Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity. (9) Plasmid super-coiling assay; and (10) Nucleosome assembly on a fragment of 5S DNA

doi:10.1038/nprot.2007.339

Method Article

Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity. (9) Plasmid super-coiling assay; and (10) Nucleosome assembly on a fragment of 5S DNA

https://doi.org/10.1038/nprot.2007.339

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Genetics

Biochemistry

Computational biology and bioinformatics

histone chaperone

nucleosome assembly

inhibition of HAT

transcription factor

AP-1

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Plasmid Super-Coiling Assay^1,2

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.
Incubate the reaction mixture at 30 °C for 30 min.
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).
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.
Incubate the mixture at 37 °C for 1 h.
Add an equal volume of 2x stop buffer and continue incubation at 37 ʰC for 30 min.
Extract the plasmid DNA with phenol, chloroform and isoamyl alcohol.
Precipitate the DNA in cold ethanol.
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

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).
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-1or GST-JDP2.
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).
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.
Transfer proteins to an ImmobilonTM membrane (PVPF membrane; Millipore Corp., Bedford, MA, USA).
Analyze proteins by Western blotting with antibodies against histone H3 (Figure 2).

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.
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.
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.
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.

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).
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).
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).

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Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity. (9) Plasmid super-coiling assay; and (10) Nucleosome assembly on a fragment of 5S DNA

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