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

Summary

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's disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Abstract

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson'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.

Introduction

Neurodegenerative diseases are devastating illnesses with little to no treatment options available. Among these, amyotrophic lateral sclerosis (ALS) and Parkinson'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 mutation^1,2. Interestingly, both of these diseases are associated with protein mislocalization and aggregation^3,4,5<....

Protocol

1. Transforming S. cerevisiae with neurodegenerative proteinopathy-associated protein constructs

1. Grow wild type (WT) 303 yeast in yeast extract peptone dextrose (YPD) broth overnight with shaking (200 rpm) at 30 °C.
2. After 12−16 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.......

Discussion

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.......

Materials

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