Question

What approaches can be used to generate chromatin in vitro? What approach is usually taken to study single nucleosomes on a short DNA fragment, and which is best for assembly arrays of physiologically spaced nucleosomes?

Answer 1

Hi,

The answer for your questions are as follows.

Highly defined nucleosome and chromatin samples are essential for studying the detailed mechanisms of chromatin.

Therefore, defined chromatin templates becomes a pre-requisite. The In vitro assembly can be performed at any level of complexity in case of chromatin assembly. The minimal chromatin unit should be used for structural, biochemical, and biophysical applications.

Also another essential pre-requisite to assembling chromatin is the availability of pure recombinant histone proteins that are free of post-translational modifications, and defined DNA templates.

Histone proteins are refolded into octamer as a first step towards assembling chromatin. Octamer is assembled by mixing each unfolded histone at equimolar amounts and refolding in 2M salt. The refolded complex contains a mix of octamer, H3-H4 and H2A-H2B and excess histones. Histone octamer is further purified by size exclusion column.

The various approaches for chromatin generation invitro are as follows.

1. Fluorescent labeling of the histones and refolding into octamer

Here, comprehensive in vitro biophysical and structural characterization of chromatin complexes is greatly aided by the usage of labeled components. Particularly, wide usage of fluorescently labeled histones to assemble nucleosomes that were used for solution-state binding affinity measurements is utilized.

2. End-labeling of DNA

When ever labelling of histones is not preferred, such as if labeling of histones disrupts functional interactions, or to confirm the presence of DNA in a complex with increased sensitivity, the DNA can also be labeled. Hence in this approach, DNA is labelled. Fluorescently labeled primers are used to amplify double stranded labeled DNA by PCR amplification. DNA can also be labeled internally, by using mutated bases at strategic positions.

3. Nucleosome assembly

Nucleosome assembly via salt gradient is traditionally employed to obtain large quantities of nucleosomes for crystallization and other biochemical as well as biophysical assays. With strongly positioning of DNA sequences this method results in high yields of homogenous mononucleosomes.

4. Trinucleosome assembly

Normally, a typical higher order chromatin templates used in vitro was composed of an array of 12 nucleosomes, assembled on a ~2400 bp DNA with 12 repeats of 601 DNA. These substrates are useful for a variety of biophysical assays, but not ideal for binding studies due to the fact that they can engage in multiple binding events.

Trinucleosomes are a minimalistic model for higher order chromatin that are more amenable to biochemical and biophysical studies. The central nucleosome represents a nucleosome in a chromatin context, as it is flanked on either side by a nucleosome.

5. “Micro-scale” or step dilution method for nucleosome reconsitution

Unlike the earlier methods for the small-scale reconstitution of nucleosomes using radioactively labeled DNA by simply assembling the octamer and DNA in 2 M salt and then diluting it with a no salt buffer, This technique can be used for unlabeled DNA or labeled DNA, or labeled histones.

This method is very much apt for studying single nucleosomes on a short DNA fragment.

and Triple nucleosome or Trinucleosome assembly is the best approach for assembly arrays of physiologically spaced nucleosomes.

Hope you are satisfied with the answer.

Expecting a feedback and rating.

Regards,

Dr.VMK