Protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination

Summary The histone lysine methyltransferase SUV420H1 preferentially targets the H2A.Z-containing nucleosome core particle (H2A.Z-NCP) and catalyzes the H4K20me2 modification at replication origins. Here, we present a protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination. We describe steps for the installation of S-ethyl-cysteine (Ecx), nucleosome and complex preparation, and performing the cryoelectron microscopy (cryo-EM) sample check. This protocol substitutes lysine 20 in histone H4 with S-ethyl-cysteine (H4K20Ecx), which enhances the stability of the interaction between SUV420H1 and nucleosomes. For complete details on the use and execution of this protocol, please refer to Huang et al.1

The large-scale production of 147-bp Widom 601 DNA, used for nucleosome reconstitution, was purified following previously described methods. 4Transform pUC19-147x12 plasmid into Top10 competent cells as per the manufacturer's instructions.a. Thaw 50 mL of Top10 competent cells on ice.b.Add 1 mL pUC19-147x12 plasmid (about 100 ng) to competent cells, moving the pipette through the cells while dispensing.Gently tap tubes to mix.
Note: Limit the volume of DNA to no more than one-tenth of the volume of competent cells.
c. Incubate cells on ice for 30 min.d.Perform heat shock at 42 C for 90 s in a water bath.e. Quickly transfer the centrifuge tube to an ice bath to cool for 3 min.f.Add 500 mL of LB media and incubate at 37 C for 1 h in the incubator at 220 rpm.g.Plate 200 mL of the transformed cells evenly onto an LB agar plate with ampicillin (100 mg/mL) and incubate at 37 C for 12-18 h. 2. Pick 3-4 colonies into separate 3 mL LB medium (100 mg/mL ampicillin) and incubate at 37 C with shaking at 220 rpm for 12-18 h. 3. Extract plasmids from 2 mL cell cultures using TIANprep rapid Mini plasmid Kit.4. Digest 200 ng of the plasmids with EcoRV enzymes according to the following system at 37 C for 3 h.

5.
Run 1% agarose gel to analyze the digestion samples.The correct plasmid should produce 147-bp DNA and pUC19 vector bands (refer to Figure 1A).
Note: If multiple bands are produced, it indicates incomplete digestion.
6. Select clones with verified plasmids and scale up by adding 200 mL of cell culture from step 2 into 200 mL LB medium (100 mg/mL ampicillin).Incubate at 37 C with shaking at 220 rpm for 12-18 h. 7. Add 15 mL cell cultures from step 6 into flasks with 800 mL of LB medium (100 mg/mL ampicillin), total 12 flasks.Incubate at 37 C at 220 rpm for 20 h. 8. Centrifuge the cells at 5000 g and 4 C for 30 min to harvest, then proceed with large-scale plasmid extraction as described in the published protocol. 4The plasmid from 12 L of cell culture is typical 100-150 mg.
Pause point: The purified pUC19-147x12 plasmid can be stored at À80 C for up to 2 years.9. Digest the plasmids in large quantities using EcoRV enzymes as per the following protocol.Taking the digestion of 20 mg of DNA as an example.b.Incubate of the mixture at 37 C for 30 min and then put the tubes on ice for 30 min.Centrifuge at 31000 g at 4 C for 30 min to pellet the plasmid backbone DNA.PEG6000 final concentration (%) (mL) c.Resuspend the precipitate in 50 mL TE 10/0.1 (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer and analyze the pellets and supernatants by running a 1% agarose gel.d.The plasmid backbone was precipitated at 6.5% PEG 6000 conditions as shown in Figure 1B.12. Mix the digestion samples with 6.5% PEG 6000 and 0.5 NaCl as before.Incubate of the mixture at 37 C for 30 min, and then put the tubes on ice for 30 min.Centrifuge at 31000 g at 4 C for 30 min to pellet the plasmid backbone DNA and collect the supernatant.13.Add 1/5 volume of PCIA (phenol: chloroform: isoamyl alcohol, 25:24:1) to the DNA, and shake gently to mix, then centrifuge at 4 C, 31000 g for 15 min.Carefully remove the upper aqueous phase to new tubes and discard the discard the lower organic phase with enzyme and PEG 6000 (Figure 1C).14.Precipitate the DNA using ethanol: Add 2.5 times the DNA volume of ice-cold anhydrous ethanol and 1/10 volume of ice-cold 3 M NaAc (pH 5.2).Place the mixture at À20 C for at least 2 h, during which some white precipitate forms (Figure 1D).Then centrifuge at 4 C, 31000 g for 30 min and discard the supernatant.15.Dissolve the DNA pellets in an appropriate volume of TE 10/0.1 (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer.Ensure the final 147-bp DNA concentration is at least 2 mg/mL.Typically, yields are 40% of the total plasmid.16.Analyze the 147-bp DNA by running a 1% agarose gel (Figure 1E) and store the 147-bp DNA at À80 C for further use.
Pause point: The purified 147-bp DNA can be stored at À80 C for up to 2 years.
Purification of histone H2A.Z/H2B Timing: 5-6 days 17.Histone H2A.Z and H2B were subcloned into the expression plasmids pQlink featuring a 63His tag at the N-terminal of H2A.Z. 5 a.The H2A.Z coding sequence was cloned into pQLinkH and the H2B coding sequence into pQLinkN using BamHI and HindIII restriction sites.b.Digest 200-500 ng of pQLinkH-H2A.Z plasmid with SwaI in a 10 mL reaction at 25 C. Similarly, digest pQLinkN-H2B with PacI in a 10 mL reaction at 37 C.After digestion, inactivate the enzymes by incubating at 65 C for 20 min, then place the tubes on ice to cool.c.Treat 10 mL of the DNA with 1 mL T4 DNA polymerase in a 20 mL reaction mixture (containing 13buffer 2, 5 mg/mL BSA, and 2.5 mM dCTP for the PacI digest or 2.5 mM dGTP for the SwaI digest).Incubate for 60 min at 25 C, then heat inactivate at 65 C for 20 min.d.Mix the two plasmids, heat to 65 C, and then cool to 25 C for 30 min to allow for annealing.e. Add 2 mL of 25 mM EDTA to stop the reaction and transform the mixture into Top10 competent cells.f.Screen the transformants using primers pQTEV3U (5 0 -TATAAAAATAGGCGTATCACGAGG-3 0 ) and pQTEV3L (5 0 -CCAGTGATTTTTTTCTCCATTTT-3 0 ), which are positioned upstream of the LINK1 and downstream of the LINK2 sequences, respectively.The expected inserts are both H2A.Z and H2B.Select clones for sequencing.18. Introduce 1 mL of pQlinkH-H2A.Z-N-H2B plasmid into 50 mL of BL21(DE3) RIPL cells, as the guidelines in step 1. 19.Pick a single colony into 100 mL LB medium with ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL).Incubate at 37 C with shaking at 220 rpm for 12-18 h.20.Grow 4.8 L LB in six bottles.Distribute 800 mL LB containing ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL) into 6 flasks and then dilute 15 mL cell culture into each of them.21.Incubate the cell culture at 37 C with shaking at 220 rpm until it reaches an OD 600 of 0.5, then cool the culture to 16 C. 22. Add 400 mL of 1 M IPTG to achieve a final concentration of 0.5 mM and induce H2A.Z-H2B expression for 18 h.
Alternatives: If a high-pressure homogenizer is not available, cell lysis can be effectively achieved through sonication.
25. Centrifuge the lysed cells at 31000 g for 1 h at 4 C. Transfer the clear supernatant with H2A.Z-H2B to a new beaker.26.Equilibrate 3 mL of packed Ni-NTA beads with 60 mL of histone lysis buffer and mix with the clarified lysate.If using new beads, wash them with distilled water to remove the ethanol.27.Incubate the beads and the lysate at 4 C for 1 h, then process through a gravity flow column and wash with 150 mL of histone lysis buffer.28.Elute H2A.Z-H2B protein with about 30 mL histone elution buffer (50 mM Tris-HCl pH 8.0, 2 M NaCl, 250 mM imidazole, 5 mM b-mercaptoethanol).Run 15% SDS-PAGE to analyze the sample (Figure 2, lanes 1).29. Add TEV Protease with 6 3 his tag to the protein sample (0.06 mg TEV/ mg protein).Dialyze the protein sample against 2 L of histone lysis buffer using 3 kDa cut-off dialysis bag at 4 C for 18 h.30.Equilibrate 1 mL of packed Ni-NTA beads with 60 mL of histone lysis buffer and reload the dialyzed sample onto equilibrated Ni-NTA beads to remove protein not cleaved and TEV Protease, wash with histone lysis buffer, and collect the flow-through samples (Figure 2  Note: The molar ratio of histone H2A.Z to H2B in the H2A.Z-H2B dimer deviates from 1:1.Make sure the culture is cooled to 16 C and the OD 600 of the cell culture is not higher than 0.6 at inducing.
Pause point: the purified H2A.Z-H2B protein can be stored at À80 C for up to 1 year.
Alternatives: H2A.Z protein and H2B protein can also be prepared as inclusion bodies following the detailed protocol for the purification of H4K20C/H3 proteins, and subsequently form H2A.Z-H2B dimers as described in the protocol for reconstituting (H3-H4K20C) 2 tetramers.

Purification of H4K20C / H3 proteins
Timing: 5-6 days The preparation of histone H4K20C / H3 proteins and nucleosomes were previously described. 4,6.Introduce 1 mL of pET3a-H4K20C plasmid into 50 mL of BL21(DE3) RIPL cells as the manufacturer's instructions in step 1. 33.Inoculate a single colony into 100 mL LB medium containing ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL) and grow at 37 C with shaking at 220 rpm for 12-16 h.34.Grow 4.8 L 2 3 YT medium in six bottles.Distribute 800 mL 23YT medium containing ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL) into 6 flasks and then dilute 15 mL cell culture into each of them.35.Incubate the cell culture at 37 C until it reaches an OD 600 of 0.6, then add 400 mL of 1 M IPTG to induce H4K20C expression at a final concentration of 0.5 mM at 37 C for 2 h.36.Centrifuge the cells at 5000 g for 30 min at 4 C, discard the supernatant, and store the pellet at À80 C for later use.

Pause point:
The cell pellets can be stored at À80 C for up to 2 months.
37. Resuspend the cell pellet collected from 4.8 L LB with 180 mL histone wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol).Lyse the cells using a highpressure homogenizer for approximately 8 cycles.38.Centrifuge the lysed cells at 31000 g for 1 h at 4 C and discard the supernatant.39.Resuspend the inclusion body in 150 mL histone wash buffer containing 1% Triton X-100, disaggregate using a high-speed disperser (Methods video S2), then centrifuge and discard the supernatant.
Alternatives: If a high-speed disperser is not available, inclusion body can be disaggregated effectively through sonication.
40.Wash the inclusion body twice with histone wash buffer containing 1% Triton X-100 and once with the buffer alone.Collect the inclusion body.41.Dissolve the inclusion bodies in 50 mL of histone unfolding buffer (20 mM Tris-HCl pH 8.0, 7 M guanidine hydrochloride, 5 mM b-mercaptoethanol), disaggregate the pellet with a high-speed disperser, and allow to dissolve at 25 C for 1 h.42.Centrifuge the protein sample at 210,100 g for 30 min at 25 C and collect the supernatant.43.Dialyze the sample against 2 L Urea buffer 0 (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 8 M urea, 5 mM b-mercaptoethanol) for at least 3 h, replacing the dialysis buffer twice.
Note: Guanidine hydrochloride (GuHCl) is generally considered a stronger denaturant compared to urea.GuHCl is a salt composed of guanidine and hydrochloric acid, commonly used to denature histone pellets.Guanidine hydrochloride is a salt which can affect the binding of proteins to ion exchange columns.Urea, an organic compound, which can not affect the binding of proteins to ion exchange columns, is frequently used in the purification of histones.
44. Equilibration 5 mL SP HP cation exchange column with Urea buffer 0 without NaCl and then load the sample into the 5 mL SP HP cation exchange column (GE Healthcare) and elute with a 0-2 M NaCl gradient.45.Pool the peak fractions and dialyze thoroughly against distilled water.46.Lyophilize the proteins and store at À80 C. 47. Prepare the histone H3 proteins using the established histone inclusion body purification protocol similar to how H4K20C proteins were made.
Pause point: The lyophilized H4K20C or H3 protein can be stored at À80 C for up to 2 years.

Final concentration Amount
Tris

Final concentration Amount
Tris

Final concentration Amount
Tris

Final concentration Amount
Tris ( In vitro methyltransferase assays demonstrate that SUV420H1 (1-393) not only mediates H4K20me2 deposition into H2A or H2A.Z nucleosomes but also shows a preference for H2A.Z nucleosomes.Secondary structure analysis indicates that the C-terminus of SUV420H1 (1-393) is largely unstructured except for residues 352-367 (Figure 3B).For purification, a 63His tag was added in-frame at the C-terminus of SUV420H1 and the protein was cloned into the pET17b vector using NdeI and XhoI sites.
1. Transform 1 mL of pET17b-SUV420H1 plasmid into 50 mL of BL21(DE3) RIPL cells as directed by the manufacturer, and plate the cells on LB agar with 100 mg/mL ampicillin and 34 mg/mL chloramphenicol.Incubate at 37 C for 12-16 h. 2. Inoculate a single colony into 100 mL LB medium with ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL), and incubate at 37 C with shaking at 220 rpm for 12-16 h. 3. Dilute 15 mL cell culture into 800 mL LB medium containing ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL), total 4.8 L. 4. Incubate the cell culture at 37 C until it reaches an OD 600 of 0.6, then cool the culture to 16 C. 5. Add 400 mL of 1 M IPTG to the medium to achieve a final concentration of 0.5 mM and induce SUV420H1 expression for 18 h.23.Dilute the sample in a buffer with 850 mM NaCl, load onto a 5 mL SP HP cation exchange column equilibrated in histone 34 buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 850 mM NaCl, 5 mM b-mercaptoethanol), and elute with a 0.85-2 M NaCl gradient.Run 15% SDS-PAGE to analyze the peak fractions (Figure 4B).24.Pool the fractions and store at À80 C for further use.
Pause point: The purified (H3-H4K20Ecx) 2 protein can be stored at À80 C for up to 1 year.

Timing: 3 days
The histone octamer is mixed with DNA in equimolar ratios under high salt conditions, followed by a gradual reduction to low salt conditions, allowing for the stable formation of nucleosomes.
Note: Purified histone octamer must be added at 2 M NaCl condition to prevent disassembly at low salt.

Pause point:
The purified nucleosomes can be stored at 4 C for up to 3 months.

Protocol
Note: The SUV420H1 protein must be added at low concentration of nucleosome to prevent largely aggregation.
34. Add 2 mL of 100 mM S-adenosylmethionine (SAM) to into the complex, and incubate for 1 h on ice.
Note: In methylation reactions, SUV420H1 catalyzes the transfer of the methyl group from SAM to H4K20.SAM serves as a methyl donor in methylation reactions.The addition of SAM along with the mutation of H4K20Ecx collectively stabilizes the complex, so the SAM should be added to get sample of high-quality.SAM must be added at low concentration of complex to prevent largely aggregation.
35.Concentrate the sample to achieve a final concentration of 0.5 mg/mL.36.Create a gradient using 6.2 mL of low sucrose buffer (10 mM HEPES, pH 7.5, 2 mM DTT, 5% sucrose) and 6.2 mL of high sucrose buffer (10 mM HEPES, pH 7.5, 2 mM DTT, 20% sucrose) with 0.15% glutaraldehyde using a gradient master (BioComp) to establish a continuous density and glutaraldehyde gradient (Figure 5).37. Load 200 mL of complexes into the top of the tube.Ultracentrifuge at 4 C for 20 h at 210,100 g using a Beckman SW-41Ti rotor.38.Collect fractions every 200 mL and analyze them using electrophoresis at 150 V for 60 min on 6.5% native TBE polyacrylamide gels, keeping the gels on ice.39.Stain the gels with ethidium bromide (EB).Choose the best fractions, dialyze them in HEPES buffer (20 mM HEPES pH 7.5, 2 mM DTT), and concentrate the samples to 0.8 mg/mL for cryo-EM preparation.
CRITICAL: Ethidium bromide (EtBr) is highly toxic and can be readily absorbed through the skin.It is essential to wear gloves when handling EtBr solutions.
DNA according to the concentration to achieve a final concentration of 1 mg/mL and add 1/10 volume of 103H buffer.b.Add Takara EcoRV enzyme to achieve a final concentration of 0.6 U/mg, and finally add TE10/ 0.1 (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer to make up the remaining volume according to the following system.c.Incubate at 37 C for 16 h.10.Examine the EcoRV-digested plasmid on a 1% agarose gel.If multiple bands appear, indicating incomplete digestion, add 50% more enzyme and incubate for another 18 h.11.Upon complete digestion, test the experimental conditions of removing the plasmid backbone with a DNA concentration of 1 mg/mL.a. Precipitate the plasmid backbone using 40% PEG6000 to a final concentration of 4-8% PEG 6000 and use 4 M NaCl to bring the final concentration of NaCl to 0.5 M.

Figure 1 .
Figure 1.Preparation of 147-bp DNA (A) pUC19-147x12 plasmid, ''plasmid+E 00 means the plasmid digested by restriction enzymes EcoRV.(B) The 147-bp DNA was then isolated by PEG precipitation.''E'' means the plasmid digested by restriction enzymes EcoRV.''S'' means the supernatants and ''P'' means the pellets after PEG precipitation.(C) The picture of DNA extraction by PCIA.(D) The picture of DNA precipitate by ethanol.(E) The analysis of 147-bp DNA.
, lanes 2).31.Purify the H2A.Z-H2B protein by SP HP cation exchange column.a. Dilute the sample with histone buffer 0 (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 5 mM b-mercaptoethanol) to lower the NaCl concentration to 500 mM NaCl. b.Equilibration 5 mL SP HP cation exchange column with histone buffer A with 500 mM NaCl and then load the sample into a 5 mL SP HP cation exchange column (GE Healthcare) and elute with a 0.5-2 M NaCl gradient.c.Check the purified protein by SDS-PAGE and collect the protein of high quality (Figure 2, lanes 7-14).d.Measure the protein concentration by NanoDrop 2000c and store the protein at À80 C for future use.Typically, yield of H2A.Z-H2B protein form 6 bottles is about 15-20 mg.

1 L
Adjust pH of medium to 8.0 at 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.

1 L 1 L 1 L 1 L 1 L( 1 L 1 L 1 L 1 L 1 L
Adjust pH of medium to 8.0 at 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 8.0 at 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 8.0 at 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Continued on next page) STAR Protocols 5, 103295, September 20, 2024 Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 6.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.

1 L 1 L
Adjust pH of medium to 8.0 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 7.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.

1 L 1 L
Adjust pH of medium to 7.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 7.5 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one week.Adjust pH of medium to 8.0 at about 25 C, and store at 4 C.This buffer should be freshly prepared.Add to 50 mL Adjust pH of medium to 7.5 at about 25 C, and store at 4 C.This buffer should be freshly prepared.STAR Protocols 5, 103295, September 20, 2024 Protocol Note: The b-mercaptoethanol or DTT should be added before the use of the buffer.STEP-BY-STEP METHOD DETAILS Preparation of SUV420H1 Timing: 1.5 weeks Human SUV420H1 has two isoforms, SUV420H1 (1-898) and SUV420H1 (1-393), 7 as shown in Figure 3A.

TABLE REAGENT
Adjust pH of medium to 8.0 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.Adjust pH of medium to 8.0 at about 25 C, and store at 4 C.The buffer can be stored at 4 C for one month.
OligonucleotidesThe guide sequence for K219A/R220A: 5 0 -GACAAAATAGAATTACTGGT-3 0 This paper N/A (Continued on next page) Sterilize at 121 C for 15 min, and store at 25 C.The media can be stored at 25 C for 2 weeks.STAR Protocols 5, 103295, September 20, 2024