Protocol for in vitro lysine deacetylation to test putative substrates of class III deacetylases

Summary Lysine acetylation is an important post-translational modification that is used in multiple cellular pathways, such as the regulation of gene expression at the histone level. The purpose of this assay is to test for putative substrates of class III deacetylases using an in vitro method. The in vitro analysis helps circumvent confounding variables when assessing for a direct relationship between deacetylase and substrate, such as the effects of other cellular deacetylases or acetyltransferases that modify the substrate in vivo. For complete details on the use and execution of this protocol, please refer to Minten et al. (2021).

Alternatives: Other cell lines can be substituted for HEK 293T, such as HeLA (ATCC CCL-2). See troubleshooting: Problem 3 if purification of the deacetylase is low. Different acetyltransferases than P300, CPB, and pCAF may be needed for optimal acetylation signal. See troubleshooting: Problem 1. The secondary antibodies needed for dual labeling may also differ depending on the species of any primary antibodies used.

MATERIALS AND EQUIPMENT
CRITICAL: Many of the chemicals listed in this protocol may be hazardous or toxic. For all chemicals, safety guidelines should be consulted before use or disposal. Note: Inhibitors should be added fresh before each lysis due to their short half-lives.
Note: Store at room temperature (23 C) for up to one year.
Note: Store at 4 C. The buffer can be kept for up to one year. Note: This master mix should be made fresh each time.
Note: Store aliquots at À20 C for up to a year. For longer-term storage, store at À80 C for up to two years.
CRITICAL: SDS causes respiratory irritation. Weigh in a fume hood and use personal protective equipment, including a face mask.
Alternatives: The same concentration of b-mercaptoethanol can be used in place of DTT.

STEP-BY-STEP METHOD DETAILS
Enzyme and substrate preparation: Day 1

Timing: 30 min
Transfection of cells should be done in the hood.
1. Transfecting of the putative substrate and the deacetylase 10 mg of DNA of the substrate with 250 mL of Opti-MEM. To increase baseline substrate acetylation levels, it's recommended to co-transfect 2-3 mg of DNA of the acetyltransferases known to target the substrate.
Note: We recommend, as a starting point if the specific acetyltransferases are unknown, using a combination of p300, pCAF, and CBP (see troubleshooting: Problem 1 for further details). Separately, mix gently by tube inversion 18 mL of Lipofectamine 2000 in 250 mL of Opti-MEM. Let both mixtures stand for 5 min, then combine in one tube. Let sit at room temperature (23 C) for 20 min, then add to 5-6 million HEK 293T cells in one 60 mm dish. Add dropwise, then gently mix the cells. Note: The cells should be about 90%-95% confluent for both the cells transfected with the deacetylase and the cells transfected with the substrate. The number of dead cells should be minimal, but will depend on the substrate being transfected.
5. Trpysinize the cells and split each dish equally into two 10 cm dishes for a total of four 10 cm dishes.
Enzyme and substrate preparation: Day 3 Timing: 15 min 6. Add to the cells with the substrate transfection ONLY: Tricostatin A (TSA) to a final concentration of 0.5 mM, and nicotinamide to a final concentration of 1 mM to inhibit class I and II, and class III acetyltransferases, respectively. Let cells incubate overnight for 12-16 h to maximize acetylation.

Deacetylase and substrate preparation: Day 4
Timing: 2 h 7. Trypsinize the cells and collect cells in an Eppendorf tube, combining duplicate dishes into one tube. Add an equal amount of media to the amount of trypsin used to neutralize the trypsin. 8. Centrifuge the cells at 3,500 3 g -5,000 3 g for 5 min and discard the supernatant, being careful not to discard the cell pellet. 9. For each tube, wash cells once with 1 mL of PBS and spin for another 5 min at 3,500 3 g -5,000 3 g. Remove the supernatant after the wash, leaving behind only the cell pellet. 10. Lyse each tube of cells with about 400 mL-500 mL cell lysis buffer (described under materials and equipment). Gently rotate the tubes or place on a rocker at 4 C for 30-40 min. 11. Spin the cells down at maximum speed ($17,000 3 g) for 15 min at 4 C. 12. While the cells are spinning, prepare the beads that will be used for pre-clearing the lysate. 30 mL of a slurry of 50% beads will be used for every 500 mL of lysate. Spin the beads down, then wash the beads 2 times with 500 mL lysis buffer, then resuspend the beads in lysis buffer using a volume equal to 1 / 2 the initial volume of the bead slurry, to bring back up to the original volume.
Note: To spin and wash the beads, spin the beads for 30 s at 100 3 g, then turn the tubes 180 degrees in the centrifuge and spin again for another 30 s at 100 3 g. Remove supernatant, leaving only the beads behind. Add 500 mL lysis buffer and repeat the spin using the same conditions as just described. Remove buffer leaving only the beads behind.
13. Keep the beads on ice and centrifuge at 4 C. For FLAG-tagged substrates, CL-4B Sepharose beads can be used for pre-clearing.
Note: Protein G agarose beads can also be used if the antibody that will be used for the IP will be mouse, while protein A agarose beads can be used for preclearing if the IP antibody will be rabbit.
14. After the spin is complete from step 13, carefully transfer the supernatant to a clean tube; discard the cell debris. To preclear the lysate, add 30 mL of the cleaned pre-clearing beads per every 500 mL of lysate. Rotate the lysate and beads at 4 C for 15 min. 15. While the lysate is pre-clearing, prepare the beads for immunoprecipitation for the tags that will be used. 30 mL of a 50% slurry of conjugated beads will be used for every 500 mL of lysate. Prepare the beads as described in step 12.
Note: Protein G agarose beads should be used if the antibody that will be used for the IP will be mouse, while protein A agarose beads should be used if the IP antibody will be rabbit.
16. After the pre-clear has completed, spin the lysate and beads down using the same conditions used in step 12 for washing the beads. Move the supernatant to their respective beads for immunoprecipitation using an anti-FLAG antibody (or antibody against the tag). 17. Allow tubes to rotate at 4 C overnight (12-16 h). The amount of antibody used for the immunoprecipitation will depend on manufacturer's instructions, but a 1 mg antibody:1 mg of lysate is generally suggested as a starting point for optimization.

Deacetylase preparation: Day 5
Timing: 45 min 18. To collect the deacetylase, wash beads 4 times with lysis buffer using the same conditions as described in step 12. 19. Wash the beads 3 more times with TBS using the same conditions as described in step 12 with.
At the end, remove the supernatant, only leaving the beads. Add 100 mL of TBS for every two dishes of cells to the beads (this volume can be adjusted but should be 2 times the volume of the packed beads at minimum). 20. For elution using a FLAG-tagged deacetylase, add 3 mL of 33 FLAG Peptide stock (5 mg/mL) for every 100 mL TBS. 21. To elute, either manually agitate the beads by gentle tapping every 5 min for 30 min on ice or rotate at 4 C for 30 min, if the volume is sufficient (we recommend about no more than 2/3 of the volume of the tube be empty; e.g., for a 1.5 mL Eppendorf tube, the minimum volume would be about 500 mL). 22. After 30 min, spin down the beads as described in step 12. Transfer the supernatant, which contains the eluted deacetylase, into small aliquots for storage at À80 C, for up to 1 year. 23. To quantify the concentration of the deacetylase, run a sample of the elutant on an SDS-PAGE gel against a BSA standard curve and stain with Coomassie blue.
Note: Measurement using a Bradford protein assay can also be used, but may be less precise depending on the amount of non-specific proteins pulled down during purification. Roughly, the concentration of the deacetylase should be around 1 mg of protein/15 mL. About 1 mg of deacetylase will be needed for each reaction where the deacetylase is used.
Deacetylase assay: Day 5 Timing: 4 h 24. Wash the beads bound to the substrate 4 times with lysis buffer. 25. After the wash with lysis buffer, wash the beads 2 times with 13 deacetylation buffer. Resuspend the beads in 1 mL of deacetylation buffer and equally split the beads into N tubes, where N is the number of treatments/conditions. We recommend four reactions below to demonstrate deacetylation of the substrate of interest. 26. Spin the beads down as described in step 12 and carefully aspirate away all liquid using a vacuum or pipette, ensuring even distribution of the beads in each tube. 27. The reactions for the conditions should be set up as follows: i. This reaction should not show any change in acetylation levels compared to reaction 1 due to lack of necessary cofactor NAD + for class III deacetylases. NAD + can be added if contamination with other class III deacetylases is expected. c. Reaction 3: Deacetylase + NAD + i. A stock of 50 mM of NAD + is used for class III deacetylases for the cofactor. If the deacetylase directly targets the substrate, a decrease in acetylation should be seen. d. Reaction 4: Deacetylase + cofactor + deacetylase inhibitor.
i. A stock of 50 mM of NAD + is used for class III deacetylases for the cofactor. ii. A stock of 2 M nicotinamide is used to inhibit class III deacetylases. No change in acetylation levels of the substrate should be seen compared to Reaction 1 and 2 due to deacetylase inhibition. The volume of the deacetylase may vary depending on the concentration after purification (see step 23). 1 mg of deacetylase is suggested for use in Reactions 2-4. 28. Incubate the reactions in a 30 C water bath with gentle tapping to agitate the beads every 15 min for 3 h. Alternatively, a 30 C shaker can be used. 29. After 3 h of incubation, add $8 mL of 43 sodium dodecyl sulfate (SDS) loading buffer to stop the reaction. Boil the samples for 5 min at 100 C and run on western blot for analysis, or store at À20 C for later analysis.
Note: Transfer conditions may depend on the substrate, but 1 h at 100 V or overnight (12-16 h) at 45 V should give sufficient transfer. Blocking can be done with 5% BSA or 5% milk but may need to be optimized.
30. For the best acetyl-lysine signal, it is suggested to incubate the membrane overnight (12-16 h) on a rocker at 4 C. The deacetylase should also be probed for to ensure there was even addition to the correct samples.
CRITICAL: Dual channel labeling is most ideal to show the overlap of the acetylated protein and total protein. We suggest using differently colored fluorescent probes, which can be imaged using a LI-COR Odyssey system (LI-COR), which allows for dual channel labeling detection. We also suggest probing for the deacetylase to ensure even levels of the enzyme were added to Reactions 2-4. During the primary antibody step, for dual-labeling, the antibody to the substrate should be a different species to the acetyl-lysine antibody (see reagents) without species cross-reactivity.

EXPECTED OUTCOMES
A successful experiment should show on the final western blot clear and even inputs of the substrate being tested for deacetylation. In addition to reactions 1-4, a negative control lane can be added to ensure specific staining of the substrate and acetylation signal. For tagged substrates, the negative control can be obtained using lysate from cells that were not transfected with the tagged substrate. For endogenous substrates, the negative control can be obtained using a control antibody during the immunoprecipitation step. Reaction 1, where no deacetylase is added, should show an acetylation signal that overlaps the signal that corresponds to the protein. Reaction 2, where the deacetylase but not NAD + is added, should show comparable acetylation to reaction 1 due to the lack of necessary cofactor (if acetylation is not comparable, see troubleshooting: Problem 4). However, when NAD+ is added to the reaction, the deacetylase should be activated. If the deacetylase targets the potential substrate, a decrease in acetylation level should be seen in the putative substrate compared to Reaction 1, 2, and 4. If no decrease in acetylation is seen, this may suggest the substrate is not a direct substrate of the deacetylase (see troubleshooting: Problem 5). Reaction 4 should show comparable acetylation to reactions 1 and 2 due to inhibition of the deacetylase. The final western blot should also show even inputs of the deacetylase in the reactions it was added.
An example of a representative blot showing the outcome of the four reactions with successful deacetylation can be seen in Figure 1. Also see (Head et al., 2017;Minten et al., 2021;Zhang et al., 2013Zhang et al., , 2016.

LIMITATIONS
This protocol relies on immunoprecipitation from cells. Immunoprecipitation from cells has its own pitfalls and limitations that are outlined in depth in other protocols (Takahashi, 2015). Briefly, proteins that are transiently overexpressed can lead to artifacts, such as misfolding or aggregation. Furthermore, immunoprecipitation from cells in conditions that attempt to preserve enzymatic function can lead to the pulldown and interaction of the deacetylase with other proteins or cofactors that may affect function. In this protocol, the deacetylase has a FLAG tag, which can potentially affect function under certain circumstances.
This protocol also measures acetylation levels using western blotting. Though in the protocol, we suggest over-expressing acetyltransferases and treating the cells with deacetylase inhibitors to maximize acetylation signal, some proteins will nevertheless have low levels of acetylation. This can make signal detection difficult using a pan acetyl-lysine antibody, especially if the acetyltransferases listed in this protocol are not optimal for the tested substrate.
In in vitro experiments, the forced interaction between enzyme and substrate may also produce non-specific results. Deacetylation may not occur in normal cellular environments, but in vitro conditions may show non-specific deacetylation. Furthermore, in vitro experiments use purified proteins and force interactions between enzyme and substrate, which may not reflect what endogenously happens in cells due to due to compartmentalization. The results should be verified in cells under more physiological-like settings.
Finally, this protocol does not necessarily identify the site of acetylation and deacetylation on a protein and is a semi-quantitative measure of acetylation levels.

TROUBLESHOOTING
Problem 1 Low acetylation signal/low substrate expression.

Potential solution
Increase the dilution of the primary antibody (see steps 24-25). We suggest a 1:500 dilution for the acetyl-lysine antibody, but higher concentrations can be used. The membrane should also be ll OPEN ACCESS incubated in antibody overnight (12-16 h) at 4 C vs. for a few hours. Either a PVDF or nitrocellulose membrane can be used, but optimization may be required depending on the substrate being tested.
The acetyltransferases listed in this protocol (P300, CBP, and pCAF) are a broad suggestion for a starting point if an increase in acetylation signal is needed, but should be optimized to each substrate used (see step 1). A low acetylation signal may be due to a non-optimized choice of acetyltransferase.
Large proteins (> 200 kDa) can be difficult to transfect or tend to overexpress at lower levels than smaller proteins (see step 24). Different transfection methods may be required, or even stable expression by lentiviral transduction with selection. Larger proteins can also take a longer transfer time after SDS-PAGE has been performed, so an overnight transfer of about 16 h at 45 V is suggested as a starting point.
The immunoprecipitation of the substrate may need optimizing. Ensuring the beads used for pulldown are able to immunoprecipitate the substrate before doing the deacetylase reaction will aid in this protocol (see step 13).

Problem 2
Reaction 2 shows lower acetylation levels than Reaction 1.

Potential solution
This may suggest there is contamination in the reaction that is allowing the enzyme to function without purposeful addition of the necessary cofactor. Ensure that NAD + has not been added to this reaction and that the substrate input is even between Reaction 1 and Reaction 2 (see step 22).

Problem 3
Reaction 4 shows decreased acetylation compared to Reaction 1. Lane 1 shows a negative control with no transfection of BRCA1 or addition of the deacetylase. Lane 2 shows the addition of BRCA1 and NAD + , and represents Reaction 1. Lane 3 shows the addition of NAD + and SIRT2 to BRCA1, and represents Reaction 3. Lane 4 shows the addition NAD + and a deacetylase-dead SIRT2 mutant to BRCA1, and represents Reaction 4. Lane 5 shows the addition of SIRT2 to BRCA1 without NAD + , and represents Reaction 2. Dual channel labeling of the acetylation signal and BRCA1 are shown.

Potential solution
If Reaction 4 shows decreased acetylation, this may suggest nicotinamide is not effectively inhibiting the deacetylase (see step 27). Make sure the stock is not degraded and that the proper concentration has been added to Reaction 4.

Problem 4
Low yield of deacetylase after purification or contamination.

Potential solution
Check that the deacetylase is transfecting properly and try increased transfection time/increased DNA used to transfect cells. As mentioned previously, larger proteins tend to overexpress at lower levels (see step 23).
Make sure the elutant is functioning properly. After eluting, boil the beads and run on a western blot next to a sample taken from the elutant to ensure the enzyme is dissociating from the beads or is being properly immunoprecipitated (see step 23).
Purification of recombinant deacetylase and/or substrate can also be performed from E. coli and/or Sf9 insect cells if contaminants are co-purifying when using mammalian cells.

Problem 5
In Reaction 3, the substrate shows no change in acetylation levels.

Potential solution
Probe for the deacetylase on the final western blot to ensure that the deacetylase was correctly added to Reactions 2-4 (see step 27, and steps 29-30). Even amounts of the deacetylase should be seen in the lanes corresponding to Reactions 2-4, and about 1 mg should be added to Reactions 2-4 to ensure a sufficient amount of enzyme.
If not stored correctly, the deacetylase may denature and become non-functional (see step 22). Ensure that the eluted deacetylase has been stored properly, or reprep the deacetylase to get a new batch for use in the assay.
No acetylation may be the result of failure to properly agitate the beads during the 3 h incubation of the deacetylase with the substrate (see step 28). Be careful to ensure that the beads are not sticking to the walls of the tube above the solution that contains the deacetylase, or failing to properly mix. Improper mixing of the beads can lead to failure of the deacetylase to reach the substrate.
The substrate being tested may not be a direct substrate of the deacetylase.

RESOURCE AVAILABILITY
Lead contact Requests for further information or resources should be directed to the lead contact, D.S. Yu (dsyu@ emory.edu).

Materials availability
No new or unique regents were generated. Questions relating to any of the regents should be directed to the lead contact, D.S. Yu (dsyu@emory.edu).

Data and code availability
No data sets or code were generated.