Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

1. Reconstitution of Nucleosomes with Differentially Isotope-labeled (and Asymmetrically Modified) Sister Histones

NOTE: The current protocol describes the reconstitution of nucleosomes with differentially isotope-labeled histone H3. To this end, two pools of histone H3 were used; one was ¹⁵N-labeled and contained a 6xHis-tag at the N-terminus and the second was ¹³C-labeled and contained the Strep peptide (WSHPQFEK) fused at the N-terminus. Both tags were separated from the native H3 sequence with a Tobacco Etch Virus (TEV) protease recognition site. To prepare additional asymmetrically modified nucleosomes, one of the two H3 pools is included in a premodified form. Premodification of a histone pool can be performed by reacting the isotope-labeled histone of interest with the respective histone-modifying enzyme in the presence of the necessary for each type of reaction cofactors/PTM donors¹⁶. The efficient placement of the respective PTM can be assessed by NMR spectroscopy or mass spectrometry. The amounts of histones/DNA used here are rough recommendations to obtain a nucleosome preparation with an approximate concentration of 10 µM (measured as DNA concentration at 260 nm).This final yield is sufficient to record good quality NMR spectra with relatively short acquisition times.

1. Reconstitution of a histone octamer with differentially isotope-labeled (and asymmetrically modified) histone H3
   NOTE: Recombinant histone proteins can be produced in mg quantities in BL21(DE3)plysS cells¹⁴. To obtain isotope-labeled histones, the same expression/purification protocol is followed with the exception of using for bacterial growth a minimal medium containing ¹⁵NH₄Cl and/or ¹³C-Glucose as nitrogen and carbon sources, for ¹⁵N- or ¹³C-labeling respectively. Purified histones are extensively dialyzed against 5 mM β-mercaptoethanol and stored lyophilized before use.
   1. Dissolve lyophilized histone aliquots, containing ~5 mg of each histone in 1 mL of unfolding buffer (7 M Guanidinium-HCl, 20 mM Tris pH 7.5, 10 mM DTT). Include both types of histone H3. Mix by pipetting and do not vortex. Keep the tubes on ice for 30 min to allow complete unfolding.
   2. Determine the exact concentration of each histone by measuring the absorbance at 280 nm against unfolding buffer and using extinction coefficients calculated with the ProtParam tool of the ExPASy portal.¹⁵
   3. Mix the core histones to equimolar ratios, keeping in mind to include 50% each of the two histone H3 pools. Start by using the whole content of the less concentrated histone and add all of the others accordingly.
   4. Adjust final protein concentration to approximately 1 mg/mL using unfolding buffer.
   5. Transfer the mixture to a 6 kDa-cutoff dialysis membrane and dialyze against 1-2 L of chilled refolding buffer (10 mM Tris pH 7.5, 2 M NaCl, 1 mM EDTA, 2 mM DTT) at 4 °C. Change the buffer at least once after dialysis has proceeded for a minimum of 3 h. Perform a final dialysis O/N at 4 °C.
   6. Collect dialyzed material and remove any precipitates by centrifugation in a microfuge for 5 min at 4 °C on maximum speed (normally no precipitation should be observed). Determine octamer concentration by measuring the absorbance at 280 nm. In the calculations, use the added extinction coefficients of histones, keeping in mind to double each value.
   7. Concentrate to a final volume of ~2 mL using a 10 kDa-cutoff centrifugal filter unit. Recalculate octamer concentration and estimate loss. At this point, an octamer concentration between 50 – 100 µM should be obtained.
   8. Connect the gel filtration column (stated in the Table of Materials) to an FPLC system and equilibrate with 2 column volumes (CV) of 0.22 µm-filtered and degassed refolding buffer at a flow rate of 1 mL/min and a pressure limit of 0.5 MPa.
      NOTE: The gel filtration run should be performed in the cold room or in a cold cabinet.
   9. Inject concentrated material using a flow rate of 1 mL/min and collect 1.0 – 1.5 mL fractions.
   10. Follow the chromatographic profile and observe histone octamer elution at ~65 – 68 mL.
       NOTE: A small shoulder of the elution peak and a peak at ~80 mL indicates the existence of free H3-H4 tetramers and H2A-H2B dimers, respectively.
   11. Run 20 µL of each collected fraction in an 18% SDS-PAGE gel.
       NOTE: Dilute the samples by a factor of at least 2 with water before loading them on the gel to reduce the high salt concentration and thus, avoid distortions. The same applies for subsequent SDS-PAGE analyses of samples in refolding buffer.
   12. Pool relevant fractions containing pure octamers judged by the equal distribution of the 4 histones on the stained gel and determine concentration as before (step 1.1.7).
   13. Connect a 1 mL Ni-NTA column (Table of Materials) to an FPLC system and equilibrate with 10 CV of 0.22 µm-filtered and degassed refolding buffer at a flow rate of 1 mL/min.
       NOTE: The Ni-NTA chromatography should be performed in the cold room or in a cold cabinet.
   14. Pass purified octamer through the Ni-NTA using a flow rate of 1 mL/min.
   15. Collect flow-through and keep samples for SDS-PAGE/WB analysis (20 µL and 4 µL, respectively). Subsequently, wash column with 10 CV of refolding buffer or until a baseline signal is reached.
   16. Elute with 10 CV of refolding buffer containing 250 mM imidazole using a flow rate of 1 mL/min. Keep samples for SDS-PAGE/WB analysis (20 µL and 4 µL, respectively).
   17. Equilibrate a 1 mL affinity column of a commercial streptavidin (Table of Materials) with 10 CV refolding buffer containing 250 mM imidazole using a batch/gravity flow setup.
       NOTE: This chromatographic step should be performed in the cold room.
   18. Pass Ni-NTA elution through the commercial streptavidin affinity column.
   19. Collect flow through and keep samples for SDS-PAGE/WB analysis (20 µL and 4 µL, respectively). Subsequently, wash with 10 CV of refolding buffer.
   20. Elute with 10 CV of refolding buffer containing 2.5 mM desthiobiotin. Keep samples for SDS-PAGE/WB analysis (20 and 4 µL respectively).
   21. Measure octamer concentration, add 10 times less TEV protease and let reaction proceed overnight at 4 °C in a standard plastic centrifuge tube.
       NOTE: The necessary amount of TEV to obtain complete digestion (removal of the affinity tags) depends on the origin (construct) and on the activity (freshly made, recombinant TEV is more active compared to the one stored for prolonged periods at – 80 °C). Try initially adding 10 times less TEV compared to the octamer (estimated as protein concentrations) and adjust accordingly.
   22. Check for the efficient digestion by running 20 µL of the mixture prior and after TEV addition on an 18% SDS-PAGE gel. If digestion is not complete, add more TEV protease and continue incubation for another 3-4 h.
   23. Dialyze sample against refolding buffer using a 50 kDa-cutoff dialysis membrane to remove TEV protease and adjust buffer composition.
   24. Concentrate the purified asymmetric octamer using a 10 kDa-cutoff centrifugal filter unit (the same filter unit from step 1.1.7 can be also used) to a volume of 1.0-2.0 mL. Measure the final concentration. Either use soon after for nucleosome reconstitution or adjust to 50% (v/v) glycerol to store at -20 °C for longer periods. At this point and expecting at least 70% loss during the previous steps, the octamer concentration should be in the range of 20 – 50 µM.
2. Nucleosome reconstitution
   1. If octamer was stored in 50% glycerol, dialyze overnight at 4 °C against refolding buffer before proceeding with nucleosome reconstitution.
   2. Adjust DNA (containing a nucleosome-positioning sequence) salt concentration to 2 M NaCl by using a 5 M NaCl stock solution.
      NOTE: The purified DNA either isolated after purification of a plasmid that contains multiple copies of the desired sequence or synthesized by PCR amplification should be stored concentrated to ~50 µM in water or a standard Tris-EDTA buffer.
   3. Mix octamer and DNA using the optimum ratio determined from a small-scale test. Use refolding buffer to adjust the final volume to ~3.0 mL. Always add the octamer last to prevent any possibility of mixing the octamer and the DNA at <2 M salt concentrations.
      1. Perform initial small-scale reconstitutions to determine the octamer:DNA ratio that leads to optimum nucleosome yields. For this, assemble three reactions of 0.9:1.0, 1.0:1.0, and 1.1:1.0 DNA:octamer ratios in a volume between 50-100 µL. Typically, use a concentration of 5 µM DNA.
      2. Proceed with reconstitution as described below (steps 1.2.4-1.2.8) and at the end, estimate the amount of soluble and good quality reconstituted nucleosome by measuring the DNA concentration at 260 nm and by running a 6% native-PAGE DNA gel, stained with ethidium bromide, respectively (Figure 5A).
   4. Transfer the mix to a 6 kDa-cutoff dialysis membrane and dialyze against 1 L of refolding buffer overnight at 4 °C.
   5. Reduce the salt concentration of the dialysis buffer in a step-wise manner from 2, to 0.85, 0.64, and 0.2 M NaCl respectively. Keep sample in each buffer for at least 3 h. Sometimes precipitate is formed after the last transition. In this case continue dialysis as planned and remove precipitate by microfuge centrifugation at the end of the next step.
   6. Transfer dialysis membrane in a beaker with 1 L of assay buffer (25 mM NaH₂PO₄/Na₂HPO₄ pH 6.8, 25 mM NaCl, 2 mM DTT) and continue dialysis O/N at 4 °C.
   7. Collect sample, remove any precipitate formed at step 1.2.5 by centrifugation in a microfuge for 5 min at 4 °C on maximum speed and concentrate using a 30 kDa-cutoff centrifugal filter unit to a final volume of ~300 µL.
   8. Measure DNA concentration at 260 nm, run an 18% SDS-PAGE protein gel (4 µL of sample) and a 6% native-PAGE DNA gel (0.2 µL of sample) and store sample at 4 °C for several weeks. The final concentration should be in the range of 10-20 µM.

2. NMR Analysis of Modification Reactions on the Two Sister Histones

NOTE: Assignment of 2D ¹H-¹⁵N correlation spectra of nucleosomal histone tails can be found at references^16,17,18. Additionally, assignments of CH_x groups of lysines, serines and threonines on 2D ¹H-¹³C correlation spectra can be found at reference¹⁶.

1. NMR setup and recording of reference spectra
   NOTE: Enzymatic assays were performed using 140 µL of the nucleosome sample in assay buffer into a 3-mm NMR tube at 300 K. Different NMR tubes with other sample volumes can be also used. The aforementioned temperature is appropriate to obtain good quality ¹H-¹⁵N correlation spectra of nucleosomal histone tails and good enzymatic activities. 2D-SOFAST-HMQC ¹H-¹⁵N and 2D-HSQC ¹H-¹³C spectra were recorded respectively with a setup that confers similar acquisition times. Typical acquisition parameters are 128 transients with 1.024 (¹H) x 128 (¹⁵N) complex points and 32 transients with 1.024 (¹H) x 64 (¹³C) complex points for the two different types of correlation spectra. For both 2D spectra, the acquisition time was ~30 min.
   1. Set 300 K as the sample temperature in the spectrometer. Add 10% D₂O (v/v) to the sample to be used for the spectrometer frequency lock.
   2. Place the sample in the spectrometer and perform basic operations (locking, tuning, shimming). Additionally, determine optimal pulse lengths and general acquisition parameters.
   3. Record 1D and 2D ¹H-¹⁵N and ¹H-¹³C reference spectra.
   4. Process reference spectra and ensure that the signals to be used for mapping the modification reactions are well resolved and have good intensities.
   5. Recover sample and transfer it to a microfuge tube.
2. Setup of the enzymatic reaction, NMR monitoring and quantitative analysis
   1. Copy and arrange a series of interleaved 2D ¹H-¹⁵N and ¹H-¹³C spectra. Aim for a total acquisition time of several hours and adjust accordingly in a new run based on the enzymatic rates obtained from the first reaction.
   2. Add to the sample the necessary cofactors for the respective type of modification reaction (1 mM ATP/2 mM MgCl₂ for phosphorylation, 1 mM acetyl-CoA for acetylation, 1 mM SAM for methylation, etc.).
   3. Add the enzyme of interest, mix by pipetting, transfer the sample back in the NMR tube and subsequently in the spectrometer (perform these operations quickly).
      NOTE: If there is no data about the expected enzymatic activity, adjust the substrate-to-enzyme molar ratio to 10:1. Perform a first trial run and adjust accordingly for the real run.
   4. Perform a fast automatic shimming and use the rest of parameters from the reference spectra.
   5. Initiate the series of the interleaved 2D ¹H-¹⁵N and ¹H-¹³C spectra.
   6. Follow the progression of the reaction in real-time and stop acquisition when the reaction reaches completion or a desired level.
   7. Process spectra as before with exactly the same parameters.
   8. Repeat the whole run using a different acquisition order for the two types of correlation spectra. If in the first experiment a ¹H-¹⁵N correlation was first in the series, start now with a ¹H-¹³C correlation. By this and having same acquisition time for both spectra, it is possible to plot and compare PTM reactions on both differentially isotope-labeled histones on the same time scale. Additionally, it is possible to calculate averages and plot error bars.
      NOTE: A thorough description of the methodologies for NMR signal analysis and subsequent quantification of enzymatic reactions can be found here¹⁹.
   9. Select NMR signals from each time-course NMR spectrum that report the progression of the reaction and calculate their relative signal intensities using a visualization and analysis software for NMR spectra. Do this for signals that report the unmodified as well as the modified state. Extract modification levels.
   10. Plot the calculated values of modification levels against the reaction time.