This protocol allows us to easily explore the epigenetic features of neurodegenerative disease. The main advantage of this technique is that it exploits yeast as a model system, compared to other cellular and animal models of neurodegenerative disease, yeast is expedient and cost-efficient. This technique allows us to map out the epigenetic changes that arise in neurodegenerative disease, which may lead to novel markers for diagnosis, as well as new targets for therapy.
This method allows further investigation of the role of epigenetic marks in neurodegenerative disease and can be modified for the assessment of human fibroblasts and induced pluripotent stem cells. This particular method contains a number of details that need to be demonstrated visually, such as the proper loading of the Western blot apparatus. Demonstrating the procedure will be Michel Fallah and Navin Rana, both undergraduate researchers in the Torrente laboratory.
Begin by restreaking yeast from frozen glycerol stocks onto histidine 2%glucose agar selective medium plates, for a two to three day incubation at 30 degrees Celsius. At the end of the incubation, inoculate restreaked yeast for each over expression model and control in five milliliters of histidine selective liquid medium. Supplemented with 2%raffinose at 30 degrees Celsius and 200 rotations per minute, overnight.
The next morning, grow 100 milliliters of liquid culture for each of the overexpression models and controls in selective mediums supplemented with 2%galactose overnight. And measure the optical density at 600 nanometers or OD600 to determine the volume of overnight culture needed to render over expression cultures at a starting OD600 of 0.3. To induce protein overexpression, grow the yeast on galactose medium with shaking at 30 degrees Celsius for the appropriate experimental time period.
Then measure the OD600 of each culture at the end of the induction period. And standardize all of the cell counts to the lowest OD600 value. Harvest the cultures in 50 milliliter centrifuge tubes by centrifugation and resuspend the pellets in one milliliter of sterile stilled water for every 10 milliliters of culture grown.
Aliquot one milliliter of cell resuspension into individual microcentrifuge tubes for an additional centrifugation and aspirate the supernatants. Then snap freeze the cell pellets in liquid nitrogen for storage at minus 80 degrees celsius. To lyse the cells for Western blot analysis, thaw the yeast cell pellets on ice before resuspension in 100 microliters of distilled water.
Add 300 microliters of 0.2-molar sodium hydroxide and 20 microliters of 2-Mercaptoethanol to each cell sample. And resuspend the pellets by pipetting. After a 10 minute incubation on ice, pellet the cells by centrifugation and resuspend the samples in 100 microliters of loading dye.
Then boil the samples for 10 minutes on a heating block. While the samples are being lysed, place two gels in a gel holder and fill the inner chamber to the top with running buffer and the outside chamber to the two-gel line mark. When the samples are ready, load 15 microliters each, cell lysis solution, into each well of the 10 well, 12%polyacrylamide gel and add five microliters of protein ladder to the protein ladder lane well.
Run the gel for approximately 45 minutes at 150 volts or until loading dye front reaches the bottom of the gel. While the gel is running, soak two fiber pads per gel in transfer buffer and one polyvinylidene fluoride, or PVDF, membrane per gel in methanol. When the gel is ready, rinse membrane in transfer buffer and place one presoaked fiber pad on the bottom of a semi-dry transfer apparatus cell.
Followed by the PVDF membrane, the gel and the second presoaked fiber pad. Then set the power to 150 milliamps for one hour. At the end of the protein transfer, remove the membrane from the transfer apparatus for a brief rinse with distilled water.
Place the rinsed membrane, protein side up in a small staining box and cover the membrane with tris-buffered saline or TBS, blocking buffer for a one hour incubation at room temperature with gentle rocking. At the end of the blocking incubation, incubate the membrane overnight with an anti-yeast histone and modification-specific antibody in TBS at four degrees Celsius. And a proper nuclear loading control antibody.
The next morning, wash the membrane four times in TBS, supplemented with 0.1%polysorbate 20 for five minutes with rocking at room temperature per wash. After the last wash, incubate the blot with the appropriate secondary antibodies for one hour at room temperature. Followed by four, five minute washes in TBST and one five minute wash in TBS alone with rocking.
Then image the blot on a fluorescent Western blot imagining system for two minutes. Here, growth suppression in solid and liquid cultures is shown with significant effects on yeast growth observed in the presence of galactose-infused in sarcoma overexpression models. A significant decrease in histone levels are apparent in the fused in sarcoma overexpression model and significant increases in the levels of histone acetylation in the TAR binding protein 43 overexpression model, are observed that are not seen in either the fused in sarcoma or alpha synuclein overexpression model.
There are also significant decreases in the histone levels in the alpha synuclein overexpression model. In this sucrose-tuning experiment, the lower the amount of galactose used to induce fused in sarcoma overexpression, the lower toxicity that is observed in both solid and liquid culture. Importantly, the lower the level of fused in sarcoma overexpression, the smaller the magnitude of the reduction in histone levels.
While performing this protocol, it is imperative to standardize the amount of yeast in each sample to enable a proper comparison in the levels of histone post-translational modification. 2-Mercaptoethanol should be used under a hood to avoid inhalation. If Ponceau stain is used, it must be discarded properly.
