Abstract
Histone variants such as H3.3, macroH2A, H2A.Z, and CENP-A are important epigenetic modifiers of the chromatin state in eukaryotic genomes. The centromeric histone H3 variant CENP-A/CENH3 epigenetically marks centromeres and is required for assembly of the kinetochore complex, a region of the chromosome that is responsible for proper genome segregation during mitosis. Several diverse techniques using biochemical, cell biology, and biophysical approaches have been utilized to study the nature of the CENP-A nucleosome across the cell cycle. In this chapter, we describe methods for CENP-A nucleosome purification and separation of CENP-A from other core histones using traditional SDS-PAGE and more resolving techniques such as Triton acid urea (TAU) and two-dimensional gels. We also discuss methods for observation of CENP-A on chromatin fibers using immunofluorescence. Finally, we provide a detailed description of analysis of chromatin structures using atomic force microscopy.
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Acknowledgements
We thank Dr. Rajbir Gill for expert advice on chromatin extraction, TAU gel preparation, and FISH protocols, and Dr. Emilios Dimitriadis for helpful advice on automated AFM image analysis.
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Walkiewicz, M.P., Bui, M., Quénet, D., Dalal, Y. (2014). Tracking Histone Variant Nucleosomes Across the Human Cell Cycle Using Biophysical, Biochemical, and Cytological Analyses. In: Noguchi, E., Gadaleta, M. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 1170. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0888-2_34
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DOI: https://doi.org/10.1007/978-1-4939-0888-2_34
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