Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity: 2. Isolation and Labeling of DNA Fragments
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
Assembly of Chromatin in vitro (see Note 1)
There are several methods for assembling chromatin on DNA fragments or plasmids. Typical simple methods include (1) the salt dialysis method, in which DNA and purified core histones are mixed and then dialyzed against a series of buffers, starting at 2 M NaCl and moving to lower concentrations of NaCl; and (2) the histone octamer transfer method, in which histone octamers are redistributed from purified donor chromatin to specific and radiolabeled DNA fragments during incubation in buffer that contains 1 M NaCl, with subsequent reduction of the concentration of NaCl either by dialysis or by stepwise dilution1. The composition of the reconstituted chromatin is well defined and such chromatin is useful for the analysis of nucleosome positioning due to intrinsic sequence-dependent DNA structure and for assays of the binding of sequence-specific transcription factors or structural proteins to nucleosomal DNA.
In vertebrates, core histones are strongly conserved and, to date, the limited interspecies variations that have been identified have not been found to be of any functional significance with respect to the analysis of chromatin. Therefore, cultured cells and chicken erythrocytes are convenient sources of core histones and chromatin. Moreover, contamination by proteases and nucleases of such preparations is relatively low.
The above-described and other methods of salt-mediated assembly of nucleosomes fail to separate nuclesomes by the physiological linker distance of approximately 200 bp on general DNA fragments1. The multiple nucleosomes reconstituted by such methods are generally rather closely packed, with one nucleosome every 150 bp or so with little or no linker DNA. However, salt dialysis methods can be used to produce physiologically spaced oligonucleosomes when DNA templates with tandem repeats of strong nucleosome-positioning sequences are used. For example, a template containing ~200-bp tandem repeats of a DNA fragment that includes a Lytechinus gene for 5S rRNA has been used to assemble properly spaced oligonucleosomes2. Such oligonucleosomes have proved to be very useful for studies of higher-order chromatin structure and transcription3.
Alternative methods for the assembly of chromatin make use of crude extracts of cell or of histone chaperones1,4 at physiological ionic strength. We will not describe these methods in detail here. However, it is worth nothing that assembly of chromatin using cell extracts derived from Xenopus eggs5 or Drosophila embryos6 does produce regularly spaced chromatin in vitro. However, the composition of the reconstituted nucleosomes is complicated.
Experiments with Mononucleosomes
Isolation of 5'-End-Radiolabeled Fragments of pB100-Uless/strider DNA (see Note 2)
A 197-bp fragment of pB100-Uless/strider is long enough to accommodate mononucleosomes and contains two tandem repeats of the 5S nucleosome-positioning element from Xenopus. Templates to be used for nucleosome reconstitution are typically radiolabeled to allow easy monitoring of the extent of reconstitution on gels, isolation of particles from sucrose gradients, and for subsequent footprinting analysis.
DRE and CRE Elements
For our model experiments, we isolated [32P]-radiolabeled 147-bp-long DNA fragments that contained either wild-type or mutated DRE7 or CRE8,9 sequences in triplicate by electrophoresis on a 10% polyacrylamide gel in 1x TBE buffer as described above.
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
Plasmid DNA (pB100-Uless/strider, which contains the 197-bp fragment of 5S rDNA from Xenopus borealis)
・Oligodeoxynucleotides synthesized “in house” and corresponding to the differentiation response element (DRE) and a mutated DRE7, and to the cyclic-AMP response element (CRE) and a mutated CRE8,9. CRE sequence: 5’-AGCTCCGTGACGTCCCG-3’, corresponding to the CRE that extends from nucleotide (nt) –177 to nt –162 in the promoter of the mouse gene for fibronectin9. DRE sequence: 5’-TTACCTCATCCCGTGAGCCT-3’, that corresponding to the DRE that extends from nt –194 to nt –175 in the promoter of the mouse c-jun gene7. Mutated CRE and mutated DRE were 5’-AGCTCCGTTATTTCCCG-3’ and 5’-TTACCTTTTCCGAAAGCCTT-3’, respectively.
・ _Ec_oR 1 (New England Biolabs Inc., Beverly, MA, USA; cat. no. R0101)
・ Calf intestinal alkaline phosphatase (Toyobo Co., Osaka, Japan; cat. no. CAP-101)
・ T4 polynucleotide kinase (Toyobo Co.; cat. no. PNK-111)
・ [γ-32P]ATP (6000 Ci/mmol; GE Healthcare Biosciences Co.)
・ Sephadex G-50 spin column (GE Healcare Biosciences Co.; cat. no. 17-0041-01)
・ 6% (w/v) Polyacrylamide gel (acrylamide:bisacrylamide, 19:1) prepared with 1x TBE (10 × 10 x 0.1 cm3)
・ 1x TBE: 89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA (pH 8.3)
・ Vertical gel-electrophoretic apparatus for fractionation of DNA and power supply
・ Equipment for autoradiography
・ Extraction buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% sodium sulfate (SDS; Sigma-Aldrich Corp., cat. no. L3771)
・ Micropore separator (Amicon, Millipore Corp., Bedford, MA, U.S.A; 0.22 mm pore size)
・ Tris-EDTA (TE) buffer: 10 mM Tris-Cl (pH 8.0), 1 mM EDTA
Equipment
Procedure
1. Digest ~20 mg of pB100-Uless/strider plasmid DNA with EcoR 1 and treat the digest with calf intestinal alkaline phosphatase (Takara Biotech. Co.) at 37 ℃ for 60 min. Purify the DNA by ethanol precipitation and remove excess salt by gently rinsing the pellet with ice-cold 70% ethanol.
2. Radiolabel the 5'-end of the site of restriction cleavage using T4 polynucleotide kinase and [γ-32P]ATP by standard methods. Remove unincorporated radiolabeled ATP with a Sephadex G-50 microcolumn, with subsequent ethanol precipitation as above.
3. Digest radiolabeled DNA with EcoR l, to liberate the 197-bp end-labeled DNA fragment.
4. Isolate the end-labeled DNA fragment on a preparative 5% nondenaturing polyacrylamide gel in 1x TBE.
5. After autoradiography of the wet gel, excise the radiolabeled band and crush the gel slice in an Eppendorf tube with a siliconized glass rod or pestle.
6. Add ~300 mL of elution buffer to the crushed gel, resuspend the fragments of gel in TE buffer and incubate the mixture at 37 ℃ to elute the end-labeled DNA fragment.
7. Filter the mixture with a MicropureTM separator (Sartorius AG, Göttingen, Germany) to remove gel pieces. After extraction of DNA with a mixture of phenol, chloroform and isoamyl alcohol, precipitate the radiolabeled DNA fragment in ethanol. Resuspend the fragment in TE buffer.
Troubleshooting
Notes
1. It is important to note that salt-mediated nuclesome-assembly methods fail to space nucleosomes with the physiological nucleosomal spacing of approximately 200 bp on general DNA fragments. Multiple nucleosomes reconstituted by these methods are generally “closely packed”, with one nucleosome every ~150 bp and little or no intervening linker DNA. However, salt-dialysis methods can be used to produce physiologically spaced oligonucleosomes if the DNA template contains tandem repeats of strong nucleosome-positioning sequences.
2. For mononucleosome reconstitution, DNA of 200 to 270 bp is probably most suitable1. The 3'-end of the DNA is labeled using Klenow DNA polymerase and [α-32P]-NTPs.
Anticipated Results
References
1. Rhodes, D. & Laskey, R. A. Assembly of nucleosomes and chromatin. Methods Enzymol. 170, 575-585 (1989)
2. Simpson, R. T., Thoma, F. & Brubaker, J. M. Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones; a model system for study of higher-order structure. Cell 42, 799-808 (1985).
3. Steger, D. J., Eberharter, A., John, S., Grant, P. A. & Workman, J. L. Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays. Proc. Natl. Acad. Sci. U.S.A. 95, 12924-12929 (1998).
4. Ito, T., Bulger, M., Pazin, M. J., Kobayashi, R. & Kadonaga, J. T. ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 90, 145-155 (1997).
5. Almouzni, G. Assembly of chromatin and nuclear structures in Xenopus egg extracts, in Chromatin: a Practical Approach (Gould, H., ed.), Oxford University Press, New York.
6. Becker, P. B., Tsukiyama, T. & Wu, C. Chromatin assembly extracts from Drosophila embryos. Methods Cell Biol. 44, 207-223 (1994).
7. 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).
8. 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).
9. Polly, P. & Nicholson, R.C. Sequence of the mouse fibronectin-encoding gene promoter region. Gene 137, 353-354 (1993).
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