Protocol for an in vitro assay to study HIV-1 Tat methylation

Summary

A critical virus-encoded regulator of HIV-1 transcription is the Tat protein, which is required to potently activate transcription. Tat is regulated by a wide variety of post-translational modifications. This protocol describes an in vitro assay to study Tat methylation. We describe steps for incorporation of radioactive methyl groups into Tat protein, visualization by gel analysis, Coomassie blue stain, gel drying, and detection by autoradiography. This protocol can also be used to assess methylation in other proteins such as histones.

For complete details on the use and execution of this protocol, please refer to Boehm et al. (2023).¹

Graphical abstract

Highlights

• •Protocol for an in vitro assay to study HIV-1 Tat protein methylation
• •Incorporation of methyl groups detected using radioactive S-adenosyl-methionine (³H-SAM)
• •Gel analysis and detection by autoradiography to detect methylated lysine in Tat
• •Protocol can also be used to assess methylation on other proteins such as histones

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

A critical virus-encoded regulator of HIV-1 transcription is the Tat protein, which is required to potently activate transcription. Tat is regulated by a wide variety of post-translational modifications. This protocol describes an in vitro assay to study Tat methylation. We describe steps for incorporation of radioactive methyl groups into Tat protein and visualization by gel analysis, Coomassie blue stain, gel drying, and detection by autoradiography. This protocol can also be used to assess methylation on other proteins such as histones.

Before you begin

Institutional permissions

The protocol below describes the specific steps to detect methylation of recombinant HIV-1 Tat protein. Incorporation of methyl groups is detected using radioactive S-adenosyl-L-[methyl-³H]-methionine (³H-SAM). Prior to proceeding with this protocol, it is necessary to obtain Radiation Use Authorization (RUA) by the institution for work with radioactive materials.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Adenosyl-L-methionine, S-[methyl-3H] (SAM[3H]), 50 μCi	PerkinElmer	Cat# NET155V250UC
S-adenosylmethionine (SAM)	New England Biolabs	Cat# B9003S; Lot# 1221803, 10153874
Surfact-Amps (Tween 20)	Thermo Fisher Scientific	Cat# 28320
DL-dithiothreitol (DTT)	Thermo Fisher Scientific	Cat# R0861
Acetic acid glacial	Thermo Fisher Scientific	Cat# A38SI-212
Methanol	Thermo Fisher Scientific	Cat# A433P-4
Brilliant Blue G-250	Thermo Fisher Scientific	Cat# BP100-25
Glycerol	Thermo Fisher Scientific	Cat# BP229-4
Tris, pH 9.0, 1 M buffer solution	Thermo Fisher Scientific	Cat# J62085.K2
4x Laemmli sample buffer	Bio-Rad	Cat# 161-0747
NAMP100 Amersham Amplify	GE Healthcare	Cat# LNAMP100V/AC; Lot# 9452993
Amersham Hyperfilm	GE Healthcare	Cat# 28906839
CDK9/cyclin T1, human, recombinant	Thermo Fisher Scientific	Cat# PV4131; Lot# 1860128B
Histone H1, human, recombinant	New England Biolabs	Cat# M2501S; Lot# 0061508
Histone H2A, human, recombinant	New England Biolabs	Cat# M2502S; Lot# 0131508
Histone H2B, human, recombinant	New England Biolabs	Cat# M2505S; Lot# 0031508
Histone H3.1, human, recombinant	New England Biolabs	Cat# M2503S; Lot# 0041504
Histone H3.2, human, recombinant	New England Biolabs	Cat# M2506S; Lot# 0021504
Histone H3.3, human, recombinant	New England Biolabs	Cat# M2507S; Lot# 0021508
Histone H4, human, recombinant	New England Biolabs	Cat# M2504S; Lot# 0061508
HIV-1 Tat aa1-72, recombinant	German Cancer Research Center Pagans et al.2	N/A
NF-κB p65, human, recombinant	Active Motif	Cat# 31102; Lot# 12312015
SMYD5, human, recombinant	Active Motif	Cat# 31409; Lot# 06013001, 16314003
SP1, human, recombinant	Active Motif	Cat# 31136; Lot# 20011003
Other
Whatman paper, 3 MM CHR	Cytiva	Cat# 3030-917; Lot# 17356903
Kodak BioMax TranScreen-LE intensifying screen	PerkinElmer	Cat# 8660789
1.7 mL Microfuge tube (Posi-Click)	Denville	Cat# C2170

Materials and equipment

Buffers

5x SMYD5 Methyltransferase Buffer

Reagent	Final concentration	Amount
Tris pH 9.0 (1 M)	250 mM	250 μL
DL-Dithiothreitol (DTT) (100 mM)	2.5 mM	25 μL
Tween 20 (10%)	0.1%	10 μL
ddH2O	Up to 1 mL	715 μL

Make fresh before the assay preparation. Buffer can be stored at 20°C−25°C on the day of the assay. Buffer without DTT can be stored at ‒20°C for up to 3 months.

Fixing Buffer

Components	Final concentration	Amount
Acetic Acid Glacial (17.4 M)	10%	10 mL
Methanol (100%)	50%	50 mL
ddH2O	40%	40 mL

Buffer closed tightly can be stored at 20°C−25°C up to 3 months.

Coomassie Blue Staining Buffer

Components	Final concentration	Amount
Acetic Acid Glacial (17.4 M)	10%	10 mL
Brilliant Blue Powder	0.012%	12 mg
ddH2O	89.988%	89.988 mL

Buffer can be stored at 20°C−25°C up to 1 year.

Coomassie Destain Buffer

Components	Final concentration	Amount
Acetic Acid Glacial (17.4 M)	10%	10 mL
Methanol (100%)	7.5%	7.5 mL
ddH2O	82.5%	82.5 mL

Buffer closed tightly can be stored at 20°C−25°C up to 3 months.

Anti-Crack Buffer

Components	Final concentration	Amount
Acetic Acid Glacial (17.4 M)	7%	7 mL
Methanol (100%)	7%	7 mL
Glycerol (100%)	3%	3 mL
ddH2O	83%	83 mL

Buffer closed tightly can be stored at 20°C−25°C up to 3 months.

CRITICAL: Methanol and its vapors are flammable. Methanol is also toxic. 100% methanol should be pipetted in a chemical fume hood. Methanol is considered hazardous waste and requires collection of hazardous waste and disposal by the Office of Environment, Health and Safety (EH&S). Glacial acetic acid is flammable and corrosive and can cause severe skin burns. Always handle glacial acetic acid in a chemical fume hood, wear protective glasses, don’t add water to glacial acetic acid, and keep sources of heat, sparks or flame away.

Step-by-step method details

In vitro methylation assay

Timing: 3 h

In this section we provide a step-by-step in vitro assay to study Tat methylation. This protocol can also be used to assess methylation in other proteins such as histones.

• 1.
  In-vitro methylation assay

  • a.
    Each reaction is performed in a 1.7-mL microcentrifuge tube.
  • b.
    The assay can be prepared at 20°C−25°C. To each tube, add 5 μL of freshly prepared 5× SMYD5 methyltransferase buffer.
  • c.
    Add desired Tat peptides, proteins or histone substrates.

    Note: Substrates can be any protein or peptide that contains lysines which are subject to methylation. To enable detection by SDS-Page and Coomassie staining use: 2 μg of each Tat peptide, protein or recombinant histone, 2–5 μg of histone peptide, 5–10 μg of core histones, or 5–10 μg of nucleosomes as substrates for the assay.

  • d.
    Add 1–2 μg of recombinant SMYD5 methyltransferase to each tube except the no-methyltransferase control reactions.

    CRITICAL: Enzymes are very sensitive to freeze – thaw cycles. Always thaw the methyltransferase on ice and avoid freezing and thawing several times to avoid a reduction in enzymatic activity.

    Note: A negative control reaction without enzyme and positive control reaction using a known methyltransferase is recommended.

  • e.
    Add ddH₂O to a final volume of 20 μL to each tube.

    CRITICAL: Until this point, the entire reaction can be pipetted together without shielding. However, the next component, ³H-SAM, is radioactive and needs to be added in an area designated for work with radioactive materials. The stock vial of ³H-SAM is stored at ‒20°C. About 30 min before the experiment take the vial out of the freezer and let it thaw at 20°C−25°C behind shielding.

  • f.
    Add 2 μL of ³H-SAM stock to each tube.

    CRITICAL: From this point, the rest of the experiment needs to be performed in the area designated for work with radioactive materials, such as a RAD-room. All waste needs to be collected and disposed of properly by EH&S. Geiger counters cannot detect ³H. Instead, regular swipe tests using a scintillation counter should be performed to check for ³H contamination.

  • g.
    Centrifuge for 15 s at 200 × g to collect the reaction at the bottom of the microcentrifuge tube.
  • h.
    Incubate the reactions for 2–18 h at 22°C–37°C.

    Note: Incubations for longer periods and above room temperature (20°C−25°C) should be performed while shaking to reduce condensation at the lid of the tube. The optimal temperature and incubation time is always enzyme dependent. For SMYD5 incubation for 2 hours at 22°C or 32°C was used.

  • i.
    Stop the reaction with 4x Laemmli sample buffer and fractionate it in a 15% SDS-PAGE gel for larger proteins or a 4%–20% Tris-Tricine gel for peptides and histones.

    Note: Store these samples at −20°C for up to one week or ‒80°C for up to 6 months until you are ready for gel analysis.

Gel analysis, Coomassie blue stain, gel drying, and autoradiography

Timing: 2 h for gel analysis, 1 day for Coomassie blue stain, 2 h to dry the gel, and 1–7 days to expose the film

In this section we provide a step-by-step description for gel analysis, Coomassie Blue stain, gel drying and autoradiography.

• 2.
  Gel analysis

  • a.
    For gel analysis, run the samples in a 10% or 15% SDS-PAGE gel for larger proteins or a 4%–20% Tris-Tricine gel for peptides and histones at 120 V for about 1–1.5 h.

Note: As peptides and histones can be very small, make sure to not let the dye front run off the gel. The running buffer will be contaminated with ³H-SAM and should be discarded as radioactive waste. The gel box will also be contaminated with ³H-SAM.

• 3.
  Coomassie Blue stain

  • a.
    Fix the gel for 5 min in Fixing Buffer.
  • b.
    Cover the gel with Coomassie Blue Staining Buffer and incubate for 1–18 h.
  • c.
    Remove the Coomassie Blue Staining Buffer and incubate the gel with Coomassie Destain Buffer until the substrate bands of interest are visible.

    Note: The gel will destain faster if some clean paper tissue is placed inside the box. Make sure to place the tissue at the edge of the box so the gel does not tear when the box is shaking.

  • d.
    Remove the gel from the Coomassie Destain Buffer and carefully place it into a Hybri-Bag or any other hard plastic bag.
  • e.
    Take scans of the bands.

    Note: Scans of the bands can also be taken later of the dry gels. However, incubation with Amersham Amplify solution and Anti-Crack Buffer will destain the gels further and the drying process often cracks the gels. Therefore, it is recommended to take scans of the Coomassie stain at this point. Be careful not to contaminate the scanner with radioactive ³H.

  • f.
    Take the gel out of the plastic bag and place it into a clean plastic tray.
    To increase the radioactive signal, incubate the gel in Amersham Amplify solution for 30 min while shaking. Add 10 mL or more to completely cover the gel.

    Note: The Amersham Amplify solution will destain the gel further.

  • g.
    Incubate the gel in Anti-Crack Buffer for 5 min.
• 4.
  Gel drying

  • a.
    Place the gel onto 2 layers of Whatman filter paper and cover it with saran wrap.
  • b.
    Dry the gel on a vacuum gel dryer for 2 h at 60°C.
  • c.
    Remove the plastic wrap and tape the dried gel inside a Kodak BioMax TranScreen-HE intensifying screen. In the dark, insert one or several Amersham Hyperfilms in an autoradiography cassette and place it at −80°C.
  • d.
    Expose the film for 12 h or longer.

Note: The intensity of the radioactive signal will vary depending on the methyltransferase, the substrate and the ³H-SAM. It can take several weeks of incubation at ‒80°C before a signal can be detected.

Expected outcomes

To identify the lysine residues methylated by a protein, a methyltransferase and target protein are incubated in the presence of ³H-SAM. After gel electrophoresis, proteins and peptides can be visualized by Coomassie staining (Figure 1). Methylation as measured by incorporation of methyl groups from ³H-SAM can be detected by autoradiography (Figure 2). Besides Histones, any protein or peptide that contains lysines can be subject to methylation. Figure 3 shows in vitro assays using the HIV-1 Tat protein. Tat was prominently methylated by SMYD5 while no methylation was detected on NF-κB RelA, Sp1, Cyclin T1, and CDK9 (Figure 3).

Figure 1.

Coomassie Staining of in vitro methylation assays

In vitro methylation of recombinant histones in the absence (left panel) and presence (right panel) of the methyltransferase SMYD5. For complete details, please refer to Figure 4 of Boehm et al.¹

Figure 2.

Autoradiographs of in vitro methylation assays

In vitro methylation of recombinant histones in the absence (left panel) and presence (right panel) of the methyltransferase SMYD5. SMYD5 methylated histones H1, H2B, H3 and weakly H4, but not histone H2A in vitro. For complete details, please refer to Figure 4 of Boehm et al.¹

Figure 3.

SMYD5 methylates Tat in vitro

In vitro methylation of synthetic Tat peptide (aa 1-72), recombinant NF-κB RelA, Sp1, Cyclin T1 or CDK9 incubated with recombinant full-length SMYD5 enzyme and radiolabeled ³H-S-adenosyl-L-methionine (SAM). Coomassie stain of control reactions without SMYD5 enzyme (left panel) and with SMYD5 enzyme (middle panel) and autoradiograph (right) is shown. For complete details, please refer to Figure 4 of Boehm et al.¹

Quantification and statistical analysis

All in vitro methylation assays on isolated histones, Tat peptides, NF-κB, SP1 and pTEFb were repeated at least three times, and representative Coomassie stain and corresponding autoradiographs are shown. To quantify or calculate the methylation status mass spectrometry should be performed.

Limitations

This protocol can be used to detect lysine methyltransferase activity. However, to identify the precise amino acid further investigation is required. To identify the target amino acid in Tat, MALDI coupled to time-of-flight mass spectrometry (MALDI-TOF MS) can be performed. To sequence Tat, MALDI-TOF works best as compared to other mass spectrometry methods. Alternatively, recombinant proteins or peptides containing mutated lysines can be tested in the assay.

Troubleshooting

Problem 1

No signal detected (Step 1c).

Potential solution

Tat and histones are very small proteins. Make sure the proteins are not running out of the gel. The signal can also be increased by using larger amounts of substrate. Sometimes, histones are already methylated, for example when isolated from E.coli. These substrates cannot be methylated further. Try using substrates from a different source.

Problem 2

Methyltransferase activity is weak or not detected (Step 1d).

Potential solution

Enzymes are very sensitive to freeze – thaw cycles. Always thaw and keep the methyltransferase on ice and avoid freezing and thawing several times to avoid a reduction in enzymatic activity. Also, try using an enzyme that was purified from a different source (Sf9 insect cells vs. E.coli).

Problem 3

Methylation is weak or not detected (Step 1f).

Potential solution

³H-SAM is not very stable and should only be used for about 6 months. Avoid repeat freeze-thaw cycles and keep the radioactive ³H-SAM stock undiluted in its highly acidic buffer. Diluted ³H-SAM will degrade faster.

Problem 4

Too much signal detected without single distinguishable bands (Step 4L).

Potential solution

The incubation time of the dried gels on film might be too long. Try exposing the gel on film without Kodak BioMax TranScreen-HE intensifying screen. Alternatively, place several films in the autoradiography cassette. The signal will reduce in strength from the film placed immediately on top of the gel to the outermost film on top of the films. Also, the film incubation time can be shortened to a few minutes or few hours at room temperature (20°C−25°C).

Problem 5

Too much signal detected with multiple distinguishable bands (Steps 1c and 1d).

Potential solution

The methyltransferase might not be pure enough and potentially contains other methyltransferases which were not removed during the enzyme purification process. Try using a methyltransferase from a different source or a catalytically inactive methyltransferase mutant.

The Tat or histone substrate might be degraded which results in multiple methylated bands. Use a fresh aliquot of substrate.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Melanie Ott (mott@gladstone.ucsf.edu).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

Data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.