Biopolym. Cell. 2018; 34(6):426-434.
http://dx.doi.org/10.7124/bc.00098D
Structure and Function of Biopolymers
DNA loop organization in glioblastoma T98G cells at their different functional states
1Afanasieva K. S., 1Semenova A. Y., 2Lukash L. L., 1Sivolob A. V.
  1. Educational and Scientific Center "Institute of Biology and Medicine",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143

Abstract

The loop domain organization of chromatin, which plays an important role in transcription regulation, may depend on the cell functional state. Aim. To investigate DNA loop reorganization upon functional transitions in the glioblastoma T98G cells. Methods. Single cell gel electrophoresis (a comet assay) was used to analyze the kinetics of the DNA loop migration from the nucleoids obtained from the lysed cells. Results. The cells arrested in the G1 phase of the cell cycle were characterized by a relatively low amount of DNA in the comet tails due to a low content of DNA in the loops which may be resolved by the comet assay (up to ~300 kb). After cell reactivation, the contour length of the loops essentially increased in parallel with a decrease in the linear loop density along the genome. Conclusions. An increase in the loop size and a respective decrease in the loop density may be a general feature of activated cells as we earlier observed similar effects upon activation of human lymphocytes.
Keywords: DNA loops, T98G cell line, comet assay, cell functional state.
Full text: (PDF, in English)

References

[1] Dekker J, Marti-Renom MA, Mirny LA. Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data. Nat Rev Genet. 2013;14(6):390-403.
[2] Gibcus JH, Dekker J. The hierarchy of the 3D genome. Mol Cell. 2013;49(5):773-82.
[3] Dekker J, Mirny L. The 3D Genome as Moderator of Chromosomal Communication. Cell. 2016;164(6):1110-1121.
[4] Dixon JR, Gorkin DU, Ren B. Chromatin Domains: The Unit of Chromosome Organization. Mol Cell. 2016;62(5):668-80. Review.
[5] Kadauke S, Blobel GA. Chromatin loops in gene regulation. Biochim Biophys Acta. 2009;1789(1):17-25.
[6] Sexton T, Cavalli G. The role of chromosome domains in shaping the functional genome. Cell. 2015;160(6):1049-59.
[7] Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009;326(5950):289-93.
[8] Rao SS, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665-80.
[9] Sanborn AL, Rao SS, Huang SC, Durand NC, Huntley MH, Jewett AI, Bochkov ID, Chinnappan D, Cutkosky A, Li J, Geeting KP, Gnirke A, Melnikov A, McKenna D, Stamenova EK, Lander ES, Aiden EL. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc Natl Acad Sci U S A. 2015;112(47):E6456-65.
[10] Fudenberg G, Imakaev M, Lu C, Goloborodko A, Abdennur N, Mirny LA. Formation of Chromosomal Domains by Loop Extrusion. Cell Rep. 2016;15(9):2038-49.
[11] Vian L, PД™kowska A, Rao SSP, Kieffer-Kwon KR, Jung S, Baranello L, Huang SC, El Khattabi L, Dose M, Pruett N, Sanborn AL, Canela A, Maman Y, Oksanen A, Resch W, Li X, Lee B, Kovalchuk AL, Tang Z, Nelson S, Di Pierro M, Cheng RR, Machol I, St Hilaire BG, Durand NC, Shamim MS, Stamenova EK, Onuchic JN, Ruan Y, Nussenzweig A, Levens D, Aiden EL, Casellas R. The Energetics and Physiological Impact of Cohesin Extrusion. Cell. 2018;173(5):1165-1178.e20.
[12] Olive PL. The comet assay. An overview of techniques. Methods Mol Biol. 2002;203:179-94.
[13] Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol. 2004;26(3):249-61.
[14] Afanasieva K, Zazhytska M, Sivolob A. Kinetics of comet formation in single-cell gel electrophoresis: loops and fragments. Electrophoresis. 2010;31(3):512-9.
[15] Afanasieva K, Chopei M, Zazhytska M, Vikhreva M, Sivolob A. DNA loop domain organization as revealed by single-cell gel electrophoresis. Biochim Biophys Acta. 2013;1833(12):3237-3244.
[16] Afanasieva K, Chopei M, Sivolob A. Single nucleus versus single-cell gel electrophoresis: kinetics of DNA track formation. Electrophoresis. 2015;36(7-8):973-7.
[17] Afanasieva K, Chopei M, Lozovik A, Semenova A, Lukash L, Sivolob A. DNA loop domain organization in nucleoids from cells of different types. Biochem Biophys Res Commun. 2017;483(1):142-146.
[18] Afanasieva K, Sivolob A. Physical principles and new applications of comet assay. Biophys Chem. 2018;238:1-7.
[19] Stein GH. T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol. 1979;99(1):43-54.
[20] Kiseleva LN, Kartashev AV, Vartanyan NL, Pinevich AA, Samoilovich MP. Characteristics of A172 and T98G cell lines. Tsitologiia. 2016;58(5):349-55. English, Russian.
[21] Elso CM, Roberts LJ, Smyth GK, Thomson RJ, Baldwin TM, Foote SJ, Handman E. Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway. Genes Immun. 2004;5(2):93-100.
[22] Phipson B, Smyth GK. Permutation P-values should never be zero: calculating exact P-values when permutations are randomly drawn. Stat Appl Genet Mol Biol. 2010;9:Article39.