This method allows for precise quantification of core histones H3 and H4 and their post-translational modifications from cell and tissue extracts consequently leading to the generation of high quality, reproducible results. The presented technique has three main advantages. First, it preserves native post-translational modifications of histones.
Secondly, it bypasses expensive low-throughput methods. Finally, it is fully compatible with downstream medium-throughput applications. Demonstrating the procedure will be Karolina Janczura a talented graduate student from my laboratory who fully optimized this protocol and employed the technique to answer important questions regarding epigenetic regulation in Alzheimer's disease.
To begin plate the cells in 10-centimeter tissue culture-treated dishes with the appropriate cell culture media according to the text protocol. Allow the cells to grow until they reach about 90%confluency. After the cells have reached the desired confluency gently aspirate the culture media and wash the cells twice with prewarmed serum-free media.
After this, remove the serum-free media from the cells and place the 10-centimeter dishes on ice. Add one milliliter of ice-cold extraction buffer to each dish. Using a plastic cell scraper collect the cells in the extraction buffer and transfer them to a labeled 1.5-milliliter tube.
Then place the tubes on ice. If frozen tissue is used, place the tissue in a pre-chilled 1.5-milliliter tube and briefly thaw it on ice. Next homogenize the brain tissue with a handheld homogenizer using the recommended amount of extraction buffer and number of strokes.
Homogenization is complete when there are no visible tissue fragments and the solution takes on a milky appearance. After this, use a single-channel 1, 000-microliter pipette to transfer the homogenate to a pre-chilled 1.5-milliliter tube and place the tubes on ice. Place the tube containing the cell lysates and homogenized brain tissue on a rotating platform rotating at 15 RPMs at four degrees Celsius.
After this, centrifuge the tube at maximum speed for 10 minutes at four degrees Celsius. Next transfer the supernatant to a new pre-chilled 1.5-milliliter tube and discard the pellet. Next neutralize the histones with a 1/4 volume of neutralization buffer, and mix by pipetting up and down.
Use pH strips to check the pH of the mixture, and further adjust the pH with neutralization buffer if needed. If acidic histones are not neutralized during this step, they will not bind to the column membrane properly, will pass through it and will be detected in the flow-through. Add 37.5 microliters of the sample to 12.5 microliters of sample buffer.
After a brief centrifugation denature the mixture at 99 degrees Celsius for 10 minutes. After this, place the tubes on ice and spin them briefly to collect condensate. Load the sample onto an SDS-PAGE gel and run the gel at 100 volts for one hour.
Then stain the gel overnight. And destain it during three consecutive washes. First add 500 microliters of equilibration buffer to each spin column.
Centrifuge the column for three minutes. Discard the flow-through and repeat centrifugation one more time. After this add 500 microliters of the sample to the column.
Centrifuge the column for three minutes and collect the flow-through. Then analyze the column flow-through as previously described. Add 500 microliters of wash buffer to the column.
Centrifuge the column for three minutes and collect the flow-through. Transfer the column to a new labeled 1.5-milliliter tube. Add 50 microliters of histone elution buffer to the column.
After centrifuging the column save the flow-through containing histone proteins. Under a chemical hood add perchloric acid to the purified histones to achieve a final concentration of 4%perchloric acid. Pipette up and down six times to mix.
If the histones in the sample are very concentrated, the solution will become cloudy after the addition of perchloric acid. After this, incubate the tube at four degrees Celsius for 24 hours. If the overnight histone precipitation is successful, a small white pellet will be visible on the bottom of the vial after the centrifugation step.
When washing the pellet in the consecutive steps assure the pellet is not disturbed as it can easily detach from the vial bottom. The next day centrifuge the samples for 75 minutes in pre-chilled microcentrifuge tubes. Carefully aspirate the supernatant and add 500 microliters ice-cold perchloric acid to the pelleted sample.
After centrifuging the sample for 10 minutes at maximum speed, aspirate the supernatant. Add 500 microliters of ice-cold acetone to the sample taking care not to disturb the pellet. Next remove the supernatant and allow the tubes to dry uncapped on ice for 30 minutes.
Then remove the tubes from the ice and allow the sample to dry at room temperature for five minutes. After the pellet is dry resuspend it in 30 microliters of sterile water. Cap the tubes and allow the histones to reconstitute on ice for 30 to 50 minutes.
After checking that the pellet is resuspended, recap the tubes and remove them from the ice. In this protocol histones from human microglial BV-2 cells were purified and the extract composition was analyzed. The stained gel demonstrates that extraction times between 15 minutes and 24 hours did not affect the overall composition of the crude histone extracts.
The efficiency of histone binding to the column's matrix was analyzed via staining of the gel. Column efficiency was approximately 100%as evidenced by the absence of detectable histone proteins in the column flow-through. Regardless of the extraction time duration, i.e.
15 minutes, two hours or 24 hours. first column wash eliminates the largest amount of non-histone proteins from the extract. The purified histone protein fractions from the 24-hour extraction group contained more H3 and H4 histones when compared to 15-minute and two-hour extraction times.
The first column elution was approximately 100%efficient as evidenced by the lack of histone proteins in the second eluate. Crude histones were then extracted from whole mouse brain and post-translational modifications were measured. In response to the broadly-acting HADC inhibitor, tributyrin, H4K12 acetylation increased in the extract.
However, the specificity of the antibody decreased due to the presence of impurities in the crude extract. The histone purification protocol was completed with the prefrontal cortex of triple transgenic A/D mice treated with the Class I and IIb HDAC inhibitor, M344, and the single pure core histone fraction was evaluated for changes in H4K12 acetylation. Total histones were detected and a significant increase of H4K12 acetylation was observed in response to M344.
While attempting this procedure it is important to remember to perform the multiple checkpoints described within the protocol to validate successful histone purification throughout the process. Following this procedure, additional questions regarding histone phosphorylation and methylation status can be addressed. Development of this technique paved the way for researchers in our lab to analyze the genetic mechanisms involved in Alzheimer's disease.
However it can be used in a variety of disease models where chromatin modification is hypothesized to play a significant role.
