SUMMARY. At higher order levels chromatin is organized into loops, and this looping plays an important role in transcription regulation. In our previous works we investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) of nucleoids obtained from lysed cells. It was shown that there are three parts of DNA in nucleoids: DNA in rather small loops which migrate rapidly; DNA in the loops up to ~150 kb, the migration of which is retarded; and larger loops that cannot migrate. Here we applied, for the first time, the pulse-field electrophoresis in the comet assay. Our results show that the first rapid step during the usual comet assay can be attributed to loops on the nucleoid surface while the second slow component represents loops inside the nucleoid.
Keywords:
Full text and supplemented materials
References
Afanasieva, K., Zazhytska, M., and Sivolob, A., Kinetics of comet formation in single–cell gel electrophoresis: Loops and fragments, Electrophoresis, 2010, vol. 31, no. 3, pp. 512–519. https://doi.org/10.1002/elps.200900421
Afanasieva, K., Chopei, M., Zazhytska, M., et al., DNA loop domain organization as revealed by single-cell gel electrophoresis, Biochim. Biophys. Acta, Mol. Cell Res., 2013, vol. 1833, no. 12, pp. 3237–3244. https://doi.org/10.1016/j.bbamcr.2013.09.021
Afanasieva, K., Chopei, M., Lozovik, A., et al., Redistribution of DNA loop domains in human lymphocytes under blast transformation with interleukin 2, Ukr. Biochem. J., 2016, vol. 88, no. 6, pp. 45–51. https://doi.org/10.15407/ubj88.06.045
Afanasieva, K., Chopei, M., Lozovik, A., et al., DNA loop domain organization in nucleoids from cells of different types, Biochem. Biophys. Res. Commun., 2017, vol. 483, no. 1, pp. 142–146. https://doi.org/10.1016/j.bbrc.2016.12.177
Afanasieva, K. and Sivolob, A., Physical principles and new applications of comet assay, Biophys. Chem., 2018, vol. 238, pp. 1–7. https://doi.org/10.1016/j.bpc.2018.04.003
Aughey, G. and Southall, T., Dam it’s good! DamID profiling of protein-DNA interactions, Wiley Interdiscip. Rev.: Dev. Biol., 2015, vol. 5, no. 1, pp. 25–37. https://doi.org/10.1002/wdev.205
Chopei, M., Afanasieva, K., and Sivolob, A., Protein intercalation in DNA as one of main modes of fixation of the most stable chromatin loop domains, Ukr. Biochem. J., 2014, vol. 86, no. 4, pp. 110–118.
Cook, P. and Brazell, I., Super coils in human DNA, J. Cell Sci., 1975, vol. 19, no. 2, pp. 261–279.
Cook, P., A model for all genomes: the role of transcription factories., J. Mol. Biol., 2010, vol. 395, no. 1, pp. 1–10. https://doi.org/10.1016/j.jmb.2009.10.031
Défontaines, A.D. and Viovy, J.L., Gel electrophoresis of an end-labeled DNA I. Dynamics and trapping in constant fields, Electrophoresis, 1993, vol. 14, no. 1, pp. 8‒17. https://doi.org/10.1002/elps.1150140103
Dekker, J., Marti-Renom, M., and Mirny, L., Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data, Nat. Rev. Genet., 2013, vol. 14, no. 6, pp. 390–403. https://doi.org/10.1038/nrg3454
Dekker, J. and Misteli, T., Long-range chromatin interactions, Cold Spring Harbor Perspect. Biol., 2015, vol. 7, no. 10, art. ID a019356. https://doi.org/10.1101/cshperspect.a019356
Kadauke, S. and Blobel, G., Chromatin loops in gene regulation, Biochim. Biophys. Acta, Gene Regul. Mech., 2009, vol. 1789, no. 1, pp. 17–25.https://doi.org/10.1016/j.bbagrm.2008.07.002
Kieffer-Kwon, K., Nimura, K., Rao, S., et al., Myc regulates chromatin decompaction and nuclear architecture during B cell activation, Mol. Cell, 2017, vol. 67, no. 4, pp. 566–578. https://doi.org/10.1016/j.molcel.2017.07.013
Kind, J., Pagie, L., de Vries, S., et al., Genome wide maps of nuclear lamina interactions in single human cells, Cell, 2015, vol. 163, no. 1, pp. 134–147. https://doi.org/10.1016/j.cell.2015.08.040
Krietenstein, N., Abraham, S., Venev, S., et al., Ultrastructural details of mammalian chromosome architecture, Mol. Cell, 2020, vol. 78, pp. 554–565. https://doi.org/10.1016/j.molcel.2020.03.003
Liao, W., McNutt, M., and Zhu, W., The comet assay: A sensitive method for detecting DNA damage in individual cells, Methods, 2009, vol. 48, no. 1, pp. 46–53. https://doi.org/10.1016/j.ymeth.2009.02.016
Lieberman-Aiden, E., van Berkum, N., Williams, L., et al., Comprehensive mapping of long-range interactions reveals folding principles of the human genome, Science, 2009, vol. 326, no. 5950, pp. 289–293. https://doi.org/10.1126/science.1181369
Maston, G., Evans, S., and Green, M., Transcriptional regulatory elements in the human genome, Ann. Rev. Genomics Hum. Genet., 2006, vol. 7, pp. 29–59. https://doi.org/10.1146/annurev.genom.7.080505.115623
Meuleman, W., Peric-Hupkes, D., Kind, J., et al., Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence, Genome Res., 2012, vol. 23, no. 2, pp. 270–280. https://doi.org/10.1101/gr.141028.112
Nagano, T., Lubling, Y., Stevens, T., et al., Single-cell Hi-C reveals cell-to-cell variability in chromosome structure, Nature, 2013, vol. 502, no. 7469, pp. 59–64. https://doi.org/10.1038/nature12593
Ong, C. and Corces, V., CTCF: An architectural protein bridging genome topology and function, Nat. Rev. Genet., 2014, vol. 15, no. 4, pp. 234–246. https://doi.org/10.1038/nrg3663
Olive, P., The comet assay: an overview of techniques, Methods Mol. Biol., 2002, vol. 203, pp. 179–194. https://doi.org/10.1385/1-59259-179-5:179
Rao, S., Huntley, M., Durand, N., et al., A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping, Cell, 2014, vol. 159, no. 7, pp. 1665–1680. https://doi.org/10.1016/j.cell.2014.11.021
Rodríguez-Ubreva, J. and Ballestar, E., Chromatin Immunoprecipitation, Methods Mol. Biol., 2014, vol. 1094, pp. 309–318. https://doi.org/10.1007/978-1-62703-706-8_24
Sanborn, A., Rao, S., Huang, S., et al., Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes, Proc. Natl. Acad. Sci. U. S. A., 2015, vol. 112, no. 47, pp. E6456–E6465. https://doi.org/10.1073/pnas.1518552112
Shaposhnikov, S., Salenko, V., Brunborg, G., et al., Single-cell gel electrophoresis (the comet assay): Loops or fragments?, Electrophoresis, 2008, vol. 29, no. 14, pp. 3005–3012. https://doi.org/10.1002/elps.200700921
Van Bortle, K. and Corces, V., Nuclear organization and genome function, Ann. Rev. Cell Dev. Biol., 2012, vol. 28, pp. 163–187. https://doi.org/10.1146/annurev-cellbio-101011-155824
Wagner, L. and Lai, E., Separation of large DNA molecules with high voltage pulsed field gel electrophoresis, Electrophoresis, 1994, vol. 15, no. 1, pp. 1078–1083. https://doi.org/10.1002/elps.11501501161
Yáñez-Cuna, J., and van Steensel, B., Genome–nuclear lamina interactions: from cell populations to single cells, Curr. Opin. Genet. Dev., 2017, vol. 43, pp. 67–72. https://doi.org/10.1016/j.gde.2016.12.005