Advertisement

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

  • Roopali Pradhan
  • Kundan SenguptaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

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.

Key words

3D-FISH Polyacrylamide matrices Stiffness Chromosome territories Gene loci 

Notes

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.

References

  1. 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:e157CrossRefGoogle Scholar
  2. 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 162:809–820CrossRefGoogle Scholar
  3. 3.
    Sun HB, Shen J, Yokota H (2000) Size-dependent positioning of human chromosomes in interphase nuclei. Biophys J 79:184–190CrossRefGoogle Scholar
  4. 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 6:44CrossRefGoogle Scholar
  5. 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 326:289–293CrossRefGoogle Scholar
  6. 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. 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 30:90–98CrossRefGoogle Scholar
  8. 8.
    Acevedo-Acevedo S, Crone WC (2015) Substrate stiffness effect and chromosome missegregation in hIPS cells. J Negat Results Biomed 14:22CrossRefGoogle Scholar
  9. 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 9:541–567CrossRefGoogle Scholar
  10. 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 145:1119–1131CrossRefGoogle Scholar
  11. 11.
    Georges PC, Janmey PA (2005) Cell type-specific response to growth on soft materials. J Appl Physiol 98:1547–1553CrossRefGoogle Scholar
  12. 12.
    Miller RT, Janmey PA (2015) Relationship of and cross-talk between physical and biologic properties of the glomerulus. Curr Opin Nephrol Hypertens 24:393–400PubMedPubMedCentralGoogle Scholar
  13. 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 99:4424–4429CrossRefGoogle Scholar
  14. 14.
    Parada LA, McQueen PG, Misteli T (2004) Tissue-specific spatial organization of genomes. Genome Biol 5:R44CrossRefGoogle Scholar
  15. 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 166:815–825CrossRefGoogle Scholar
  16. 16.
    Meaburn KJ, Gudla PR, Khan S, Lockett SJ, Misteli T (2009) Disease-specific gene repositioning in breast cancer. J Cell Biol 187:801–812CrossRefGoogle Scholar
  17. 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 145:433–446CrossRefGoogle Scholar
  18. 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 91:12378–12382CrossRefGoogle Scholar
  19. 19.
    Caron JM (1990) Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol 10:1239–1243CrossRefGoogle Scholar
  20. 20.
    Assoian RK, Klein EA (2008) Growth control by intracellular tension and extracellular stiffness. Trends Cell Biol 18:347–352CrossRefGoogle Scholar
  21. 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 6:55–65CrossRefGoogle Scholar
  22. 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 8:383–391CrossRefGoogle Scholar
  23. 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 28:1997–2009CrossRefGoogle Scholar
  24. 24.
    Pradhan R, Ranade D, Sengupta K (2018) Emerin modulates spatial organization of chromosome territories in cells on softer matrices. Nucleic Acids Res 46:5561–5586CrossRefGoogle Scholar
  25. 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 7:2056–2066CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biology, Main BuildingIndian Institute of Science Education and Research (IISER)PuneIndia

Personalised recommendations