Abstract
ATP-dependent remodeling factors regulate chromatin structure by catalyzing processes such as nucleosome repositioning or conformational changes of nucleosomes. Predominantly, their enzymatic properties have been investigated using mononucleosomal substrates. However, short nucleosomal arrays represent a much better mimic of the physiological chromatin context. Here, we provide a protocol for the enzyme-free reconstitution of regularly spaced nucleosomal arrays. We then explain how these arrays can serve as substrates to monitor ATP-dependent nucleosome movements and changes in the accessibility of nucleosomal DNA.
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Chioda, M., and Becker, P. B. (2010) Soft skills turned into hard facts: nucleosome remodelling at developmental switches, Heredity 105, 71–79.
Gangaraju, V. K., and Bartholomew, B. (2007) Mechanisms of ATP dependent chromatin remodeling, Mutat Res 618, 3–17.
Huynh, V. A., Robinson, P. J., and Rhodes, D. (2005) A method for the in vitro reconstitution of a defined “30 nm” chromatin fibre containing stoichiometric amounts of the linker histone, J Mol Biol 345, 957–968.
Robinson, P. J., An, W., Routh, A., Martino, F., Chapman, L., Roeder, R. G., and Rhodes, D. (2008) 30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction, J Mol Biol 381, 816–825.
Robinson, P. J., Fairall, L., Huynh, V. A., and Rhodes, D. (2006) EM measurements define the dimensions of the “30-nm” chromatin fiber: evidence for a compact, interdigitated structure, Proc Natl Acad Sci U S A 103, 6506–6511.
Routh, A., Sandin, S., and Rhodes, D. (2008) Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure, Proc Natl Acad Sci U S A 105, 8872–8877.
Maier, V. K., Chioda, M., Rhodes, D., and Becker, P. B. (2008) ACF catalyses chromatosome movements in chromatin fibres, EMBO J 27, 817–826.
Becker, P. B., Tsukiyama, T., and Wu, C. (1994) Chromatin assembly extracts from Drosophila embryos, Methods Cell Biol 44, 207–223.
Bulger, M., Ito, T., Kamakaka, R. T., and Kadonaga, J. T. (1995) Assembly of regularly spaced nucleosome arrays by Drosophila chromatin assembly factor 1 and a 56-kDa histone-binding protein, Proc Natl Acad Sci U S A 92, 11726–11730.
Fyodorov, D. V., and Kadonaga, J. T. (2003) Chromatin assembly in vitro with purified recombinant ACF and NAP-1, Methods Enzymol 371, 499–515.
Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Chromatin assembly in Xenopus oocytes: in vitro studies, Cell 37, 33–41.
Loyola, A., and Reinberg, D. (2003) Histone deposition and chromatin assembly by RSF, Methods 31, 96–103.
Sandaltzopoulos, R., and Becker, P. B. (2009) Analysis of reconstituted chromatin using a solid-phase approach, Methods Mol Biol 523, 11–25.
Schwarz, P. M., Felthauser, A., Fletcher, T. M., and Hansen, J. C. (1996) Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains, Biochemistry 35, 4009–4015.
Nightingale, K., Dimitrov, S., Reeves, R., and Wolffe, A. P. (1996) Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin, EMBO J 15, 548–561.
Acknowledgments
Work in the laboratory of P. Becker on nucleosome remodeling is supported by the Deutsche Forschungsgemeinschaft (SFB594). We thank Mariacristina Chioda, Henrike Klinker, and Felix Müller-Planitz for helpful discussion.
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Maier, V.K., Becker, P.B. (2012). A Defined In Vitro System to Study ATP-Dependent Remodeling of Short Chromatin Fibers. In: Morse, R. (eds) Chromatin Remodeling. Methods in Molecular Biology, vol 833. Humana Press. https://doi.org/10.1007/978-1-61779-477-3_16
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DOI: https://doi.org/10.1007/978-1-61779-477-3_16
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