Detecting Histone Modifications in Yeast Cells with Neurodegenerative Protein Overexpression

Abstract

Source: Bennett, S. A., et al., Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models. J. Vis. Exp. (2019).

This video demonstrates the procedure of assessing histone modifications in yeast cells overexpressing neurodegenerative proteins. The steps include cell lysis, centrifugation, electrophoresis, membrane transfer, and antibody-based detection of modified histones.

Protocol

1. Cell lysis and western blotting to detect histone post-translational modifications

1. Cell lysis
   1. Thaw yeast cell pellets on ice and resuspend cell pellets in 100 µL of dH₂O.
   2. To the cell resuspension, add 300 µL of 0.2 M NaOH and 20 µL of 2-mercaptoethanol. Resuspend by pipetting up and down.
   3. Incubate cells on ice for 10 min and then centrifuged at 3,200 x g on a tabletop centrifuge for 30 s at RT. Discard supernatant.
   4. Resuspend the cell pellet in 100 µL of 1x loading dye and boil for 10 min on a heating block.
      NOTE: The recipe for 6x loading dye can be found in the Table of Materials.
2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and membrane transfer
   1. Prepare the gel chamber by placing two gels in a gel holder filling the inner chamber to the top with running buffer and filling the outside chamber to the two-gel line mark.
   2. Load 15 µL of sample per well into a 10-well 12% polyacrylamide gel. For the protein ladder lane, load 5 µL.
   3. Run the gel for approximately 45 minutes at 150 V, or until the loading dye front reaches the bottom of the gel.
   4. While the gel is running, prepare for membrane transfer by soaking fiber pads (two per gel) in transfer buffer (Table of Materials) and soaking polyvinylidene fluoride (PVDF) membrane in methanol. Rinse the membrane in the transfer buffer before transfer.
      NOTE: The PVDF membrane should be cut to the size of the gel and use one PVDF membrane for every gel being transferred.
   5. Assemble, on a semi-dry transfer apparatus cell, a transfer ‘sandwich’: from the bottom electrode, place (1) a presoaked fiber pad, (2) presoaked and rinsed PVDF membrane, (3) gel, and (4) a second presoaked fiber pad.
      NOTE: To avoid air bubbles in the transfer ‘sandwich, ' gently roll the ‘sandwich’ with a serological pipet to force out bubbles. Once the gel has been placed on top of the PDVF membrane, do not move it.
   6. To carry out protein transfer, set the power pack to 150 mA for 1 h (for one ‘sandwich’).
3. Detection of histone post-translational modifications (PTMs) with modification-specific antibodies
   1. Remove the membrane from the transfer apparatus. Rinse briefly with dH₂O.
      1. Optionally, check the transfer of proteins with Ponceau-S stain by pouring enough Ponceau-S stain to cover the membrane in a small plastic box and incubating at RT with gentle shaking for 30−60 s. Remove the Ponceau-S stain and rinse with dH₂O until the background stain disappears and protein bands are visible to the naked eye. Continue to rinse with dH₂O until all stain is removed from the membrane.
   2. Block the membrane by incubating the blot for 1 h at RT with gentle rocking in tris buffered saline (TBS) blocking buffer (Table of Materials) in a small staining box. Use enough blocking buffer to cover the blot.
      NOTE: Be careful to place the membrane upright in the staining box so that the membrane side on which proteins lie is not facing down.
   3. Incubate blot in the staining box overnight with a histone modification-specific antibody reactive towards yeast at 4 °C. Dilute the antibody in TBS blocking buffer according to manufacturer specifications. Also include a proper nuclear loading control, such as anti-total H3 raised in a different host species from the modification-specific antibody. For instance, if probing for H3S10ph with an anti-H3S10ph antibody raised in rabbits, use an anti-total H3 antibody raised in mice as a loading control. Repeat this for every blot as necessary.
      NOTE: Antibody dilutions can be reused for a total of three times within a month. Store them at 4 °C.
   4. Wash the membrane 4x in house-made TBS + 0.1% polysorbate 20 (TBST) for 5 min with rocking at RT.
   5. Incubate blot with fluorescent secondary antibodies at manufacturer-specified dilutions (donkey anti-rabbit 680 and donkey anti-mouse) for 1 h at RT.
      NOTE: Fluorescent secondary antibodies must be protected from light during both storage and usage. Carry out incubation in dark plastic boxes or cover with aluminum foil.
   6. Wash membrane 4x with TBST for 5 min and wash with TBS for 5 min while rocking at RT.
   7. Visualize blot on a fluorescent Western blot imaging system for 2 min. Visualize the anti-rabbit 680 and anti-mouse 800 secondary antibodies in the 800 nm and 700 nm channels, respectively.
      NOTE: Replicates are independently conducted starting from section 1. For appropriate data interpretation in these experiments, it is necessary to verify that the antibody signal response is within the range over which the signal response is linear.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 (Lys16) 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
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
Immobilon-FL Transfer Membranes	MilliporeSigma	IPFL00010
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
Nuclease Free Water	Qiagen	129114
Odyssey Fc Imaging System	LI-COR Biosciences	2800-03
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
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.
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