Abstract
Modifications in chromatin structure are traditionally monitored by biochemical assays that provide average measurements of static events in a population of cells. Microscopy provides a method by which single cells or nuclei can be observed. Traditionally, microscopy has been used to image the nucleus by the application of immunostaining to chemically fixed samples or the use of exogenously expressed fluorescent proteins. This method represents an approach to observe changes in endogenous proteins relating to chromatin structure in real time. Here we describe a method for isolating transcriptionally and enzymatically active nuclei from live cells and visualizing events using fluorescently labeled antibodies. This method allows the observation of real time changes in chromatin architecture and can be used to observe the effects of drugs on nuclei while under microscopic observation.
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References
Heitz E (1928) Das Heterochromatin der Moose. I Jahrb Wiss Bot 69:762–818
Harr JC, Gonzalez-Sandoval A, Gasser SM (2016) Histones and histone modifications in perinuclear chromatin anchoring: from yeast to man. EMBO Rep 17:139–155
Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080
Benedetti R, Conte M, Altucci L (2015) Targeting histone deacetylases in diseases: where are we? Antioxid Redox Signal 23:99–126
Brien GL, Valerio DG, Armstrong SA (2016) Exploiting the epigenome to control cancer-promoting gene-expression programs. Cancer Cell 29:464–476
Milavetz BI, Balakrishnan L (2015) Viral epigenetics. Methods Mol Biol 1238:569–596
Zhu J, Adli M, Zou JY et al (2013) Genome-wide chromatin state transitions associated with developmental and environmental cues. Cell 152:642–654
Lakadamyali M, Cosma MP (2015) Advanced microscopy methods for visualizing chromatin structure. FEBS Lett 589:3023–3030
Kimura H, Hayashi-Takanaka Y, Stasevich TJ et al (2015) Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo. Histochem Cell Biol 144:101–109
Ricci MA, Manzo C, García-Parajo MF et al (2015) Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo. Cell 160:1145–1158
Smeets D, Markaki Y, Schmid VJ et al (2014) Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci. Epigenetics Chromatin 7:8
Wombacher R, Heidbreder M, van de Linde S et al (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7:717–719
Sardo L, Lin A, Khakhina S et al (2017) Real-time visualization of chromatin modification in isolated nuclei. J Cell Sci 130:2926–2940
Farrell RE (2010) Analysis of nuclear RNA. In: RNA methodologies. A laboratory guide for isolation and characterization, 4th edn. Elsevier/Academic Press, San Diego
Acknowledgments
This work was supported by NIH grant 1DP2DA044550 and the W. W. Smith Charitable Trust.
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Li, Y., Klase, Z., Sardo, L. (2020). Visualizing Chromatin Modifications in Isolated Nuclei. In: Hancock, R. (eds) The Nucleus . Methods in Molecular Biology, vol 2175. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0763-3_3
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DOI: https://doi.org/10.1007/978-1-0716-0763-3_3
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0762-6
Online ISBN: 978-1-0716-0763-3
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