Abstract
In order to study the functions and activities of chromatin remodeling enzymes in vitro, it is necessary to be able to reconstitute nucleosomes on DNA templates. In this chapter, we describe procedures for purification of histones from E. coli, formation of octamers, and reconstitution of nucleosomes that can be further modified by chromatin modifiers. In addition, we describe methods to purify nucleosomes from human cells. Finally, we also describe assays to test binding and remodeling activities of chromatin remodelers.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Clapier CR, Cairns BR (2009) The biology of chromatin remodeling complexes. Annu Rev Biochem 78:273–304
Dutta A et al (2014) Swi/Snf dynamics on stress-responsive genes is governed by competitive bromodomain interactions. Genes Dev 28(20):2314–2330
Chatterjee N et al (2011) Histone H3 tail acetylation modulates ATP-dependent remodeling through multiple mechanisms. Nucleic Acids Res 39(19):8378–8391
Gutierrez JL et al (2007) Activation domains drive nucleosome eviction by SWI/SNF. EMBO J 26(3):730–740
Owen-Hughes T et al (1999) Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes. Methods Mol Biol 119:319–331
Simon MD et al (2007) The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128(5):1003–1012
Yun M et al (2012) Reconstitution of modified chromatin templates for in vitro functional assays. Methods Mol Biol 833:237–253
Dyer PN et al (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375:23–44
Neumann H et al (2009) A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation. Mol Cell 36(1):153–163
Shogren-Knaak M et al (2006) Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311(5762):844–847
Kouzarides T (2007) Chromatin modifications and their function. Cell 128(4):693–705
Luger K et al (1999) Expression and Purification of Recombinant Histones and Nucleosome Reconstitution. Methods Mol Bio 119:1–16
Acknowledgments
We thank current and former members of the Workman laboratory for help with developing these protocols and many useful discussions. We acknowledge Malini Natarajan for critical reading of the manuscript. This work was supported by National Institutes of Health grant R01GM047867 to J.L.W. and funding from the Stowers Institute for Medical Research.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Dutta, A., Workman, J.L. (2017). In Vitro Assembly of Nucleosomes for Binding/Remodeling Assays. In: Guillemette, B., Gaudreau, L. (eds) Histones. Methods in Molecular Biology, vol 1528. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6630-1_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6630-1_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6628-8
Online ISBN: 978-1-4939-6630-1
eBook Packages: Springer Protocols