Abstract
The accumulating discoveries of new posttranslational modifications (PTMs) and the increasing relevance of histone variants within the frame of epigenetics demand the availability of methods for a rapid and efficient nucleosome reconstitution to analyze their structural and functional implications. Here we describe a method suitable for this purpose, starting from bacterially expressed histones, solubilized by acid and purified by reversed-phase high-performance liquid chromatography. This method allows the preparation of micrograms to milligram amounts of in vitro-assembled nucleosomes. Finally, we demonstrate the efficiency of this method for the structural analysis of nucleosomes in the analytical ultracentrifuge.
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
van Holde KE (1988) Chromatin. Springer, NY
Ausio J (2006) Histone variants—the structure behind the function. Brief Funct Genomic Proteomic 5:228–243
Patel DJ, Wang Z (2013) Readout of epigenetic modifications. Annu Rev Biochem 82:81–118
Gurard-Levin ZA, Almouzni G (2014) Histone modifications and a choice of variant: a language that helps the genome express itself. F1000Prime Rep 6:76
Chen P, Zhao J, Li G (2013) Histone variants in development and diseases. J Genet Genomics 40:355–365
Huang H, Sabari BR, Garcia BA, Allis CD, Zhao Y (2014) SnapShot: histone modifications. Cell 159, 458–458.e451
Tessarz P, Kouzarides T (2014) Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol 15:703–708
Tatchell K, van Holde KE (1977) Reconstitution of chromatin core particles. Biochemistry 16:5295–5303
Song F, Chen P, Sun D, Wang M, Dong L et al (2014) Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344:376–380
Ausió J, Dong F, van Holde KE (1989) Use of selectively trypsinized nucleosome core particles to analyze the role of the histone “tails” in the stabilization of the nucleosome. J Mol Biol 206:451–463
Peterson CL, Hansen JC (2008) Chicken erythrocyte histone octamer preparation. CSH Protoc 2008: pdb.prot5112.
Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251–260
Luger K, Rechsteiner TJ, Richmond TJ (1999) Expression and purification of recombinant histones and nucleosome reconstitution. Methods in Molecular Biology 119:1–16
Ausió J, Moore SC (1998) Reconstitution of chromatin complexes from high-performance liquid chromatography-purified histones. Methods 15:333–342
Arents G, Moudrianakis EN (1995) The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization. Proc Natl Acad Sci U S A 92:11170–11174
Li A, Yu Y, Lee SC, Ishibashi T, Lees-Miller SP et al (2010) Phosphorylation of histone H2A.X by DNA-dependent protein kinase is not affected by core histone acetylation, but it alters nucleosome stability and histone H1 binding. J Biol Chem 285:17778–17788
Ishibashi T, Dryhurst D, Rose KL, Shabanowitz J, Hunt DF et al (2009) Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome. Biochemistry 48:5007–5017
Shogren-Knaak M, Ishii H, Sun JM, Pazin MJ, Davie JR et al (2006) Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311:844–847
Shogren-Knaak MA, Peterson CL (2004) Creating designer histones by native chemical ligation. Methods Enzymol 375:62–76
Simon MD, Chu F, Racki LR, de la Cruz CC, Burlingame AL et al (2007) The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128:1003–1012
Thakar A, Gupta P, Ishibashi T, Finn R, Silva-Moreno B et al (2009) H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies. Biochemistry 48:10852–10857
Levenstein ME, Kadonaga JT (2002) Biochemical analysis of chromatin containing recombinant Drosophila core histones. J Biol Chem 277:8749–8754
Simpson RT, Thoma F, Brubaker JM (1985) Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. Cell 42:799–808
Ausió J (2000) Analytical ultracentrifugation and the characterization of chromatin structure. Biophys Chem 86:141–153
van Holde KE, Weischet WO (1978) Boundary analysis of sedimentation velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 17:1387–1403
Simon RH, Felsenfeld G (1979) A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxyapatite. Nucleic Acids Res 6:689–696
Thastrom A, Lowary PT, Widlund HR, Cao H, Kubista M et al (1999) Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences. J Mol Biol 288:213–229
Ausió J, Seger D, Eisenberg H (1984) Nucleosome core particle stability and conformational change. Effect of temperature, particle and NaCl concentrations, and crosslinking of histone H3 sulfhydryl groups. J Mol Biol 176:77–104
Acknowledgement
This work was supported by a Canadian Institutes of Health Research (CIHR) [MOP-130417] grant.
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
Cheema, M.S., Ausió, J. (2017). Analytical Ultracentrifuge Analysis of Nucleosomes Assembled from Recombinant, Acid-Extracted, HPLC-Purified Histones. 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_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6630-1_6
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