The overall goal of this biochemical protocol is to generate nucleosomes with differentially isotope-labeled sister histones. For use in mapping post translational modification reactions simultaneously on both sister histones. The overall goal of this biochem protocol is to generate nucleosomes with differentially isotope-labeled sister histones for use in mapping post translational modifications simultaneously on both sister histones.
This method in combination with high resolution techniques to map post translational modifications such as in mass spectroscopy addresses key questions in chromatin biology like what mechanisms contribute to the formation of a symmetrically modified nucleosomes? The main advantage of this technique is that it can be easily established in a biochemical laboratory because it combines standard nucleosome reconstitution protocols with common protein purification methodologies. Two pools of histone H3 will be used for octamer reconstitution.
Nitrogen 15 labeled H3 that contains a polyhistidine-tag at the amino terminus, and carbon 13 labeled H3 that contains a streptavidin affinity tag at the amino terminus. Begin this procedure by dissolving the lyophilized histone aliquots including the two types of histone H3 in unfolding buffer. To each tube that contains about five milligrams of histones add one milliliter of unfolding buffer and mix by pipetting, but do not vortex.
Keep the tubes on ice for 30 minutes to allow complete unfolding. Next, measure the absorbance at 280 nanometers to determine the exact concentration of each histone. Mix the core histones to equally molar ratios, keeping in mind to include 50%each of the two histone H3 pools.
Use unfolding buffer to adjust the final protein concentration to approximately one milligram per milliliter. Transfer the mixture to a six kilodalton cutoff dialysis membrane. And dialyze against one to two liters of chilled refolding buffer at four degrees Celsius.
On the following day, collect the dialyzed material and remove any precipitates by centrifugation in a microfuge for five minutes at four degrees Celsius on maximum speed. After determining the octamer concentration, use a 10 kilodalton cutoff centrifugal filter unit to concentrate the octamer to a final volume of about two milliliters. An octamer concentration between 50 to 100 micromolar should be obtained.
Prepare for gel filtration by connecting the gel filtration column to an FPLC system. Equilibrate with two column volumes, or CV of 0.22 micrometer filtered and degassed refolding buffer. Inject the concentrated material at a flow rate of one milliliter per minute and collect one to 1.5 milliliter fractions.
To begin this procedure, pool the relevant fractions containing pure octamers and determine the concentration as described previously. Connect a one milliliter nickel nitrilotriacetic acid or nickel NTA column to the FPLC system. And equilibrate with 10 CV of 0.22 micrometer filtered and degassed refolding buffer at a flow rate of one milliliter per minute.
Pass the purified octamer through the nickel NTA using a flow rate of one milliliter per minute. Collect the flow through. Elute with 10 CV of refolding buffer containing 250 millimolar imidazole using a flow rate of one milliliter per minute.
Collect the nickel NTA elution. The streptavidin based purification should ideally be performed in a cold room. Equilibrate a one milliliter affinity column of a commercial streptavidin with 10 CV refolding buffer containing 250 millimolar imidazole using a batch gravity flow setup.
Pass the nickel NTA elution through the streptavidin affinity column. Collect the flow through. Elute with 10 CV of refolding buffer containing 2.5 millimolar desthiobiotin.
Collect the elution. After measuring the octamer concentration in the elution, add 10 times less TEV protease and let the reaction proceed overnight at four degrees Celsius. On the following day, transfer the mixture to a 50 kilodalton cutoff dialysis membrane and dialyze against refolding buffer overnight at four degrees Celsius.
Concentrate the purified asymmetric octamer using a 10 kilodalton cutoff centrifugal filter unit to a volume of one to two milliliters. The expected octamer concentration is 20 to 50 micromolar. Nucleosome reconstitution requires DNA containing a nucleosome positioning sequence.
Adjust the DNA salt concentration to two molar sodium chloride using a five molar sodium chloride stock solution. Mix the DNA with octamer and refolding buffer using the optimum octamer to DNA ratio determined from a small scale test. Add the octamer last to prevent any possibility of mixing the octamer and the DNA at a salt concentration lower than two molar.
Transfer the mix to a six kilodalton cutoff dialysis membrane and dialyze against one liter of refolding buffer overight at four degrees Celsius. On the following day, reduce the salt concentration of the dialysis buffer in a stepwise manner. Keeping the sample in each sodium chloride buffer for at least three hours.
Next, transfer the dialysis membrane to a beaker with one liter of assay buffer and continue dialysis overnight at four degrees Celsius. On the following day, collect the dialyzed sample. Remove any precipitate formed by centrifugation in a microfuge for five minutes at four degrees Celsius on maximum speed.
Concentrate the sample using a 30 kilodalton cutoff centrifugal filter unit to a final volume of about 300 microliters. Measure the DNA concentration at 260 nanometers. The final concentration should be in the range of 10 to 20 micromolar.
Store the sample at four degrees Celsius for up to several weeks. A gel filtration elution profile of the refolded histone mixture shows the histone octamers eluting at about 70 milliliters. An SDS page gel shows the correct histone stoichiometry.
The reconstituted octameric pool containing the three types of octamers is subjected to a tandem affinity purification scheme. The isolation of asymmetric species is verified by the presence of the affinity tag. The affinity tags are subsequently removed with TEV protease.
A small scale nucleosome reconstitution using different molar ratios of DNA to octamer showed that a ratio of one to one was optimal. A large scale nucleosome reconstitution was performed using the optimized DNA to octamer molar ratio, and the correct stoichiometry of the four core histones was observed. NMR spectroscopy of differentially isotope-labeled nucleosomes confirmed the presence of nitrogen 15 labeled and carbon 13 labeled sister H3 histones.
In this application example, nucleosomes that were differentially isotope-labeled and asymmetrically phosphorylated on C ring 10 of histone H3 were reacted with GCN5 acetyltransferase. And acetylation of histone H3 lysine 14 on both H3 copies was followed in real time by NMR spectroscopy. The analysis revealed that C ring 10 of histone H3 phosphorylation stimulates histone H3 lysine 14 acetylation by GCN5 in cis compared to in trans.
Once mastered, this technique can be done in five days if it's performed properly. After watching this video you should have a good understanding of how to reconstitute nucleosomes containing sister histones with different features.
