(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.
(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":http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.php