Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity: 5. Preparation of Core Histones
Lab/Group: Yokoyama Lab (RIKEN BRC), Nagata Lab (Tsukuba University)
Related Journal & Article Information
Journal: Nature Structural & Molecular Biology
Article Title: Regulation of histone acetylation and nucleosome assembly by transcription factor JDP2
Introduction
At present, the best-defined and most useful source of material for reconstitution of chromatin consists of purified core histones. These core histones can be prepared from either solubilized chromatin or H1-depleted chromatin by column chromatography on hydroxyapatite. To maintain the correct stoichiometry of all histones, we recommend preparation from H1-depleted chromatin by single-step elution with 2 M NaCl (see Note 1).
For a detailed introduction to assays of nucleosome assembly and the inhibition of histone acetyltransferase activity, please go here:
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.php
Materials
Reagents
Preparation of Core Histones by Fast-Performance Liquid Chromatography (FPLC)
・ Buffer A: 50 mM Na-phosphate (pH 6.8), 0.1 mM PMSF
・ Buffer B: 50 mM Na-phosphate (pH 6.8), 2 M NaCl, 0.1 mM PMSF
・ Dialysis membrane (MWCO 12,000-14,000; Spectrum Laboratories Inc.)
・ Centricon (MWCO, 30,000 and MWCO 10,000; Millipore Corp.)
・ 20-mL hydroxyapatite column (Bio-Gel HTP Gel, Bio-Rad Laboratories, Inc.)
・ FPLC system (Amersham Biosciences Co., Buckinghamshire, UK)
Further Purification and Concentration of Core Histones
・ Dialysis buffer: 0.2 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM PMSF
・ Elution buffer: 2 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM PMSF
・ 2-mL CM52 column (5 mg histone/mL bed volume; Whatman International Ltd., Maidstone, England)
・ Dialysis membrane (MWCO, 6,000 to 8,000; Millipore Corp.)
Equipment
Procedure
1. Dialyze the pooled H1-depleted chromatin overnight against buffer A at 4 °C.
2. Equilibrate a 20-mL hydroxyapatite column with buffer A and apply H1-depleted chromatin at a flow rate of 2 mL/min in 0% buffer B (see Note 2). Wash the column with the same buffer under the same conditions for 30 min.
3. Elute non-histone proteins with 25% buffer B (0.5 M NaCl), at a flow rate of 2 mL/min over 30 min. Elute the core histones as octamers wth 100% buffer B (2 M NaCl) over 15 min (collect 2-mL factions). Determine the concentration of core histones in each fraction from the absorbance (OD230nm = 4.2 corresponds to 1 mg/mL). Analyze the proteins in each fraction on an 18% polyacrylamide gel by SDS-PAGE (see Note 3).
4. Pool fractions that contain core histones. Concentrate proteins to greater than 0.1 mg/mL for reconstitution of chromatin, if necessary, using a concentrator (MWCO, 10,000) or a CM52 column. Store at －20 °C in siliconized tubes.
Further Purification and Concentration of Core Histones
1. Dialyze core histones against dialysis buffer at 4 °C.
2. Prepare a 2-mL CM52 column (5 mg histone/mL bed volume) and equilibrate with dialysis buffer.
3. Apply core histones to the column, wash thoroughly with 15 mL of dialysis buffer and elute with 5 mL of elution buffer. Measure the absorbance of a small aliquot in water at 230 nm to determine the concentration of histones. Store at －20 °C in siliconized tubes.
Troubleshooting
Notes
1. When solubilized chromatin is applied to the hydroxyapatite column, wash out H1 first with 0.6 M NaCl in buffer. During this washing, some H2A/H2B is lost, destroying core-histone stoichiometry. Therefore, it is necessary to elute H2A/H2B and H3/H4 separately, with 1 M NaCl and with 2 M NaCl, respectively1, and to re-adjust the histone stoichiometry for reconstitution of chromatin.
2. The total volume of the preparation of H1-depleted chromatin obtained by protocol A should be large (~50 mL). In this case, apply sample chromatin to a column using a 50-mL super loop (Amersham Pharmacia) or after concentrating H1-depleted chromatin to 1 mg/mL with a concentrator (MWCO, 30,000).
3. Acrylamide:bisacrylamide, 37.5:1; SDS-polyacrylamide separating gel [18% polyacrylamide (w/v)] containing 0.375 M Tris-HCl (pH 8.8); and SDS-polyacrylamide stacking gel [4% polyacrylamide (w/v)] containing 0.125 M Tris-HCl (pH 6.8).
Anticipated Results
References
1. Kawase, H., Okuwaki, M., Miyaji, M., Handa, H., Ishimi, Y., Fujii-Makata, T., Kikuchi, A. & Nagata, K. NAP-1 is functional homologue of TAF-1 that is required for replication and transcription of the adenovirous genome in a chromatin-like structure. Gene Cells 1, 1045-1056 (1996).
Related Protocols
This protocol is one of nine related Network Protocols by Yamasaki et al. This is the complete list:
Inhibition of Histone Actyltransferase (HAT) Activity
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_1.php
Isolation and labeling of DNA fragments (includes information on Assembly of Chromatin in vitro, Experiments with Mononucleosomes, Isolation of 5'-End-Radiolabeled Fragments of pB100-Uless/strider DNA, and DRE and CRE Elements)
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_2.php
Preparation of Nuclei from HeLa Cells
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_3.php
Preparation of Histone H1-Depleted Chromatin
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_4.php
Preparation of Core Histones (Includes Preparation of Core Histones by FPLC and Further Purification and Concentration of Core Histones)
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_5.php
Reconstitution of Chromatin, Salt Dialysis Using Purified Core Histones, Octamer Transfer from Donor Chromatin, Analysis of Nucleoproteins on an Agarose Gel, Purification of Reconstituted Chromatin on a Sucrose Gradient, and Binding of Linker Histones to Reconstituted Chromatin
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_6.php
Plasmid Super-Coiling Assay and Nucleosome Assembly on a Fragment of 5S DNA
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_7.php
Digestion of Chromatin in Permeabilized Cells with Micrococcal Nuclease (MNase), Permeabilization of cells and digestion with MNase, Purification and Characterization of DNA after Digestion of Chromatin, and Nuclease Cleavage and Mapping Strategies
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_8.php
Ligation-Mediated Single-Sided PCR (LMPCR)
(including: First-strand Synthesis, Ligation-Mediated PCR for Nucleosome Mapping in vivo, and Ligation-Mediated Polymerase Chain Reaction (LM-PCR))
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_9.php
Acknowledgements
Keywords
KeyWords; Histone Chaperone, Nucleosome assembly, Inhibition of HAT, Transcription factor, AP-1