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

Summary

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.

Abstract

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.

Introduction

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" struct....

Protocol

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.

1. Purify and lyophilize recombinant core histones (H2A, H2B, H3, H4) as described¹³.
2. Dissolve approximately 5 mg of each lyophilized core histone in 3 ml of unfolding buffer (6 M guanidinium HCl, 20 mM Tris pH 7.5, 5 mM DTT). Allow each aliquot to d....

Results

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.......

Discussion

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 modifications^14-16,36. Here we describe a gene.......

Materials

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