Introduction

Whilst great insights into the structure and properties of chromatin have been gained using chromatin samples extracted from native sources, analyses of such material have limitations because of their inherent heterogeneity. Native chromatin samples contain an ensemble of different core histones, linker histones and their variants, and other chromatin-associated proteins, all of which are adorned by a plethora of post-translational modifications. The DNA from these sources is also highly variable, prescribing varying and irregular nucleosome repeat lengths. Indeed, this heterogeneity is a consequence of the critical role that chromatin plays in the regulation of DNA transcription and replication, where local chromatin environments are tailored to suit the particular needs of a given DNA locus.

To overcome the limitations of using native chromatin samples, we have developed an in vitro nucleosome array reconstitution system that produces very long, highly regular and soluble nucleosome arrays, or folded ‘30nm’ chromatin fibres, with a stoichiometry of one histone octamer and up to one linker histone per nucleosome. The production of long nucleosome arrays of high homogeneity, with both a prescribed histone content and nucleosome repeat length is critical for obtaining biochemical, biophysical and structural data of high quality and reproducibility and permits the investigation of the oftentimes subtle determinants of chromatin compaction and function.

Our reconstitution system is based on DNA arrays constructed from the strong nucleosome positioning 601 DNA sequence and purified histones. The 601 DNA sequence was identified in a SELEX experiment by Widom which set out to find a DNA sequence that binds the histone octamer with high affinity and with a unique position (Lowary & Widom, 1998). The high affinity of the 601 DNA nucleosome positioning sequence for histones permits the use of Competitor DNA (crDNA) that controls nucleosome assembly, allowing the saturation of the 601 DNA array with the Histone octamer and Linker histones whilst preventing oversaturation (Huynh et al, 2005; Robinson et al, 2006; Routh et al, 2008). The assembly of the nucleosome arrays is by the classical and simple salt reconstitution system in which histones and DNA are mixed together in high salt followed by a slow dialysis to reduce the salt and to permit nucleosome formation.

Nucleosome array assembly Basic principles of the reconstitution protocol: DNA arrays are constructed using the Widom 601 nucleosome positioning DNA sequence. DNA arrays containing between 12 and 80 tandem 601 DNA repeats with different nucleosome repeat lengths (NRLs) (167 to 237bp) have been constructed (Huynh et al, 2005; Robinson et al, 2006; Routh et al, 2008). Competitor DNA (crDNA) 147 bp in length is included in the reconstitution to prevent the super-saturation of the 601 DNA arrays with excess histone octamer or linker histone. Histone octamers and linker histones: Histone octamers can be purified from native sources (Thomas & Butler, 1977) or assembled from recombinant histones (Luger et al, 1999). The latter method allows the incorporation of modified histones, or histone variants. Linker histones H1 and H5 isolated from native sources or recombinant linker histones can be used (Wellman et al, 1997), again permitting incorporation of a specified linker histone.

The starting point for the successful reconstitution of nucleosome arrays is the production of DNA arrays containing the 601 nucleosome positioning DNA sequence arranged in tandem. The use of a unique sequence DNA confers the advantage of specifying both the number of nucleosomes in the array and the NRL (Routh et al, 2008). The key to the reconstitution protocol is that the 601 DNA binds histone octamers with very high affinity (Lowary & Widom, 1998) so that any histone octamers that are added to the reconstitution mixture bind to the 601 DNA in preference to the weakly-binding Competitor DNA. The histone octamers will only bind to the crDNA once all the 601 DNA positions have been occupied, resulting in the saturation of the 601 DNA but not super-saturation. Without the crDNA, excess histone octamers would bind non-specifically to the 601 nucleosome arrays causing them to aggregate and to precipitate. This creates a ‘window’ in which the 601 DNA array is saturated but the crDNA is not, which can easily be determined by native gel electrophoresis. Other DNA arrays, such as the ones based on the 5S rDNA gene are available, but these are short (contain only 12 repeats) and produce less homogeneous nucleosome arrays because of multiple nucleosome positions (Panetta et al, 1998).

The reconstitution starts with the mixing of histones and DNA in high salt. Here, the exact molar input of histone octamer required to saturate a DNA array must be determined empirically by a histone octamer titration. Several samples are reconstituted in parallel with increasing concentrations of histone octamer. A straight forward calculation to determine the correct stoichiometric input based upon mass is usually inaccurate. This is due to difficulty in estimations of histone octamer sample concentrations, the loss of histone octamer through aggregation and due to the ‘sticking’ (comment 1) of the highly charged histone octamers onto the reconstitution vessels. This is why the empirically determined input ratios of histone octamer to DNA template may vary greatly between seemingly identical reconstitutions if they are carried out in different vessels, reaction volumes, temperatures, etc. Once the saturation point of the histone octamer has been established, a similar titration must also be carried out (perhaps even more critically) to find the correct molar input of linker histone required to saturate the nucleosome core array.

The protocol given below describes the reconstitution using a DNA array containing 25 tandem repeats of 197bp 601 DNA (197bp x 25). The protocol is essentially split into two sections, a histone octamer titration and a linker histone titration, performed in three overnight steps.

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Procedure Day 1: Histone octamer titration A series of reactions are set up side-by-side, encompassing a molar input ratio from 0.5:1 to 3:1 of histone octamer: DNA (for example, see table 1). The histone octamer and DNA are mixed together in high salt (2M NaCl) in RNase-free, non-stick Eppendorf tubes and placed on ice. Typically, the DNA array is at a concentration of 25µg/ml (0.2 10-6 M 601 DNA repeat) but should not exceed ~250µg/ml (2.0 10-6 M 601 DNA repeat). For optimal reconstitutions, the crDNA is included at a mass ratio of 1:1 crDNA: 601 DNA array. All manipulations are carried out at +4ºC. Note: It is usually more practical and reproducible to make up a stock of the 197bp x 25 601 DNA array and crDNA aliquot the mixture. Each reaction is placed into a dialysis vessel. We prefer to use small glass tubes (siliconized to prevent sticking), which are sealed at one end with a silicone bung, and at the other with a section of dialysis membrane (7’000 MWCO). All the dialysis vessels are then placed inside another dialysis bag, filled with ~20ml reaction buffer (2M NaCl, 10mM TEA-HCl pH7.4, 1mM EDTA). This is then placed into a large volume (e.g. 4 litres) of low-salt buffer (10mM TEA-HCl (comment 2) pH 7.4, 1mM EDTA) and left to dialyse overnight at +4ºC. The TEA �HCl buffer is used in the reconstitution to permit glutaraldehyde cross-linking required in later analyses.

Such ‘double bag’ dialysis slows the dialysis to provide sufficient time for the histones to ‘find’ their 601 DNA sequence before the salt concentration drops too low forcing the proteins to stick non-specifically to any available DNA. Dialysis that is too quick can produce material that either precipitates, or creates smeary bands when run on a native agarose gel (comment 3).

Table 1: Histone Octamer Titration