Nucleosome mobilization assay

Introduction

The organization of eukaryotic genomes into nucleosome arrays restricts DNA sequence accessibility to many nuclear factors. Thus most DNA-based processes require opening (or "re-closing") of these arrays. One major class of enzymes, the "chromatin/nucleosome remodeling" factors, uses ATP hydrolysis to alter the canonical histone-DNA contacts. The term "nucleosome remodeling" can be defined and monitored in different ways (Flaus and Owen-Hughes, 2004). The simplest configuration to study one aspect of nucleosome remodeling is to use a purely reconstituted system consisting of mononucleosomes and an ATP-dependent nucleosome remodeler in the so-called "nucleosome sliding" or "nucleosome mobilization" assay. This technique was initially developed by Carl Wu and Peter Becker laboratories (Hamiche et al. , 1999; Langst et al. , 1999) by taking advantage of two nucleosome properties: Nucleosomal histones can moderately move on DNA under rather mild temperature and salt conditions (Beard, 1978; Meersseman et al. , 1991; Pennings et al. , 1991) and nucleosomes reconstituted on a short DNA fragment can adopt multiple positions that can be separated by native gel electrophoresis (Linxweiler and Horz, 1984; Pennings et al. , 1991).

In fact, the sliding assay monitors alterations in nucleosomes electrophoretic mobility in native gel that are caused by remodeling factors in an ATP-dependent manner. Repositioning of the histone octamer along a DNA fragment usually accounts for these mobility shifts. However, changes in electrophoretic mobility can also result from an altered (non-canonical) nucleosomal DNA path (Kassabov et al. , 2003; Narlikar et al. , 2001). Hence, actual repositioning of the histone octamer may need to be confirmed by mapping of the new positions for uncharacterized remodelers. It is also noteworthy that mononucleosomes do not recapitulate all chromatin properties (Hansen, 2002). Consequently, all conclusions derived from using this substrate may not always apply to nucleosome arrays (even not to other mononucleosomes using a different DNA template). Despite these caveats, the sliding assay is still a powerful tool that has greatly contributed to our understanding of how chromatin remodeling factors work.

Acknowledgements: I am grateful to Gernot Längst and Anton Eberharter for introducing me to this great technique.

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Procedure

Preparation of the nucleosome substrate

Nucleosomes can be assembled using various methods (Chromatin Protocols, 1999 and ref. therein). The central theme to all these methods is mixing histones and DNA together, roughly around a 1:1 ratio (w/w).

Before getting into the details of nucleosome assembly, it is important to prepare the two reagents (DNA and histones) with special cares as follows:

Preparation of the DNA template

See note 1

As mentioned above, the nucleosome sliding assay relies on the fact that the location of a histone octamer on a DNA fragment affects its electrophoretic mobility in native polyacrylamide (PAA) gels. Centrally positioned nucleosomes migrate slower than nucleosomes positioned at one end of a DNA fragment.

In this assay, DNA templates between 200 bp and 300 bp will be preferred for the following reasons: DNA fragments in this size range should allow the formation of essentially one nucleosome per template. Using longer DNA fragments would require performing the assembly at lower histone: DNA ratios (around 0.6:1) to avoid assembly of more than one nucleosome per DNA template. This would result in lower mononucleosome assembly yields. More importantly, having a limited number of nucleosome positions (ideally two positions) greatly simplifies the readout of this assay (Note that a very strong positioning sequence on a long DNA fragment may lead to having one position after assembly and a weak positioning sequence may still lead to getting several positions on a small DNA template).

DNA fragments shorter than 200 bp can also be used however some remodelers require a minimal length of DNA protruding from the nucleosome core to perform the remodeling reaction (typically, ISWI subfamily members, (Aalfs et al. , 2001; Zofall et al. , 2004)) and separation of the various nucleosome positions is less apparent with such fragments. It is also worth mentioning that not all DNA sequences can be efficiently assembled into nucleosome (Cao et al. , 1998).

For all these reasons, it is convenient to use the 247 bp (or 248 bp) murine rDNA promoter fragment.

PCR cycles (see comment 1 and note 2)

94°C for 2 minutes / (94°C for 15 seconds / 60°C for 30 seconds / 72°C for 30 seconds) x30 cycles / 72°C for 5 minutes.

DNA precipitation

1. Precipitate PCR product by adding 1/10 volume of 3M Na-Acetate pH 5.2 and 2.5 volumes of ice-cold ethanol;
2. Leave on ice for 20 minutes;
3. Collect DNA by centrifugation at 13000Xg for 30 minutes at 4°C;
4. Wash pellet with ice-cold 70% ethanol;
5. Dry (partially) and resuspend DNA in 100µl volume of TE.

Gel purification of the DNA (see comment 2)

1. Pour a 4.5% PAA (0.5X TBE) mini-gel (classic protein gel dimensions) with at least 2 wells. A large one that allows loading of the PCR (≥ 110µl) and an additional well to load running dyes (orange G, bromophenol blue and xylene cyanol) to follow the migration;
2. Rinse wells (with a syringe) and pre-run gel at 80-100V for about 45 minutes;
3. Add 10% glycerol (v/v) to the PCR product, mix and load on the 4.5% PAA gel;
4. Run until orange G reaches the bottom of the gel. Using TBE, there is no need for buffer recirculation like for TE gels;
5. From now on, handle the gel behind a Plexiglas screen. Open gel (leave on one plate) and cover with Saran™ wrap;
6. Stick phosphorescent position markers on 2 opposite sides of the gel, place a film on the gel and put a small glass-plate on top to maintain film against the gel for 10-15 minutes (in a dark room);
7. Develop film and make a hole around the band of interest BEFORE overlaying the film on the gel;
8. Place the film according to the position markers and cut through the Saran™ wrap into the gel following the outline of the hole using a clean scalpel;
9. Place the gel slice in 1.5ml tube containing 1ml of TE (10mM TRIS-HCl pH 8.0, 1mM EDTA) and allow diffusion of the DNA out of the gel for about 2 hours;
10. Recover the supernatant and repeat DNA elution with 1ml of TE for at least 2 hours (preferably overnight);
11. Combine supernatants and roughly monitor elution efficiency with a Geiger counter by comparing supernatants to gel slice (take into consideration that the aqueous solution reduces the radioactive signal);
12. Optional: If a 'significant' amount of DNA is left behind in gel slice, repeat DNA elution with another 1ml of TE overnight;
13. Combine all eluates and precipitate the DNA in 15ml tube as described above (in the DNA precipitation section);
14. Resuspend DNA in about 100µl volume of TE containing 150mM NaCl (TE150);
15. Determine DNA concentration by using a spectrophotometer or by running 1µl and 2µl of DNA on a 1.5% agarose gel along with a cold DNA fragment of known concentration (e.g. DNA size markers);

Depending on the efficiency of all the previous steps and the option you choose, the final DNA concentration may range from 100-800ng/µl. (see note 3)

Preparation of the Histone-PGA (HP)-mix

As mentioned above, mononucleosomes can be assembled in different ways. However, when handling radioactive DNA and/or small amounts of material, it is very convenient to use the following method developed by Stein and colleagues (Stein et al. , 1979).

Poly-L-glutamic acid (PGA) is a negatively charged polymer, which can force histones to form octamers in low salt conditions. It can also prevent histones from precipitating onto DNA when both are mixed under these ionic conditions. The PGA polymers then progressively exchange with DNA molecules allowing nucleosome assembly (see note 4).

HP-mix

1. Prepare a 10mg/ml PGA (Sigma P4886; MW: 50,000-100,000) stock solution in water (keep aliquots at -20°C);
2. Add a 2-fold weight excess of PGA to histones. Flick tube about 6-10 times;
3. Adjust salt concentration to 150mM NaCl with TE (pH 8.0); gradually fill up with TE150 to a final histone concentration of 50-100ng/µl.
   Typical reaction
   25 µl histones (2µg/µl, in 1M NaCl/50% glycerol) = 50µg histones
   10µl PGA (10µg/µl) = 100µg PGA
   132µl TE
   833µl TE150;
4. GENTLY pipette up and down 3-4 times and leave at room temperature for 1 hour;
5. Spin down possible aggregates at 13000Xg for 10 minutes and transfer into fresh tubes;
6. Aliquot supernatant (200µl each) and store at -20°C (see note 5)

Nucleosome assembly

The optimal nucleosome assembly is usually obtained around a histone:DNA ratio of 0.9:1 (w/w). However, the affinity of DNA sequences for the histone octamer can vary significantly (Thastrom et al. , 2004; Wu and Travers, 2005). Furthermore, histone preparations of slightly different quality may also affect optimal assembly concentrations. Therefore it is important to carefully determine the most advantageous histone:DNA ratio experimentally (for every new DNA or new histone/HP-mix preparation) before setting up a large assembly for the best results. A meticulous titration is especially important here since preparative nucleosome reconstitutions often require to scale up test assemblies about 200 folds.