We thank Royena Tanaz, Huda Yousuf, and Sadiqa Taasen for technical help. We are very grateful to Prof. James Shorter for the generous provision of reagents and intellectual assistance in the design of sucrose tuning experiments. Yeast plasmids were a generous gift from Prof. Aaron Gitler (including 303Gal-FUS; Addgene plasmid # 29614). Brooklyn College and the Advanced Science Research Center (CUNY) as well as an NIH NINDS Advanced Postdoctoral Transition Award (K22NS09131401) supported M.P.T.

1. Transforming S. cerevisiae with neurodegenerative proteinopathy-associated protein constructs
<ol>
	<li>Grow wild type (WT) 303 yeast in yeast extract peptone dextrose (YPD) broth overnight with shaking (200 rpm) at 30 &#176;C.</li>
	<li>After 12&#8722;16 h of growth, dilute yeast to an optical density at 600 nm (OD600) of 0.25 with YPD. As 10 mL of yeast liquid culture will be needed for each transformation, prepare 50 mL of yeast liquid culture for five transformations corresponding to FUS, TDP...

<ol>
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The authors have nothing to disclose.

The protocol described here provides a straightforward, expedient, and cost-effective way of categorizing genome-wide histone PTM changes correlated with neurodegenerative proteinopathies. While there are other models of ALS and PD, such as in vitro human cell lines and murine models32, S. cerevisiae remains attractive because of its ease of use. For instance, yeast models do not require use of a sterile hood, nor do they require the intensive training that goes along with cell c...

Neurodegenerative diseases are devastating illnesses with little to no treatment options available. Among these, amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD) are particularly dreadful. Approximately 90% of ALS and PD cases are considered sporadic, occurring without family history of the disease, while the remaining cases run in families and are generally linked to a specific gene mutation1,2. Interestingly, both of these diseases are associated with protein mislocalization and aggregation3,4,5,6. For instance, fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA binding proteins that mislocalize to the cytoplasm and aggregate in ALS7,8,9,10,11,12, while &#945;-synuclein is the principle component of proteinaceous aggregates termed Lewy bodies in PD5,13,14,15.
Despite the extensive genome-wide association efforts in large patient populations, the overwhelming majority of ALS and PD cases remain unexplained genetically. Can epigenetics play a role in neurodegenerative disease? Epigenetics comprises changes in gene expression occurring without changes to underlying DNA sequence16. A main epigenetic mechanism involves the post translational modifications (PTMs) of histone proteins16. In eukaryotic cells, genetic material is tightly wrapped into chromatin. The base unit of chromatin is the nucleosome, consisting of 146 base pairs of DNA wrapped around a histone octamer, composed of four pairs of histones (two copies each of histones H2A, H2B, H3, and H4)17. Each histone has an N-terminal tail that protrudes out of the nucleosome and can be modified by the addition of various chemical moieties, usually on lysine and arginine residues18. These PTMs are dynamic, which means they can be easily added and removed, and include groups such as acetylation, methylation, and phosphorylation. PTMs control the accessibility of DNA to the transcriptional machinery, and thus help control gene expression18. For example, histone acetylation reduces the strength of the electrostatic interaction between the highly basic histone protein and the negatively charged DNA backbone, allowing the genes packed by acetylated histones to be more accessible and thus highly expressed19. More recently, the remarkable biological specificity of particular histone PTMs and their combinations has led to the histone code hypothesis20,21 in which proteins that write, erase, and read histone PTMs all act in concert to modulate gene expression.
Yeast is a very useful model to study neurodegeneration. Importantly, many neuronal cellular pathways are conserved from yeast to humans22,23,24. Yeast recapitulate cytotoxicity phenotypes and protein inclusions upon overexpression of FUS, TDP-43, or &#945;-synuclein22,23,24,25,26. In fact, Saccharomyces cerevisiae models of ALS have been used to identify genetic risk factors in humans27. Furthermore, yeast overexpressing human &#945;-synuclein allowed for the characterization of the Rsp5 network as a druggable target to ameliorate &#945;-synuclein toxicity in neurons28,29.
Here, we describe a protocol exploiting Saccharomyces cerevisiae to detect genome-wide histone PTM changes associated with neurodegenerative proteinopathies (Figure 1). The use of S. cerevisiae is highly attractive because of its ease of use, low cost, and speed compared to other in vitro and animal models of neurodegeneration. Harnessing previously developed ALS and PD models22,23,25,26, we have overexpressed human FUS, TDP-43, and &#945;-synuclein in yeast and uncovered distinct histone PTM changes occurring in connection with each proteinopathy30. The protocol that we describe here can be completed in less than two weeks from transformation to data analysis.

<table>
	<tbody>
		<tr>
			<td>-His DO Supplement</td>
			<td>Clontech</td>
			<td>630415</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Running Buffer</td>
			<td></td>
			<td></td>
			<td>Mix: 141.65 g glycine (ThermoFisher BP381-1), 30.3 g Tizma base (Sigma-Aldrich T6066), 10 g sodium dodecyl sulfate (Sigma-Aldrich L3771), and 1 L deionized water, pH 8.8.</td>
		</tr>
		<tr>
			<td>12% Polyacrylamide Gels</td>
			<td>BIO-RAD</td>
			<td>456-1041</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H3 (Lys14) Primary Antibody</td>
			<td>MilliporeSigma</td>
			<td>07-353 (Lot No. 2762291)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H4 (Lys 16) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab109463 (Lot No. GR187780)</td>
			<td>Dilution: 1/2000</td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H4 (Lys12) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab46983 (Lot No. GR71882)</td>
			<td>Dilution: 1/5000</td>
		</tr>
		<tr>
			<td>Anti-dimethyl-Histone H3 (Lys36) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab9049 (Lot No. GR266894, GR3236147)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-Histone H3 Primary Antibody</td>
			<td>Abcam</td>
			<td>ab24834 (Lot No. GR236539, GR174196, GR3194335)</td>
			<td>Nuclear Loading Control; Dilution: 1/2000</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H2B (Thr129) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab188292 (Lot No. GR211874)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H3 (Ser10) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab5176 (Lot No. GR264582, GR192662, GR3217296)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>BioPhotometer D30</td>
			<td>Eppendorf</td>
			<td>6133000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish (100 x 20 mm)</td>
			<td>Eppendorf</td>
			<td>30702118</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Plate, 96 well</td>
			<td>Eppendorf</td>
			<td>30730011</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5804/5804 R/5810/5810 R</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IRDye 800 CW</td>
			<td>LI-COR</td>
			<td>926-32212 (Lot No. C60301-05, C61116-02, C80108-05)</td>
			<td>Dilution: 1/5000</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IRDye 860 RD</td>
			<td>LI-COR</td>
			<td>926-68073 (Lot No. C60217-06, C70323-06, C70601-05, C80116-07)</td>
			<td>Dilution: 1/2500</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra thick blot paper (filter paper)</td>
			<td>BIO-RAD</td>
			<td>1703968</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Prepare 50 % w/v solution.</td>
		</tr>
		<tr>
			<td>Immobilon-FL Transfer Membranes</td>
			<td>MilliporeSigma</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium acetate dihydrate (LiAc)</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td>Prepare a 1 M solution.</td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td></td>
			<td></td>
			<td>Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue (Sigma-Aldrich B8026), 4.7 mL glycerol, 1.2 mL 0.5M Trizma base pH 6.8, 0.93 g DL-Dithiothreitol (Sigma-Aldrich D0632), and 2.1 mL deionized water.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoeresis Cell</td>
			<td>BIO-RAD</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>2231300045</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Restriction Enzyme Buffer 2.1, 10x</td>
			<td>New England Bio Labs</td>
			<td>102855-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Nhe I Restriction Enzyme</td>
			<td>New England Bio Labs</td>
			<td>101228-710</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free Water</td>
			<td>Qiagen</td>
			<td>129114</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc Imaging System</td>
			<td>LI-COR Biosciences</td>
			<td>2800-03</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniTray Cell Culture Treated w/Lid Sterile, PS (86 x 128 mm)</td>
			<td>ThermoFisher</td>
			<td>165218</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-a-synuclein-GFP</td>
			<td>Gift from A. Gitler</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-ccdB</td>
			<td>Addgene</td>
			<td>14133</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303Gal-FUS</td>
			<td>Addgene</td>
			<td>29614</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-TDP-43</td>
			<td>Gift from A. Gitler</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) (PEG)</td>
			<td>Sigma-Aldrich</td>
			<td>P4338</td>
			<td>Prepare a 50% w/v solution.</td>
		</tr>
		<tr>
			<td>Ponceau S Stain</td>
			<td>Sigma-Aldrich</td>
			<td>P3504</td>
			<td>Mix: 0.5 g 0.1% w/w Ponceau S dye, 5 mL 1% v/v acetic acid (Sigma-Aldrich 320099), and 500 mL deionized water.</td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BIO-RAD</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>R7630</td>
			<td>Prepare 10% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Agilent Tech</td>
			<td>201190</td>
			<td></td>
		</tr>
		<tr>
			<td>SD-His plates</td>
			<td></td>
			<td></td>
			<td>Mix: 20 g Agar (Sigma-Aldrich A1296), 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids (ThermoFisher 291920), and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>SGal-His plates</td>
			<td></td>
			<td></td>
			<td>Mix: 20 g Agar, 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids, 100 mL galactose solution, and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate Loading Buffer</td>
			<td></td>
			<td></td>
			<td>Store at -20 oC. 6X, Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue, 0.93 g DL-Dithiothreitol, 2.1 mL deionized water, 4.7 mL glycerol, and 1.2 mL 0.5 M Trizma base, pH 6.8.</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>Prepare 0.2 M solution.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>TBS + 0.1% Tween 20 (TBST)</td>
			<td></td>
			<td></td>
			<td>Mix: 100 mL 10X TBS, 1 mL Tween 20 (Sigma-Aldrich P7949), and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>TBS Blocking Buffer</td>
			<td>LI-COR</td>
			<td>927-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td>BIO-RAD</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Buffer</td>
			<td></td>
			<td></td>
			<td>Mix: 22.5 g glycine, 4.84 g Tizma base, 400 mL methanol, 1 g sodium dodecyl sulfate, and 1.6 L deionized water.</td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline (TBS)</td>
			<td></td>
			<td></td>
			<td>10X, 7.6 pH, Solution: Mix 24 g Trizma base, and 88 g sodium chloride (Sigma-Aldrich S7653). Fill to 1 L with deionized water.</td>
		</tr>
		<tr>
			<td>WT 303 S. cerevisiae yeast</td>
			<td>Gift from J. Shorter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Peptone Dextrose (YPD)</td>
			<td>Sigma-Aldrich</td>
			<td>Y1375</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing histone post-translational modification alterations in yeast neurodegenerative proteinopathy models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

To illustrate this method, we will take advantage of recently published results30. WT human FUS and TDP-43 were overexpressed for 5 h, while WT &#945;-synuclein was overexpressed for 8 h. A ccdB construct was used as a vector negative control. Figure 2 shows growth suppression in solid and liquid cultures. Yeast was harvested as described and Western blotting with modification-specific antibodies was performed. Anti-total H3 was used a...

Watch this Scientific Journal Video about Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models at JoVE.com

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

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.

characterizing-histone-post-translational-modification-alterations

Research

JoVE Journal

Genetics

7.4K visualizações.  Brooklyn College. Este protocolo nos permite explorar facilmente as características epigenéticas da doença neurodegenerativa. A principal vantagem dessa técnica é que ela explora a levedura como um sistema modelo, em comparação com outros modelos celulares e animais de doença neurodegenerativa, a levedura é conveniente e econômica. Essa técnica nos permite mapear as alterações epigenéticas que surgem na doença neurodegenerativa, que podem levar a novos marcadores para diagnóstico, bem como novo...