Sengupta, K. (2019). Imaging Chromosome Territory and Gene Loci Positions in Cells Grown on Soft Matrices. 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_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9674-2_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9673-5

  • Online ISBN: 978-1-4939-9674-2

  • eBook Packages: Springer Protocols

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