Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity: 9. Plasmid Super-Coiling Assay; 10. Nucleosome Assembly on a Fragment of 5S DNA
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
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
Equipment
Procedure
Plasmid Super-Coiling Assay1,2
1. In our experiments, we mixed 2 pmol of core histones from HeLa cells (see “Preparation of Core Histones by FPLC”) with 4 pmol or 8 pmol of GST or GST-JDP2, respectively, in the assembly buffer (tube A). The instructions that follow refer to a generalized assay.
2. Incubate the reaction mixture at 30 °C for 30 min.
3. In parallel, mix 100 ng of plasmid DNA (see Note 1) and 5 units of topoisomerase I in topoisomerase I reaction buffer and incubate at 37 °C for 30 min (tube B).
4. Combine the topoisomerase I-relaxed circular DNA (in tube B) and core histones (in tube A) that were pre-incubated with, in our experiments, GST or GST-JDP2 in 50 μl of assembly buffer.
5. Incubate the mixture at 37 °C for 1 h.
6. Add an equal volume of 2x stop buffer and continue incubation at 37 ʰC for 30 min.
7. Extract the plasmid DNA with phenol, chloroform and isoamyl alcohol.
8. Precipitate the DNA in cold ethanol.
9. Fractionate the purified DNA by electrophoresis on a 1.2% agarose gel (see Note 2), and visualize by staining with ethidium bromide (Figure 1).
Nucleosome Assembly on a Fragment of 5S DNA3
1. Prepare 200 ng of a 197-bp-long fragment of 5S DNA by digestion of pB100-Uless/Strider DNA with EcoR I (see Note 3).
2. Incubate the DNA in 10 μl of reaction buffer at 37 °C for 1 h with or without 200 ng of core histones that have been pre-incubated with increasing concentrations of, for example, either human His-TAF-1or GST-JDP2.
3. Fractionate samples on a 5% non-denaturing polyacrylamide gel in TBS buffer and visualize reaction products by staining with ethidium bromide or SYBR Gold (see Note 4).
4. Excise gel portions that correspond to mononucleosomes. Allow them to equilibrate at room temperature for 15 min in the standard buffer for SDS-PAGE, prepared with 0.1 % SDS.
5. Transfer proteins to an ImmobilonTM membrane (PVPF membrane; Millipore Corp., Bedford, MA, USA).
6. Analyze proteins by Western blotting with antibodies against histone H3 (Figure 2).
Troubleshooting
Notes
1. Plasmid DNA should be very pure and maintained at an appropriate molar ratio of open-circular to super-coiled DNA (normally less than 1:9). Purification of closed circular DNA by equilibrium centrifugation in CsCl gradients is recommended.
2. To avoid formation of non-specific complexes of proteins with super-coiled DNA during electrophoresis, it is critical to avoid contamination by ethidium bromide of the agarose gel, the electrophoresis buffer, and the electrophoretic apparatus.
3. Purification of a 197-bp-long fragment of 5S DNA is not always required. A mixture of a fragment of 5S DNA and a 2-kbp-long fragment derived from the pB100-Uless plasmid can also be used for nucleosome assembly.
4. In general, two bands of DNA, corresponding to 197-bp-long 5S mononucleosomes, are detected because of the presence of two (major and minor) differently positioned mononucleosomes on the fragment of DNA.
Anticipated Results
References
1. Munakata, T., Adachi, N., Yokoyama, N., Kuzuhara, T. & Horikoshi, M. A human homologue of yeast anti-silencing factor has histone-chaperone activity. Genes Cells 5, 221-233 (2000).
2. Miyaji-Yamagichi, M., Kato, K., Nakano, R., Akashi, T., Kikuchi, A. & Nagata, K. Involvement of nucleocytoplasmic shuttling of yeast Nap1 in mitotic progression. Mol. Cell. Biol. 23, 6672-6684 (2003).
3. Miyaji-Yamagichi, M., Kato, K., Nakano, R., Akashi, T., Kikuchi, A. & Nagata, K. Involvement of nucleocytoplasmic shuttling of yeast Nap1 in mitotic progression. Mol. Cell. Biol. 23, 6672-6684 (2003).
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
Histone Chaperone, Nucleosome assembly, Inhibition of HAT, Transcription factor, AP-1
Figure 1
The histone-chaperone activity of JDP2, as detected by an assay of supercoiling. In the presence of core histones from HeLa cells, 8 pmole of GST-JDP2 (lane 2) and His-TAF-1β (lane 4) specifically introduced supercoils into circular DNA. Buffer (lane 1) and GST (8 pmole; lane 3) served as negative controls. The mobilities of relaxed and nicked DNA and of supercoiled DNA are indicated on the right.
Figure 2
The dependence of histone-chaperone activity on histones. (Upper panel) Nucleosome assembly by JDP2 on a fragment of 5S DNA. 200 ng of a 197-bp-long fragment of 5S DNA were incubated without (lanes 1-3) or with 200 ng (lanes 4-9) of core histones, which had been preincubated without (lanes 1 and 4) or with 500 ng (lane 5) or 1.0 μg (lanes 2 and 6) of His-TAF-Iβ or with 300 ng (lane 7), 600 ng (lane 8) or 1.2 μg (lanes 3 and 9) of GST-JDP2, respectively. After incubation, samples were subjected to electrophoresis on a 5% non-denaturing polyacrylamide gel. The positions corresponding to the 197-bp-long fragment of 5S DNA and mononucleosomes are indicated. (Lower panel) The nucleosomes formed in the presence of JDP2 contained histone H3. Gel portions corresponding to mononucleosomes (upper panel) were excised and the proteins within them were subjected to Western blotting analysis with histone H3-specific antibodies.
