Isolation of Histones and Nucleosome Cores from Mammalian Cells

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Abstract
Table of Contents
Materials
Literature Cited

Abstract

In vitro analysis of DNA in chromatin is often important for understanding mechanisms of regulation of transcription and other processes that occur on DNA. The basic unit of chromatin is the nucleosome core, containing two copies each of the core histones H2A, H2B, H3, and H4 to form a histone octamer that wraps 145 base pairs of DNA in a left?handed superhelix. In vivo, chromatin is associated with linker histones (such as H1), which facilitate the ordered packing of nucleosomes. This linker histone?containing particle is properly termed the nucleosome (or chromatosome), while the linker histone?free particle is the nucleosome core. Pure polynucleosome cores and histones can be readily isolated from mammalian tissue culture cells. This unit describes procedures for isolation and purification of nuclei, isolation of polynucleosomes lacking linker histones from these nuclei, isolation of pure populations of mono? and dinucleosome cores from oligonucleosome fractions, and isolation of core histones from purified nuclei. The methods presented here do not denature the histones, and may yield histones that are more active for in vitro assemblies.

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Basic Protocol 1: Preparation of a Washed Nuclear Pellet
Basic Protocol 2: Solubilization and Purification of Histone H1–Depleted Oligonucleosomes
Basic Protocol 3: Purification of Mono‐ and Dinucleosomes
Basic Protocol 4: Purification of Core Histones by Hydroxylapatite Chromatography
Reagents and Solutions
Commentary

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Materials

Basic Protocol 1: Preparation of a Washed Nuclear Pellet

Materials

Mammalian tissue cell culture (e.g., HeLa cells)
PBS ( appendix 22 )
recipeLysis buffer (see recipe )
recipeBuffer B (see recipe )
2 M NaCl
Buffer B/0.6 M KCl/10% (v/v) glycerol

Dounce homogenizer with type B pestle
Light microscope

Additional reagents and equipment for mammalian cell culture ( appendix 3F ) and quantitation of DNA ( appendix 3D )

Basic Protocol 2: Solubilization and Purification of Histone H1–Depleted Oligonucleosomes

Materials

Washed nuclear pellet (see protocol 1 )
recipeMSB (see recipe )
recipeHSB (see recipe )
2 M NaCl
recipeLSB (see recipe )
0.1 M CaCl₂
recipe50 U/µl micrococcal nuclease (see recipe )
0.5 M EGTA, pH 8.0
recipeHSB without sucrose
HSB/glycerol: recipeHSB containing 10% and 40% (v/v) glycerol instead of sucrose
0.5% (w/v) SDS
0.5 mg/ml proteinase K
recipeDialysis buffer (see recipe )

Dounce homogenizer with type B pestle
6 to 8 kDa MWCO dialysis membrane
∼1.6 × 58–cm Sepharose CL‐6B column (Amersham Pharmacia Biotech; for gel filtration) and accessories
Refrigerated ultracentrafuge with appropriate rotor and tubes, e.g.
Beckman SW55 rotor with polyallomer or ultraclear tubes (for gel filtration)
Beckman SW28 rotor with 1 × 3.5–in. (2.5 × 8.9–cm) polyallomer tubes (for centrifugation)
Gradient maker (for centrifugation)
21‐G needle (for centrifugation)
Tubing (for centrifugation): e.g., 21‐G needle infusion set with 12‐in. (30.5‐cm) tubing (Abbot Laboratories)
50°C water bath
6 to 8 kDa MWCO dialysis bag

Additional reagents and equipment for quantitation of DNA ( appendix 3D ), pouring gradients (unit 10.10 ), agarose gel electrophoresis (unit 2.5 ), and SDS‐PAGE (unit 10.2 )

Basic Protocol 3: Purification of Mono‐ and Dinucleosomes

Materials

Oligonucleosomes: medium or large polynucleosome fractions (see protocol 2 )
100 mM CaCl₂
1 M MgCl₂
recipe50 U/µl micrococcal nuclease (see recipe )
0.5 M EDTA
recipe10% and 30% (v/v) glycerol gradient buffer (see recipe )

30°C water bath
Ultracentrifuge with rotor (e.g., Beckman SW55) and 0.5 × 2.5–in. (1.3 × 6.4–cm) polyallomer tubes
Gradient maker

Additional reagents and equipment for harvesting, analyzing, and concentrating fractions (see protocol 2 ) and for nondenaturing acrylamide electrophoresis (unit 10.2 )

Basic Protocol 4: Purification of Core Histones by Hydroxylapatite Chromatography

Materials

Nuclear pellet (see protocol 1 )
recipeHAP buffer (see recipe ), with and without 2.5 M NaCl
BioGel HTP powder (Bio‐Rad), with adsorption capacity 0.6 mg DNA per g dry powder
Bio‐Rad Protein Assay (optional)
2 × 15–cm column and accessories
Centriprep‐10 concentrators (Amicon; optional)

Additional reagents and equipment to determining protein concentration (unit 10.1 ) and SDS‐PAGE (unit 10.2 )

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Figures

Videos

Literature Cited

Literature Cited
	Becker, P.B., Tsukiyama, T., and Wu, C. 1994. Chromatin assembly extracts from Drosophila embryos. Methods Cell Biol. 44:207‐223.
	Carruthers, L.M., Tse, C., Walker, K.P. III, and Hansen, J.C. 1999. Assembly of defined nucleosomal and chromatin arrays from pure components. Methods Enzymol. 304:19‐35.
	Côté, J., Utley, R.T., and Workman, J.L. 1995. Basic analysis of transcription factor binding to nucleosomes. Methods Mol. Genet. 6:108‐128.
	Guyon, J.R., Narlikar, G.J., Sif, S., and Kingston, R.E. 1999. Stable remodeling of tailless nucleosomes by the human SWI‐SNF complex. Mol. Cell Biol. 19:2088‐2097.
	Hayes, J.J. and Lee, K.‐M. 1997. In vitro reconstitution and analysis of mononucleosomes containing defined DNAs and proteins. Methods 12:2‐9.
	Imbalzano, A.N., Schnitzler, G.R., and Kingston, R.E. 1996. Nucleosome disruption by human SWI/SNF is maintained in the absence of continued ATP hydrolysis. J. Biol. Chem. 271:20726‐20733.
	Ito, T., Tyler, J.K., Bulger, M., Kobayashi, R., and Kadonaga, J.T. 1996. ATP‐facilitated chromatin assembly with a nucleoplasmin‐like protein from Drosophila melanogaster. J. Biol. Chem. 271:25041‐25048.
	Ito, T., Bulger, M., Pazin, M.J., Kobayashi, R., and Kadonaga, J.T. 1997. ACF, an ISWI‐containing and ATP‐utilizing chromatin assembly and remodeling factor. Cell 90:145‐155.
	Kornberg, R.D., LaPointe, J.W., and Lorch, Y. 1989. Preparation of nucleosomes and chromatin. Methods Enzymol. 170:3‐14.
	Kwon, H., Imbalzano, A.N., Khavari, P.A., Kingston, R.E., and Green, M.R. 1997. Nucleosome disruption and enhancement of activator binding by a human SWI/SNF complex. Nature 370:477‐481.
	Luger, K., Rechsteiner, T.J., Flaus, A.J., Waye, M., and Richmond, T.J. 1997. Characterization of nucleosome core particles containing histone proteins made in bacteria. J. Mol. Biol. 272:301‐311.
	Schnitzler, G., Sif, S., and Kingston, R.E. 1998. Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state. Cell 94:17‐27.
	Sessa, G., Ruberti, I. 1990. Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes. Nucl. Acids Res. 18:5449‐5455.
	Simon, R.H. and Felsenfeld, G. 1979. A new procedure for purifying histone pairs H2A+H2B and H3+H4 from chromatin using hydroxylapatite. Nucl. Acids Res. 6:689‐696.
	Stein, A. 1989. Reconstitution of chromatin from purified components. Methods Enzymol. 170:585‐603.
	Varga‐Weisz, P.D., Wilm, M., Bonte, E., Dumas, K., Mann, M., and Becker, P.B. 1997. Chromatin‐remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 388:598‐602.
	Vettese‐Dadey, M., Adams, C.C., Côté, J., Walter, P., and Workman, J.L. 1995. Experimental analysis of transcription factor‐nucleosome interactions. Methods Mol. Genet. 6:129‐151.
	Workman, J.L., Taylor, I.C.A., Kingston, R.E., and Roeder, R.G. 1991. Control of class II gene transcription during in vitro nucleosome assembly. Methods Cell Biol. 35:419‐447.
Key References
	Côté, et al., 1995. See above.
	Basic Protocols 1, 2, and 4 are modifications of protocols in this paper.
	Wasserman, P.M. and Wolffe, A.P. (eds.) 1999. Chromatin. Methods Enzymol. Vol. 304.
	This volume contains many other useful and current protocols for the assembly and analysis of chromatin.

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Isolation of Histones and Nucleosome Cores from Mammalian Cells

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation of a Washed Nuclear Pellet

Basic Protocol 2: Solubilization and Purification of Histone H1–Depleted Oligonucleosomes

Basic Protocol 3: Purification of Mono‐ and Dinucleosomes

Basic Protocol 4: Purification of Core Histones by Hydroxylapatite Chromatography

Figures

Videos

Literature Cited