This work was supported by NIH grants GM45916 and GM66834 to J.C.H. and a fellowship from the International Rett Syndrome Foundation to A.K. This work was also supported by NIH grant GM088409 and Howard Hughes Medical Institute contributions to K.L.

1. Assembly of Recombinant Core Histones into Octamers
Rationale: The first step in nucleosomal array reconstitution is to prepare native core histone octamers from lyophilized recombinant core histones. Histone proteins are combined in equal molar amounts and assembled into histone octamers by dialyzing the samples out of a denaturing buffer into refolding buffer.
<ol>
	<li>Purify and lyophilize recombinant core histones (H2A, H2B, H3, H4) as described13.</li>
	<li>Dissolve appro...

<ol>
	<li>Luger, K., Mader, A., Richmond, R., Crystal Sargent, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 7, (1997).</li><li>Hansen, J. 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Model nucleosomal arrays are a very useful tool for the in vitro study of chromatin structure and function. For example, they have been widely used to study the mechanism of chromatin fiber condensation in solution 30-34, and made it possible to obtain an x-ray structure of a tetranucleosome 35. More recently they have proven useful in deciphering the structural effects of specific core histone variants, mutants and posttranslational modifications14-16,36. Here we describe a gene...

Eukaryotic genomes do not exist as naked DNA, but rather are compacted and organized by bound proteins. These complexes of DNA and protein are known as chromatin. The basic repeating unit of chromatin is the nucleosome. A nucleosome consists of histone octamer and 146 base pairs of DNA wrapped around the histone octamer about 1.6 times1. The histone octamer is composed of two copies each of the core histones H2A, H2B, H3, and H4. Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. The extended structure of nucleosomal arrays has been referred to as the 10 nm fiber or the "beads on a string" structure and is present in vitro under low salt conditions2. The 10 nm fiber is capable of condensing into higher order structures through intra-array compaction and/or inter-array oligomerization2. These higher order structures can be induced in the presence of salts, or can be influenced through the binding of chromatin architectural proteins to the nucleosomal array3,4. Levels of chromatin compaction are inversely correlated with rate of transcription in vivo5,6. Recent research highlights the importance of the structural organization of genomes in processes such as differentiation, cancer development, and others7,8. The use of nucleosomal arrays to study chromatin structure and function has become widespread. Here we describe a method for the assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning DNA.
Using recombinant DNA with tandem repeats of nucleosome positioning sequences allows for the reconstitution of arrays that contain regularly spaced nucleosomes. Two of the more popular positioning sequences are the 5S rRNA gene sequence and the "601" sequence9,10. The 601 sequence was derived from SELEX experiments and more strongly positions nucleosomes than the 5S sequence11. Consequently, the linker DNA length of the 601 arrays is more homogeneous. Tandemly repeated nucleosome positioning DNA is obtained by gel filtration4,12. Recombinant histones are purified from E. coli under denaturing conditions13. The use of recombinant histones allows one to carefully control the histone composition of the nucleosomal arrays. For example, core histones bearing specific mutations14 or post-translational modifications15,16 can be substituted for wild type core histones.
Sedimentation velocity experiments monitor the rate of sedimentation of macromolecules in solution under an applied centrifugal force17. This yields information about the size and shape of macromolecules in a sample. Sedimentation velocity experiments are thus an appropriate tool for studying solution state changes in chromatin fiber structure due to chromatin condensation18. Importantly, it is first necessary to use sedimentation velocity experiments as a quality control step in nucleosomal array reconstitution. If DNA and nucleosomes are not combined at the proper molar ratio, the arrays may be under- or over-saturated with core histones. Thus, the information gained from sedimentation velocity experiments is used to ensure that the DNA is properly saturated with nucleosomes. It is important to use alternative methods to estimate the saturation of DNA with nucleosomes, especially if working with a previously uncharacterized DNA template. Therefore, we also describe a method for analysis of nucleosomal arrays using atomic force microscopy (AFM). AFM is a powerful technique that allows visualization of the effects of a number of parameters, such as the level of saturation, effect of the presence of histone variants or the effects of MgCl2 19,20. AFM has also been applied to study nucleosome dynamics using time lapse imaging 21. In vitro assembled nucleosomal 12-mer arrays are particularly amenable to AFM studies because they belong in the right size range for AFM imaging 22. In the present study we have used AFM of nucleosomal arrays as a quality control as well as a means of affirming the data from AUC ("seeing is believing"). In addition to simple visualization, AFM allows measurement of height profiles of samples as an additional metric.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(3-Aminopropyl)triethoxysilane</td>
 <td>Sigma-Aldrich </td>
 <td>A3648-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>6-8 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>21-152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>HiLoad Superdex 200 16/60 Column</td>
 <td>GE</td>
 <td>17-1069-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Vivaspin 50 kDa MWCO Centrifugal Concentrator</td>
 <td>Sartorius</td>
 <td>VS2031</td>
 <td></td>
 </tr>
 <tr>
 <td>12-14 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>08-667A</td>
 <td></td>
 </tr>
 <tr>
 <td>Illustra Sephacryl S-1000 Superfine</td>
 <td>GE</td>
 <td>17-0476-01 </td>
 <td></td>
 </tr>
 <tr>
 <td>XL-A/I Analytical Ultracentrifuge</td>
 <td>Beckman-Coulter</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning dna

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

To illustrate the protocol we reconstituted nucleosomal arrays from recombinant Xenopus core histones and DNA consisting of 12 tandem 207 bp repeats of the 601 positioning sequence (601207 x 12). We first assembled native octamers from lyophilized core histones and then purified the octamers by FPLC using an S200 column (Figure 1A). Larger complexes elute earlier from the S200 column. Histones generally elute in this order: non-specific histone aggregates, histone octamer, H3/H4 tetra...

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Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

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Cells and their Components

SCIENTISTS IN ACTION

22.4K vistas.  Colorado State University.  Se presenta un método para la reconstitución del modelo nucleosomal arrays de histonas y el ADN recombinante posicionamiento de nucleosomas en tándem repetido. También describe cómo se utilizan los experimentos de velocidad de sedimentación en la ultracentrífuga analítica y microscopía de fuerza atómica (AFM) para supervisar el grado de saturación de gama nucleosomal después de la reconstitución. 