Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity: 1. Assay of the Inhibition of Histone Acetyltransferase (HAT) Activity
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
We describe the protocol here using transcription factor JDP2 as a model protein with HAT-inhibitory and nucleosome-assembly activity. Filter-binding assays are performed as described elsewhere1 with minor modifications (see Figure 1a).
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
・ 500 mM Tris-HCl (pH 8.0) (Sigma-Aldrich Corp., St. Louis, MO, USA; cat. no. 93349)
・ 0.5 mM Ethylenediaminetetraacetic acid (ETDA; Sigma-Aldrich Japan, Tokyo, Japan; cat. no. E9884)
・ 5.0 mM Dithiothreitol (DDT; Sigma-Aldrich Japan; cat. no. D0632)
・ 50% Glycerol (Sigma-Aldrich Japan; cat. no. G5516)
・ Core histones (Upstates Biotech. Co.; Charlottesville, VA, USA; cat. no. 13-107)
・ [3H]Acetyl-CoA (4.3 Ci/mmol; GE Healthcare Bioscience Co., Tokyo, Japan)
・ 0.5 mM Acetyl-CoA trilithium salt (Wako Pure Chemical Industries Ltd., Osaka, Japan; cat no. 018-10811)
・ P-81 Phosphocellulose filter paper (Upstate Biotech. Co.; cat. no. 20-134)
・ 50 mM Na2HPO4・12H2O (pH 9.0) (Wako Pure Chemical Industries Ltd.; cat. no. 194-02835)
・ Acetone (Wako Pure Chemical Industries Ltd.; cat. no. D18-01146)
・ 5x Reaction buffer; 500 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 5.0 mM DDT, 50% glycerol
・ [3H]Acetyl-CoA mix; [3H]acetyl-CoA (4.3 Ci/mmol), non-radiolabeled 0.5 mM acetyl-CoA trilithium salt and distilled water (ddH2O) combined at 1:1:3 (v/v)
・ Jun dimerization protein 2 (JDP2) purified as glutathione S-transferase-JDP2 (GST-JDP2), as described elsewhere2,3
・ p300 purified as (His)6-tagged-p300, as described elsewhere4
Equipment
Procedure
1. Incubate the sample at 30 °C for 60 min in 25 ml of assay buffer [which contains 1x reaction buffer, namely, 50 mM Tris-HCl (pH 8.0), 10% glycerol, 0.1 mM EDTA, 1.0 mM DTT] with 6 pmol of [3H]acetyl-CoA (4.3 mCi/mmol; Amersham Biosciences Co., Tokyo, Japan) and core histones as indicated:
p300 protein is used as the HAT for acetylation of core histones in vitro. In the absence of JDP2, the HAT is taken as 100%.
2. In some cases, in our model experiments, reconstituted nucleosomes that contain the cis-element CRE, DRE, mDRE or mCRE2,5, prepared as described elsewhere4,6, and JDP2, as well as bZIP/ATF-2, are incubated first at 30 °C for 15 min. Then p300 and [3H]acetyl-CoA are added and incubation is continued for a further 45 min at 30 °C.
3. After incubation, spot each reaction mixture onto P-81 phosphocellulose filter paper (Upstate Biotechnology Co.).
4. Wash filters five times for 5 min each in 50 ml of 50 mM Na2HPO4 buffer and once in 50 ml of acetone for 5 min at room temperature.
5. Measure the radioactivity on air-dried filters in a liquid scintillation counter (Figure 1b).
6. Analyze reaction mixtures by SDS-PAGE (18% polyacrylamide) after reactions have been performed as described above. In experiments for which results are shown in Figures 1c and 1d, we used 100 pmol of [14C]acetyl-CoA (55 mCi/mmol; Amersham Life Science Inc.) instead of [3H]acetyl-CoA (Figures 1c and 1d).
7. The same protocol can be applied using reconstituted nucleosomes (Figure 1e).
Troubleshooting
Critical Steps
Anticipated Results
References
1. Brownell, J. E. & Allis, C. D. An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei. Proc. Natl. Acad. Sci. U.S.A. 92, 6364-6368 (1995).
2. Jin, C., Ugai, H., Song, J., Murata, T., Nili, F., Sun, K., Horikoshi, M. & Yokoyama, K. K. Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-2. FEBS Lett. 489, 34-41 (2001).
3. Jin, C., Li, H., Murata, T., Sun, K., Horikoshi, M., Chiu, R. & Yokoyama, K. K. JDP2, a repressor of AP-1, recruits a histone deacetylase 3 complex to inhibit the retinoic acid-induced differentiation of F9 cells. Mol. Cell. Biol. 22, 4815-4826 (2002).
4. Kraus, W. L. & Kadonaga, J. p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation. Genes Dev. 12, 331-342 (1998).
5. Polly, P. & Nicholson, R.C. Sequence of the mouse fibronectin-encoding gene promoter region. Gene 137, 353-354 (1993).
6. Ito, T., Levenstein, M. E., Fyodorov, D. V., Kutach, A. K., Kobayashi, R. & Kadonaga, J. ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev. 13, 1529-1539 (1999).
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
We thank G Gachelin, R. Chiu and K. Itakura for valuable comments on the manuscript, and K. Inabe and M. Hirose for their excellent technical assistance. This work was supported by grants from the RIKEN Bioresource Project (to KKY) and the Ministry of Education, Culture, Sports, Science and Technology of Japan (to KN and KKY).
Keywords
Histone Chaperone, Nucleosome assembly, Inhibition of HAT, Transcription factor, AP-1
Figure 1
Inhibition of the HAT activity of GST-JDP2.
a) Schematic representation of the design of the experiment for measuring HAT-inhibitory activity. AcCoA, acetyl CoA. (b) Dose-dependent inhibition of the acetylation of histones by JDP2. Filter-binding assays were performed with 2 g of core histones and increasing concentrations (10 pmole and 30 pmole) of GST-JDP2 (columns 6 and 7), GST (columns 8 and 9) and BSA (columns 10 and 11). Lane 1, B buffer; lanes 5-11, 4 pmole of p300; lane 3, 30 pmole of BSA; and lane 4, 30 pmole of GST-JDP2. (c) Analysis by SDS-PAGE of HAT activity. Assays of HAT activity were performed and [14C]-acetylated histones were detected by autoradiography after separation by SDS-PAGE. Lanes 1-4, 500 ng of core histones; lanes 2-4, 4 pmole of p300; lane 3, 10 pmole of GST-JDP2; and lane 4, 5 pmole of GST. (d) Analysis by SDS-PAGE of HAT activity. Lanes 1-8, 4 pmole of p300; lane 2, with 10 pmole of GST; lanes 3-7, with 10 pmole of mutant GST-JDP2 (mutations at amino acid positions 39, 47, 49, 50 and 51 of JDP2, respectively) and lane 8, with 10 pmole of wild-type GST-JDP2. (e) JDP2 did not inhibit the HAT activity of p300 in nucleosomes. [14C]-Acetylated histones were detected by autoradiography after separation by SDS-PAGE. Lanes 1-4, 500 ng of mononucleosomes; lanes 2-4, 3 pmole of p300; lane 3, 5 pmole of GST-JDP2; and lane 4, 5 pmole of GST.
