Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

The aim of the following procedure is to measure the various biochemical activities of a TP dependent chromatin remodeling complexes. This is achieved by measuring a TP dependent sliding of a position nucleosome from one end of a DNA molecule to a more central position on the DNA by using an electrophoretic mobility shift assay or emsa. A similar EMSA based method can be used to monitor the formation of remodeling complex bound mononucleo zones to assess the nucleosome binding activity of the chromatin remodeling complexes.

Additionally, thin layer chromatography can be used to measure the APAs activity of a TP dependent chromatin remodeling complexes. Ultimately, these methods can be used to measure the a TP dependent chromatin remodeling of human in O 80 complexes. In this video, we will demonstrate biochemical assays studying NO 80 chromatin remodeling complexes.

Similar method can also be applied to study other chromatin remodeling complexes as well To perform the a TP dependent nucleosome remodeling assay. First prepare nucleosome substrates after setting up a 100 microliter PCR reaction as outlined in the table. Perform the PCR reactions in a thermal cycler and then pass the reaction products twice through nuclease free spin columns to remove any unincorporated nucleotides.

Next, mix two ole of P 32 labeled 6 0 1 DNA fragments with six micrograms of hilar nucleosomes in 50 microliters of buffer, and incubate the mixture at 30 degrees Celsius for 30 minutes. Then sequentially dilute the mixture to 0.8 molar, 0.6 molar and 0.4 molar sodium chloride by addition of 12.5 microliters, 20.8 microliters and 41.6 microliters respectively of tris HCL buffer solution supplemented with E-D-T-A-P-M-S-F and DTT incubate for 30 minutes at 30 degrees Celsius after each addition, after the last incubation at 125 and then 250 microliters of the tris hydrochloride buffer. Further supplemented with non indebt P 40 glycerol and PSA in two more, 30 minute, 30 degrees Celsius dilutions to 0.2 molar and 0.1 molar sodium chloride respectively.

Then store the mono nucleosome substrate at four degrees Celsius for up to three months before setting up the assays cast native poly acrylamide gels. Then for each reaction to be performed, combine around 20 nanomolar in oh 80 with the appropriate amount of EB 100 buffer sufficient to give a volume of 4.75 microliters in a pre chilled siliconized. 1.5 milliliter micro centrifuge tube immediately refreeze any remaining NO 80 containing fractions, imp powdered dry ice.

Next, dispense 5.25 microliters of freshly prepared master cocktail to each reaction tube, mixing well by pipetting up and down. Then incubate the reactions in a 30 degrees Celsius heat block. At the end of the incubation add 1.5 microliters of freshly prepared removing mix to each tube to d terminate the reactions, then mix each reaction tube.

Well spin them down and incubate them at 30 degrees Celsius for another 30 minutes. Meanwhile, pre-run the native poly acrylamide gel in a vertical electrophoresis unit at 100 volts for 30 minutes at four degrees Celsius, using 0.5 XTBE as running buffer with a magnetic stir bar inside the lower chamber to maintain a constant buffer circulation at the end of the incubation with removing mix, mix 2.5 microliters of loading die with each reaction. And after a brief spin, use loading tips to load the samples onto the gel.

Run the gel at 200 volts for 4.5 hours at four degrees Celsius with buffer circulation. Then transfer the gel to a stack of two sheets of filter paper and drop the filters of paper with the gel on top using clear plastic wrap to detect the signal. Expose the gel to a storage phospho screen at four degrees Celsius for the desired time, and then scan the screen with an isotope imaging scanner system and analyze the data using the appropriate software to perform the DNA and nucleosome dependent APAs assays.

Begin by combining 10 to 50 nanomolar of the immuno purified sub complexes with an amount of eeb 100 buffer sufficient to give a volume of 2.2 microliters in one pre chilled lubricated 1.5 milliliter micro centrifuge tube for each reaction, and then immediately refreeze any NO 80 containing fractions in powdered dry ice. Then gently mix 2.8 microliters of freshly prepared sub cocktail containing DNA nucleosomes or neither to the enzyme containing reaction tubes with up and down pipetting and transfer the tubes to a 30 degrees Celsius heat block. Next, use a pencil to draw a very light line at least 1.5 centimeters from the bottom edge of a 20 by 10 polyethylene.

I mean thin layer chromatography or TLC plate. Then spot 0.5 microliters of each reaction mixture onto the line at 5 15, 30, and 60 minutes after incubation immediately returning each sample to the heat block, so multiple time points can be taken from a single tube. After applying the last sample, use a hair dryer to dry the plate and then transfer the plate to a glass chamber containing enough.

0.375 molar potassium phosphate to allow the bottom 0.5 centimeters of the TLC plate to be submerged in the solution. Cover the chamber and develop the reaction until the front of the liquid phase reaches the top of the TLC plate, at which point the plate should be immediately blow dried and exposed to a storage phospho screen at room temperature. Finally, scan the screen with an isotope imaging scanner system to determine the amount of radioactive a TP substrate and a DP product.

This first experiment demonstrates that complex is purified through flag IES two flag, N-O-A-T-E and flag NO 80 delta N have similar nucleosome sliding activities indicating that the NO 80 N terminus and or its associated subunits are dispensable for nucleosome remodeling by the NO 80 complex. In contrast, complex is purified through flag NO 80 delta n delta HSA are inactive in this assay, indicating that the remodeling activity depends on the HSA domain of the NO 80 APAs and or its associated subunits. Using a modification of the NUCLEOSOME sliding assay procedure, one can measure the nucleosome binding activity of chromatin remodeling complexes.

In this experiment, the incubation of the nucleosomes with increasing amounts of intact NO 80 complexes purified through the NO 80 E subunit led to a dose dependent disappearance of the band corresponding to the free mononucleo zones and the appearance of a new shifted species that migrated near the top of the gel. In contrast, while the nucleosomes were incubated with smaller complexes that were purified through NO 80 delta N and that lacked a subset of NO 80 subunits, the shifted species migrated more rapidly suggesting that the relative mobility of the super shifted band is determined by the size of the complexes assay. In this final experiment, the results of an assay comparing the DNA and NUCLEOSOME activated APAs activities of two different NO 80 sub complexes are shown.

The more slowly migrating spots correspond to the starting alpha 32 P labeled a TP, and the more rapidly migrating species are the A DP reaction products with the areas indicating the direction of solvent migration. While using these procedures, it's important to remember to perform assays that vary in length of time and contain different concentrations of enzyme. This will ensure that measurements are being taken when the product time and dose response curves are linear.

After watching this video, you should have a good understanding how to analyze the at TPAs nucleosome binding and Nucleosome sliding activities of purified chromatin remodeling complexes. Don't forget that working with radioactive materials can be hazardous. One should take precautions like wearing a lab coat and eye protection monitoring for radioactive contamination and properly disposing of radioactive waste.