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Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
二甲基辛二硝酸盐交联后酵母核小体的生化分析   

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eLIFE
Sep 2017

Abstract

Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.

Keywords: Chromatin (染色质), Nucleosome (核小体), Histone (组蛋白), Protein-protein interaction (蛋白-蛋白相互作用), Crosslink (交联), Streptavidin affinity chromatography (链亲和素亲和层析法)

Background

Post-translational modifications of histone proteins affect every aspect of chromosome biology, including transcription, replication, repair, and recombination. Because nucleosomes contain two copies of each core histone, modifications could be symmetric (same modifications on both H3 tails, e.g., K27me on both H3 tails within a nucleosome (Voigt et al., 2012)) or asymmetric (modifications on a single H3 tail, e.g., K27me on a single H3 tail within a nucleosome (Voigt et al., 2012)). Recent studies have demonstrated that nucleosomes in mammalian cells indeed display some asymmetric modifications (Voigt et al., 2012; Shema et al., 2016). To allow experimental manipulation of nucleosomal symmetry in vivo, we designed a pair of altered histone H3 proteins that have obligate heterodimeric interactions, termed H3X (L126A, L130V) and H3Y (L109I, A110W, L130I) (Ichikawa et al., 2017). Yeast cells expressing both H3X and H3Y are viable, but inviable if cells express only H3X or H3Y.

For biochemical validation of H3X-H3Y interactions within individual nucleosomes, we generated yeast strains expressing the bacterial biotin ligase BirA, N-terminal V5-tagged H3X and N-terminal biotin-accepting epitope tagged H3Y (Beckett et al., 1999). BirA is an enzyme that attaches biotin to a specific acceptor epitope, enabling us to purify the biotinylated molecules by streptavidin affinity chromatography. We treated extracts from yeast cells with dimethyl suberimidate (DMS), a crosslinking agent that contains a primary amine reactive imidoester group at each end of an 8-atom spacer arm (Figure 1A). DMS produces well-characterized crosslinks within histone octamers, including links between the two H3 molecules (Figure 1B; Kornberg and Thomas, 1974; Thomas, 1989). Therefore, this method can be used to report on the composition of asymmetric epitope tags.

Crosslinked samples are digested with micrococcal nuclease (MNase) to generate a soluble population of chromatin fragments containing approximately mononucleosome-sized DNA molecules (we note that DMS crosslinking prevents generation of a uniform ladder of MNase-digested products, Figure 1C). Biotin-tagged, MNase-digested chromatin is then purified via streptavidin-agarose affinity purification in the presence of high salt (2 M NaCl). This salt concentration is sufficient to remove DNA from histones (Bartley and Chalkley, 1972), avoiding interference from any neighbor nucleosomes that survived the MNase digestion. Bound proteins are then analyzed by Western blotting (Figure 1D). The DMS crosslinking efficiency of the X-Y heterodimeric pairs was around 10%, nearly identical to wild-type H3 homodimeric pairs; additionally, approximately 20% of the crosslinked heterodimers in the input fractions were precipitated by streptavidin-agarose (Ichikawa et al., 2017). We applied this method to analyze the extent of homodimerization of H3X or H3Y, as well as X-Y heterodimerization (Ichikawa et al., 2017). To examine this, we quantified X-Y dimer bands rather than the monomer, because these DMS crosslinked species represent direct H3-H3 interactions within individual nucleosomes.


Figure 1. Biochemical validation of asymmetric nucleosome formation in vivo. A. Chemistry of DMS cross-linking. DMS reacts with primary amines of proteins to form amidine bonds. B. Schematic for DMS crosslink of H3X and H3Y heterodimer. Yeast strains expressed V5-tagged H3X and Biotin-tagged H3Y, as indicated. C. DNA samples purified from MNase-digested chromatin from each time point (0, 10, 20 min) were analyzed by electrophoresis on a 1.5% TAE agarose gel, and stained with ethidium bromide. Note that after DMS crosslinking, the MNase-digested DNA fragments do not display the characteristic polynucleosomal ladder of uncrosslinked chromatin. D. Immunoblot analysis of V5-H3X and biotin-H3Y interactions. The left two lanes show total uncrosslinked and DMS-crosslinked chromatin, and right lanes show MNase-digested chromatin (Input), flow through fraction (Unbound) and streptavidin-precipitated biotinylated-H3 (Bound). Samples were separated by 17% SDS-PAGE, transferred to a membrane, and probed with anti-V5 antibody.

Materials and Reagents

  1. 200 μl and 1,000 μl Pipette tips (Corning, Axygen®, catalog numbers: RFL-222-C , RFL-1200-C )
  2. 1.5 ml O-ring screw-cap tubes (Fisher Scientific, catalog number: 02-707-353 )
  3. 1.5 ml microfuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  4. 0.5 ml glass beads (Bio Spec Product, catalog number: 11079105 )
  5. 26 gauge needle (BD, catalog number: 305115 )
  6. 12 x 75 mm plastic tube (Corning, Falcon®, catalog number: 352008 )
  7. Nitrocellulose blotting membrane (GE Healthcare, catalog number: 10600004 )
  8. Examination gloves (Fisher Scientific, catalog number: 19-130-1597D )
  9. Biotin (Sigma-Aldrich, catalog number: B4501 )
  10. Dimethyl suberimidate (DMS) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20700 )
  11. Trichloroacetic acid (TCA) (Fisher Scientific, catalog number: BP555 )
  12. Magnesium chloride hexahydrate (MgCl2·6H2O) (Acros Organics, catalog number: 197530010 )
  13. Calcium chloride dihydrate (CaCl2·2H2O) (Merck, Millipore Sigma, catalog number: 102382 )
  14. Disodium ethylenediamine tetraacetate (EDTA) (Fisher Scientific, catalog number: S311 )
  15. Ethylene glycol tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E4378 )
  16. RNase A (Sigma-Aldrich, Roche Diagnostics, catalog number: 10109169001 )
  17. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
  18. Ammonium acetate (NH4Ac) (Fisher Scientific, catalog number: A637 )
  19. Phenol:Chloroform:Isoamyl Alcohol (PCI) (Thermo Fisher Scientific, catalog number: 15593031 )
  20. 2-Propanol (Fisher Scientific, catalog number: A416 )
  21. Ethanol (Decon Labs, catalog number: 2701 )
  22. TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0).
  23. 6x gel loading dye (New England Biolabs, catalog number: B7042 )
  24. Agarose (Fisher Scientific, catalog number: BP160 )
  25. Ethidium bromide (Sigma-Aldrich, catalog number: E7637 )
  26. CL2B Sepharose beads (Sigma-Aldrich, catalog number: CL2B300 )
  27. Streptavidin Sepharose beads (GE Healthcare, catalog number: 17-5113-01 )
  28. Insulin (Sigma-Aldrich, catalog number: I1882 )
  29. Clarity Western ECL Substrate (Bio-Rad Laboratories, catalog number: 1705060 )
  30. Primary antibody: anti-V5 tag (Thermo Fisher Scientific, Invitrogen, catalog number: R960-25 )
  31. Secondary antibody: anti-Mouse IgG (Thermo Fisher Scientific, Invitrogen, catalog number: 31430 )
  32. Nonfat dry milk (Walmart, Great Value)
  33. Yeast nitrogen base without amino acids (United States Biological, catalog number: Y2025 )
  34. Glucose (Merck, Millipore Sigma, catalog number: DX0145 )
  35. Micrococcal Nuclease (MNase) (Worthington Biochemical, catalog number: LS004797 )
  36. Tris hydroxymethyl aminomethane (Tris) (Fisher Scientific, catalog number: BP152 )
  37. Sodium tetraborate decahydrate (Na borate) (Sigma-Aldrich, catalog number: S9640 )
  38. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  39. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144 )
  40. Phenylmethylsulfonyl fluoride (PMSF) (RPI, catalog number: P20270 )
  41. Sodium dodecylsulfate (SDS) (RPI, catalog number: L22010 )
  42. Glycerol (Fisher Scientific, catalog number: G33 )
  43. Bromophenol blue (Sigma-Aldrich, catalog number: B7021 )
  44. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  45. Tween 20 (Sigma-Aldrich, catalog number: P2287 )
  46. Acetic acid, Glacial (Fisher Scientific, catalog number: A38 )
  47. 40% acrylamide solution (Bio-Rad Laboratories, catalog number: 1610140 )
  48. 2% Bis solution (Bio-Rad Laboratories, catalog number: 1610142 )
  49. Ammonium persulfate (Fisher Scientific, catalog number: BP179 )
  50. Tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, catalog number: 1610800 )
  51. Sodium bicarbonate (NaHCO3) (Fisher Scientific, catalog number: S233 )
  52. Sodium carbonate (Na2CO3) (Fisher Scientific, catalog number: S263 )
  53. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318 )
  54. Methanol (Fisher Scientific, catalog number: A412 )
  55. Synthetic media (see Recipes)
  56. MNase (see Recipes)
  57. Extraction (E) buffer (see Recipes)
  58. 2x SDS-sample buffer (SB) (see Recipes)
  59. Wash (W) buffer (see Recipes)
  60. 17% SDS-PAGE gel (see Recipes)
  61. 5% stacking gel (see Recipes)
  62. 1x SDS-running buffer (see Recipes)
  63. 40x Na carbonate buffer (see Recipes)
  64. Blotting buffer (see Recipes)
  65. TBST (Tris-buffered saline + Tween 20) (see Recipes)

Equipment

  1. P20, P200 and P1000 Pipettes (Gilson)
  2. Labquake Tube Shaker/Rotators (Thermo Fisher Scientific)
  3. Swing bucket rotor (Beckman Coulter, model: GH 3.8 )
  4. Shaker incubator (INFORS HT)
  5. Mini-Beadbeater-96 (Bio Spec Product)
  6. Tabletop centrifuge (Beckman Coulter, model: Allegra 6R )
  7. Centrifuge for microcentrifuge tubes (Eppendorf, model: 5415 D )
  8. ChemiDoc Touch Imaging System (Bio-Rad Laboratories, model: ChemiDoc Touch Imaging System )
  9. Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
  10. Water bath (Precision Scientific, catalog number: 66551 )
  11. Vertical Mini-Gel systems (C.B.S. Scientific, model: MGV102 )
  12. Transfer electrophoresis unit (Hoefer, model: TE22 )
  13. EASY-CAST Electrophoresis System (Thermo Fisher Scientific, Thermo ScientificTM, model: OwlTM Easy-CastTM B1 )
  14. Power Supply (Bio-Rad Laboratories, model: PowerPacTM Basic )

Software

  1. Image Lab (Bio-Rad Laboratories)

Procedure

Day 1
Pick a single colony from a plate, and inoculate an overnight culture of cells in 15 ml of synthetic media. Grow at 30 °C, 170 rpm in a shaker incubator. Do this 1-2 days beforehand, depending on growth rate of strain.

Day 2
Inoculate the overnight culture grown on day 1 into 110 ml of synthetic media supplemented with 250 nM biotin. Biotin is added to favor in vivo biotinylation of the tagged H3 proteins. The amount of cells to inoculate depends on growth rate (see below; typically, inoculate 3 OD units of cells of most X-Y strains into 110 ml media, which are then grown for 12 h). Grow at 30 °C, 170 rpm in a shaker incubator.

Day3

  1. Cross-linking H3 dimers with DMS
    1. Harvest at desired cell density. The desired cell density is 0.25 at OD600; don’t use more than 100 ml of cells at OD600 = 0.3 per Streptavidin-pull down described below, in order to assure that chromosomes are adequately digested with 20 μl MNase to generate mostly monosomes.
    2. Spin down cells in Swing bucket rotor at 2,000 x g for 10 min at 4 °C. Gently pour off supernatant.
    3. Resuspend cells in 0.5 ml E buffer at 4 °C and transfer to a 1.5 ml O-ring screw-cap tube. Spin down in microfuge 20 sec at max speed at 4 °C. Remove supernatant.
    4. Wash 3 times with 1 ml E buffer by vortex. Spin down in microfuge 20 sec at max speed at 4 °C. Remove supernatant. Thorough washing is important here to remove amine-containing compounds that will impair crosslinking.
    5. Resuspend each tube of cells completely in 900 μl E buffer at 4 °C, and add 0.5 ml glass beads.
    6. Bead beat: 3 times of 1 min beating (2,100 rpm) in the Mini-Beadbeater-96 (5 min between pulses, on ice).
    7. Heat a 26 gauge needle with a gas burner (Figure 2A), and puncture the tube bottom with the red-hot needle (Figure 2B). Place into a 12 x 75 mm plastic tube (‘FACS tube’) (Figure 2C) and spin for 2 min at 365 x g in a tabletop centrifuge at 4 °C (Figure 2D). Discard screwcap tube with glass beads (Figure 2E).


      Figure 2. Step by step photos of the procedure Day 3, Step A7

    8. Resuspend pellet in the liquid (a mixture of E buffer and cell lysate on the bottom of FACS tube) completely, and transfer to a 1.5 ml microfuge tube.
    9. Make DMS stock 11 mg/ml in E buffer (typically 1 ml) at room temperature. This stock should be made freshly every time.
    10. Remove 0 min aliquot, 100 μl for SDS-PAGE. Add 1/10 volume 100% TCA. Incubate at room temperature for 10 min. Spin down in a microfuge for 10 min at max speed at room temperature. Remove supernatant, wash pellet with 1 ml of acetone at room temperature. Leave the lid open for 30 min to air-dry the pellet. Resuspend air dried pellet in 50 μl 2x SB. Store at -20 °C.
    11. Add 1/10 volume of 11 mg/ml DMS to a final concentration of 1 mg/ml. Incubate at room temperature with rotating for 60 min.
    12. Add 1/20 volume of 1 M Tris-HCl, pH 7.5 to a final concentration of 50 mM for quenching the DMS crosslinking and further rotation for 15 min at room temperature.
    13. Remove 60 min aliquot, 100 μl for SDS-PAGE. Add 1/10 volume 100% TCA to the aliquot, process as above.
    14. Go next step (MNase digestion) immediately after the cross-linking.

  2. MNase digestion
    1. Add 1/100 volume of 1 M MgCl2 to a final concentration of 10 mM (really 8 mM final, since E buffer contains 2 mM EDTA), and add 1/100 volume of 0.1 M CaCl2 to a final concentration of 1 mM to the DMS-crosslinked sample (the remaining amount after Step A-13, approximately 800 μl) at room temperature. Take 100 μl to generate an un-digested DNA sample for gel analysis. Store on ice.
    2. Equilibrate at 37 °C in a water bath for 5 min.
    3. Add 20 μl of MNase, invert the tubes 5 times and incubate at 37 °C for 20 min.
    4. Add 1/25 volume of 0.25 M EDTA/EGTA to a final concentration of 10 mM and invert 5-10 times to inhibit MNase. Take 100 μl to generate an MNase-digested DNA sample. Spin at 8,000 x g for 1 min, 4 °C, take supernatant for Streptavidin-pull down.

  3. Gel analysis of MNase digestion
    1. For DNA purification, add 5 μl of RNase A (10 mg/ml) to the 100 μl sample aliquots and incubate at 37 °C for 30 min.
    2. Add 5 μl of 20% SDS and 2 μl of Proteinase K (20 mg/ml). Incubate at 65 °C for 3 h.
    3. Add 200 μl of 7.5 M NH4Ac and 300 μl of ddH2O.
    4. Add 600 μl of PCI and vortex. Spin for 5 min in a microfuge at max speed.
    5. Take aqueous phase, and precipitate with 1 volume of 2-propanol.
    6. Spin down for 20 min at max speed at room temperature right after isopropanol precipitation.
    7. Wash pellet with 1 ml of 70% ethanol, spin for 5 min and air dry for 1 h.
    8. Resuspend pellet in 10 μl of TE.
    9. Add 2 μl of 6x loading buffer and run on 1.5% TAE agarose gel containing 0.5 μg/ml ethidium bromide at 5 V/cm for 50 min.

  4. Streptavidin-pull down
    1. Equilibrate CL2B Sepharose beads with E buffer at 4 °C. To block the beads, add 10 μg insulin per 40 μl slurry for each sample, rotate for 30 min at 4 °C. Add MNase digested samples (described at Step B4) to 40 μl slurry preblocked CL2B Sepharose beads, rotate for 30-60 min at 4 °C.
    2. Spin at 8,000 x g for 1 min, 4 °C. Take 30 μl supernatant + 30 μl 2x SB as ‘Input’ sample. Store at -20 °C.
    3. Transfer the supernatant into a new 1.5 ml tube. Add 30 μl slurry streptavidin-Sepharose beads preblocked with insulin (use 10 μg insulin per 30 μl slurry for each sample, process as above), rotate 2 h at 4 °C.
    4. Spin at 8,000 x g for 1 min, 4 °C. Take 30 μl supernatant + 30 μl 2x SB as ‘Unbound’ sample.
    5. Wash beads three times with 1 ml W buffer at 4 °C, by rotating at 4 °C for 5 min each time. Spin at 8,000 x g for 1 min at 4 °C, then discard supernatant.
    6. After the last wash, remove all supernatant with a 26 gauge needle on a vacuum. Resuspend the beads in 50 μl 2x SB. Store at -20 °C.

  5. Western blot
    1. Boil samples at 100 °C for 10 min.
    2. Load 10 μl of 0 min and 60 min samples, 15 μl of Input samples and 8 μl of Bound samples. Run on 17% SDS-PAGE gels in 1x SDS-running buffer at constant 8 mA while bromophenol blue goes through the stacking gel, and 18 mA as it goes through the resolving gel. Continue running for 15 min after bromophenol blue runs off the bottom to improve the resolution of histones. Total run time is approximately 2 h.
    3. Transfer to Nitrocellulose membrane in blotting buffer at constant 0.5 A for 48 min.
    4. Block the membrane in 5% milk/TBST for 1 h at room temperature.
    5. Incubate membrane overnight at 4 °C with anti-V5 antibody (1:10,000 dilution in 5% milk/TBST).
    6. Wash the membrane three times each for 10 min with 5% milk/TBST.
    7. Incubate the membrane with secondary antibody (anti-Mouse IgG 1:15,000 dilution in 5% milk/TBST) at room temperature for 1 h.
    8. Wash the membrane three times each for 10 min with TBST.
    9. Remove the TBST and incubate the membrane with 1 ml ECL (enhanced chemiluminescence) solution for 10 min. Detect by using ChemiDoc Touch Imaging System (Chemiluminescent Blot mode, at the highest resolution).

Data analysis

H3-H3 crosslinked species were quantified with Bio-Rad ‘Image Lab’ software, using the ‘Volume Tools’. The area of the band was defined by surrounding it with a rectangle box (Figure 3). The same volume area was used to measure background signals, which were subtracted from the band intensity. The percentage of precipitated H3 dimer was calculated with following formulas:
Total input = ‘Band intensity of H3 dimer on input lane’ x ‘Total volume of input fraction’/‘Loading volume of input fraction’
Total bound = ‘Band intensity of H3 dimer on bound lane’ x ‘Total volume of bound fraction’/‘Loading volume of bound fraction’
Percentage of precipitated H3 dimer = (Total bound/Total input) x 100
The mean and standard deviation were calculated from the values of 3 independent replicate experiments.


Figure 3. Data analysis with Bio-Rad image Lab. Areas of the H3-H3 dimer bands and the blanks were defined by surrounding it with a rectangle box using the ‘Volume Tools’.

Notes

  1. Use fresh cells (don’t freeze cells before the crosslink).
  2. Use fresh DMS stock.
  3. Note that during bead beating, visible aggregates appear, and many of these remain in the screwcap tube with the discarded glass beads at Step A7 of the DMS cross-link section. This is normal and does not indicate a problem.
  4. 60 min of incubation during the DMS crosslinking reaction at Step A11 was sufficient for us to obtain crosslinked H3 dimers. However, you can change the incubation time depending on your purpose and sample conditions.
  5. PVDF membranes can be used for the Western blot, but in our experience Nitrocellulose membranes detect histones more sensitively.
  6. Acrylamide is toxic. Wear examination gloves when you make SDS-PAGE gels.

Recipes

  1. Synthetic media
    0.67% yeast nitrogen base without amino acids
    2% glucose
    Note: No pH adjustment.
  2. MNase
    20 U/μl in 10 mM Tris-HCl pH 7.4
  3. Extraction (E) buffer
    20 mM Na borate
    0.35 M NaCl
    2 mM EDTA
    Adjust pH to 9.00 with HCl
    1 mM PMSF (added freshly)
  4. 2x SDS-sample buffer (SB)
    0.1 M Tris-HCl pH 6.8
    2% SDS
    20% glycerol
    0.02% bromophenol blue
    1/50 volume of 2-mercaptoethanol
  5. Wash (W) buffer
    10 mM Tris-HCl pH 8.0
    1 mM EDTA
    2 M NaCl
    0.2% Tween 20
  6. 17% SDS-PAGE gel (0.75 mm thickness, 14-well comb)
    2.13 ml of 40% acrylamide
    0.18 ml of 2% bisacrylamide
    1.875 ml of 1.0 M Tris-HCl pH 8.8
    25 μl of 20% SDS
    0.78 ml of H2O
    20 μl of 10% ammonium persulfate
    5 μl of TEMED
  7. 5% stacking gel
    0.31 ml of 40% acrylamide
    0.18 ml of 2% bisacrylamide
    0.31 ml of 1.0 M Tris-HCl pH 6.8
    12.5 μl of 20% SDS
    1.69 ml of H2O
    10 μl of 10% ammonium persulfate
    5 μl of TEMED
  8. 1x SDS-running buffer
    25 mM Tris
    192 mM glycine
    0.1% SDS
  9. 40x Na carbonate buffer
    251 mM NaHCO3
    173 mM Na2CO3
    Adjust pH to 9.5 with NaOH
  10. Blotting buffer
    1x Na carbonate buffer
    20% methanol
  11. TBST
    25 mM Tris-HCl pH 8.0
    137 mM NaCl
    2.68 mM KCl
    0.1% Tween 20

Acknowledgments

This work was supported by NIH grant R01GM100164. The authors have no conflicts of interest or competing interests.

References

  1. Bartley, J. A. and Chalkley, R. (1972). The binding of deoxyribonucleic acid and histone in native nucleohistone. J Biol Chem 247(11): 3647-3655.
  2. Beckett, D., Kovaleva, E. and Schatz, P. J. (1999). A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8(4): 921-929.
  3. Ichikawa, Y., Connelly, C. F., Appleboim, A., Miller, T. C., Jacobi, H., Abshiru, N. A., Chou, H. J., Chen, Y., Sharma, U., Zheng, Y., Thomas, P. M., Chen, H. V., Bajaj, V., Muller, C. W., Kelleher, N. L., Friedman, N., Bolon, D. N., Rando, O. J. and Kaufman, P. D. (2017). A synthetic biology approach to probing nucleosome symmetry. Elife 6: e28836.
  4. Kornberg, R. D. and Thomas, J. O. (1974). Chromatin structure; oligomers of the histones. Science 184(4139): 865-868.
  5. Shema, E., Jones, D., Shoresh, N., Donohue, L., Ram, O. and Bernstein, B. E. (2016). Single-molecule decoding of combinatorially modified nucleosomes. Science 352(6286): 717-721.
  6. Thomas, J. O. (1989). Chemical cross-linking of histones. Methods Enzymol 170: 549-571.
  7. Voigt, P., LeRoy, G., Drury, W. J., 3rd, Zee, B. M., Son, J., Beck, D. B., Young, N. L., Garcia, B. A. and Reinberg, D. (2012). Asymmetrically modified nucleosomes. Cell 151(1): 181-193.

简介

核小体是真核染色体包装的基本单元,由围绕核心组蛋白H2A,H2B,H3和H4中的每一个的两个分子包裹的147bp DNA组成。 核小体是对称的,一个对称轴以H3分子的C-末端之间的同源二聚体相互作用为中心。 为了探索核小体对称性的功能性后果,我们设计了一对特异性H3异二聚体,称为H3X和H3Y,使我们能够比较具有单一或双重H3改变的细胞。 我们对异二聚体X-Y相互作用的生物化学验证包括使用二甲基琥珀三酸酯(DMS)进行的核内H3交联。 在这里,我们提供了使用DMS来分析酵母核小体的详细方案。

【背景】组蛋白的翻译后修饰影响染色体生物学的各个方面,包括转录,复制,修复和重组。因为核小体包含每个核心组蛋白的两个拷贝,所以修饰可以是对称的(在两个H3尾部上的相同修饰,例如,在核小体内的两个H3尾部上的K27me(Voigt等人
对于单个核小体内H3X-H3Y相互作用的生化验证,我们生成了表达细菌生物素连接酶BirA,N-末端V5-标记的H3X和N-末端生物素接受表位标记的H3Y的酵母菌株(Beckett等人, 1999)。 BirA是一种将生物素连接到特定受体表位的酶,使我们能够通过链霉亲和素亲和层析纯化生物素化分子。我们用二甲基辛二酰亚胺(DMS)处理酵母细胞提取物,DMS是一种交联剂,在8个原子间隔臂(图1A)的每个末端含有伯胺反应性亚氨酸酯基团。 DMS在组蛋白八聚体内产生良好表征的交联,包括两个H3分子之间的连接(图1B; Kornberg和Thomas,1974; Thomas,1989)。因此,该方法可用于报告不对称表位标签的组成。

用微球菌核酸酶(MNase)消化交联的样品以产生含有近似单核小体大小的DNA分子的可溶性染色质片段群(我们注意到DMS交联防止产生MNase消化产物的均匀梯形,图1C)。生物素标记的,MNase消化的染色质然后通过在高盐(2M NaCl)存在下的链霉抗生物素蛋白 - 琼脂糖亲和纯化来纯化。这种盐浓度足以从组蛋白中去除DNA(Bartley和Chalkley,1972),避免了在MNase消化后存活的任何邻近核小体的干扰。然后通过Western印迹分析结合的蛋白质(图1D)。 X-Y异二聚体对的DMS交联效率约为10%,几乎与野生型H3同源二聚体对相同;此外,通过链霉抗生物素蛋白 - 琼脂糖(Ichikawa等人,2017)将约20%的输入级分中的交联异二聚体沉淀。我们应用这种方法来分析H3X或H3Y同源二聚化的程度,以及X-Y异源二聚化(Ichikawa等人,2017)。为了检验这一点,我们量化了X-Y二聚体条带而不是单体,因为这些DMS交联物种代表单个核小体内的直接H3-H3相互作用。

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图1.体内不对称核小体形成的生物化学验证A. DMS化学交联。 DMS与蛋白质的伯胺反应形成脒键。 B.H3X和H3Y异二聚体的DMS交联的示意图。如所示,酵母菌株表达V5标记的H3X和生物素标记的H3Y。 C.通过在1.5%TAE琼脂糖凝胶上电泳分析从每个时间点(0,10,20分钟)从MNase消化的染色质纯化的DNA样品,并用溴化乙锭染色。请注意,DMS交联后,MNase消化的DNA片段不显示未交联染色质的特征性多核苷酸梯形。 D.V5-H3X和生物素-H3Y相互作用的免疫印迹分析。左侧两条泳道显示完全未交联和DMS交联的染色质,右侧泳道显示MNase消化的染色质(输入),流过流分(未结合)和链霉亲和素沉淀的生物素化H3(结合)。样品通过17%SDS-PAGE分离,转移至膜上,并用抗V5抗体探测。

关键字:染色质, 核小体, 组蛋白, 蛋白-蛋白相互作用, 交联, 链亲和素亲和层析法

材料和试剂

  1. 200μl和1,000μl移液枪头(Corning,Axygen ,目录号:RFL-222-C,RFL-1200-C)
  2. 1.5毫升O形环螺帽管(Fisher Scientific,目录号:02-707-353)
  3. 1.5毫升微量离心管(Corning,Axygen,目录号:MCT-150-C)
  4. 0.5 ml玻璃珠(Bio Spec产品,目录号:11079105)
  5. 26号针头(BD,目录号:305115)

  6. 12 x 75毫米塑料管(Corning,Falcon ,产品目录号:352008)
  7. 硝化纤维素印迹膜(GE Healthcare,目录号:10600004)
  8. 检查手套(Fisher Scientific,目录号:19-130-1597D)
  9. 生物素(Sigma-Aldrich,目录号:B4501)
  10. 二甲基琥珀酰亚胺酯(DMS)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:20700)
  11. 三氯乙酸(TCA)(Fisher Scientific,目录号:BP555)
  12. 氯化镁六水合物(MgCl 2·6H 2 O)(Acros Organics,目录号:197530010)
  13. 氯化钙二水合物(CaCl 2·2H 2 O)(Merck,Millipore Sigma,目录号:102382)
  14. 乙二胺四乙酸二钠(EDTA)(Fisher Scientific,目录号:S311)
  15. 乙二醇四乙酸(EGTA)(Sigma-Aldrich,目录号:E4378)
  16. RNase A(Sigma-Aldrich,Roche Diagnostics,目录号:10109169001)
  17. 蛋白酶K(Sigma-Aldrich,目录号:P2308)
  18. 乙酸铵(NH 4 Ac)(Fisher Scientific,目录号:A637)
  19. 苯酚:氯仿:异戊醇(PCI)(Thermo Fisher Scientific,目录号:15593031)
  20. 2-丙醇(Fisher Scientific,目录号:A416)
  21. 乙醇(Decon Labs,目录号:2701)
  22. TE(10mM Tris-Cl,1mM EDTA,pH 8.0)。
  23. 6x凝胶上样染料(New England Biolabs,目录号:B7042)
  24. 琼脂糖(Fisher Scientific,目录号:BP160)
  25. 溴化乙锭(Sigma-Aldrich,目录号:E7637)
  26. CL2B琼脂糖珠(Sigma-Aldrich,目录号:CL2B300)
  27. 链霉亲和素琼脂糖珠(GE Healthcare,目录号:17-5113-01)
  28. 胰岛素(Sigma-Aldrich,目录号:I1882)
  29. 澄清西方ECL底物(Bio-Rad Laboratories,目录号:1705060)
  30. 一抗:抗V5标签(Thermo Fisher Scientific,Invitrogen,目录号:R960-25)
  31. 二抗:抗小鼠IgG(Thermo Fisher Scientific,Invitrogen,目录号:31430)
  32. 脱脂奶粉(沃尔玛,超值)
  33. 没有氨基酸的酵母氮碱(美国生物,目录号:Y2025)
  34. 葡萄糖(Merck,Millipore Sigma,目录号:DX0145)
  35. 微球菌核酸酶(MNase)(Worthington Biochemical,目录号:LS004797)
  36. 三羟甲基氨基甲烷(Tris)(Fisher Scientific,目录号:BP152)
  37. 四硼酸钠十水合物(硼酸钠)(Sigma-Aldrich,目录号:S9640)
  38. 氯化钠(NaCl)(Fisher Scientific,目录号:S271)
  39. 盐酸(HCl)(Fisher Scientific,目录号:A144)
  40. 苯基甲基磺酰氟(PMSF)(RPI,目录号:P20270)
  41. 十二烷基硫酸钠(SDS)(RPI,目录号:L22010)
  42. 甘油(Fisher Scientific,目录号:G33)
  43. 溴酚蓝(Sigma-Aldrich,目录号:B7021)
  44. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  45. 吐温20(Sigma-Aldrich,目录号:P2287)
  46. 冰醋酸(Fisher Scientific,目录号:A38)
  47. 40%丙烯酰胺溶液(Bio-Rad Laboratories,目录号:1610140)
  48. 2%Bis溶液(Bio-Rad Laboratories,目录号:1610142)
  49. 过硫酸铵(Fisher Scientific,目录号:BP179)
  50. 四甲基乙二胺(TEMED)(Bio-Rad Laboratories,目录号:1610800)
  51. 碳酸氢钠(NaHCO 3)(Fisher Scientific,目录号:S233)
  52. 碳酸钠(Na 2 CO 3)(Fisher Scientific,目录号:S263)
  53. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318)
  54. 甲醇(Fisher Scientific,目录号:A412)
  55. 合成媒体(见食谱)
  56. MNase(见食谱)
  57. 提取(E)缓冲液(见食谱)
  58. 2x SDS样品缓冲液(SB)(请参阅食谱)
  59. 洗(W)缓冲液(见食谱)
  60. 17%SDS-PAGE凝胶(见食谱)
  61. 5%浓缩胶(见食谱)
  62. 1x SDS缓冲液(见食谱)
  63. 40倍碳酸钠缓冲液(见食谱)
  64. 印迹缓冲液(见食谱)
  65. TBST(高速缓冲液)+ 20(参见食谱)

设备

  1. P20,P200和P1000移液器(吉尔森)
  2. Labquake Tube Shaker / Rotators(赛默飞世尔科技)
  3. 摆臂式转子(Beckman Coulter,型号:GH 3.8)
  4. 摇床培养箱(INFORS HT)
  5. Mini-Beadbeater-96(Bio Spec产品)
  6. 台式离心机(Beckman Coulter,型号:Allegra 6R)
  7. 离心机用于离心管(Eppendorf,型号:5415 D)
  8. ChemiDoc触摸成像系统(Bio-Rad Laboratories,型号:ChemiDoc触摸成像系统)
  9. Vortex-Genie 2(Scientific Industries,型号:Vortex-Genie 2)
  10. 水浴(Precision Scientific,目录号:66551)
  11. 垂直微型凝胶系统(C.B.S. Scientific,型号:MGV102)
  12. 转移电泳单元(Hoefer,型号:TE22)
  13. EASY-CAST电泳系统(Thermo Fisher Scientific,Thermo Scientific TM,型号:Owl TM Easy-Cast TM B1)
  14. 电源(Bio-Rad Laboratories,型号:PowerPac TM TM Basic)

软件

  1. Image Lab(Bio-Rad Laboratories)

程序

第1天
从平板上挑选单个菌落,接种15ml合成培养基中的细胞过夜培养物。在摇床培养箱中在30°C,170 rpm的条件下培养。根据菌株的生长速度,预先做1-2天。

第2天
将在第1天生长的过夜培养物接种到补充有250nM生物素的110ml合成培养基中。添加生物素以有利于标记的H3蛋白的体内生物素化。接种细胞的量取决于生长速率(见下文;典型地,将大多数X-Y菌株的3个OD单位接种到110ml培养基中,然后培养12小时)。
在30°C,170转/分的摇床培养箱中培养
的第三天

  1. 将H3二聚体与DMS交联
    1. 以期望的细胞密度收获。在OD 600下所需的细胞密度为0.25;为了确保染色体用20μlMNase充分消化以产生大部分单体,在OD 600≤0.3= 0.3每链霉亲和素下拉下不使用超过100ml的细胞,以确保染色体被充分消化。 />
    2. 在4℃下以2,000 em xg 的速度在Swing斗式转子中旋转10分钟10分钟。轻轻地倒掉上清。
    3. 将细胞重悬于0.5ml E缓冲液中4°C并转移至1.5ml O形环螺帽试管中。在4°C下以最大速度离心20秒。去除上清液。
    4. 用1ml E缓冲液通过涡旋洗涤3次。在4°C下以最大速度离心20秒。除去上清液。
      。彻底清洗对于除去会损害交联的含胺化合物非常重要。

    5. 在4°C下,将每管细胞完全重悬于900μlE缓冲液中,并加入0.5 ml玻璃珠
    6. 珠击:在Mini-Beadbeater-96中(脉冲之间5分钟,在冰上),3次1分钟的跳动(2,100rpm)。
    7. 用气体燃烧器加热26号针头(图2A),并用红热针刺穿管底(图2B)。放入12×75mm塑料管('FACS管')(图2C)中,并在4℃下在台式离心机中以365×g旋转2分钟(图2D)。用玻璃珠丢弃螺帽管(图2E)。


      图2.第3天,第A7天的步骤照片

    8. 将沉淀彻底重悬于液体(F缓冲液和FACS管底部的细胞裂解液的混合物)中,并转移至1.5ml微量离心管中。
    9. 在E缓冲液(通常1ml)中室温下使DMS储备11mg / ml。
      每次都应该新鲜制作这种股票。
    10. 删除0分钟的等分,100微升的SDS-PAGE。添加1/10体积100%TCA。在室温下孵育10分钟。在室温下以最大速度在微型离心机中旋转10分钟。去除上清液,在室温下用1ml丙酮洗颗粒。保持盖子打开30分钟以风干颗粒。用50μl2x SB重悬空气干燥的颗粒。在-20°C储存。
    11. 加1/10体积的11mg / ml DMS至最终浓度1mg / ml。在室温下孵育60分钟。
    12. 加入1/20体积的1M Tris-HCl,pH7.5至终浓度为50mM,以淬灭DMS交联并在室温下进一步旋转15分钟。
    13. 取出60分钟的等分试样,100μl用于SDS-PAGE。将1/10体积的100%TCA加入等分试样中,如上所述处理。
    14. 在交联后立即进行下一步(MNase消化)。

  2. MNase消化
    1. 加入1/100体积的1M MgCl 2至最终浓度为10mM(最终确实为8mM,因为E缓冲液含有2mM EDTA),并加入1/100体积的0.1M CaCl 2接着在室温下向DMS-交联的样品(步骤A-13之后的剩余量,大约800μl)添加1mM的终浓度至1mM的终浓度。取100μl产生未消化的DNA样品进行凝胶分析。存放在冰上。

    2. 在37℃水浴中平衡5分钟
    3. 加入20μlMNase,倒置管5次,37°C孵育20分钟。
    4. 加入1/25体积的0.25M EDTA / EGTA至终浓度为10mM,颠倒5-10次以抑制MNase。取100μl产生MNase消化的DNA样品。在8,000×g g / min下旋转1分钟,4℃,取上清液进行抗生蛋白链菌素下拉。

  3. 凝胶分析的MNase消化
    1. 为了进行DNA纯化,将5μlRNase A(10 mg / ml)加入到100μl样品中,37°C孵育30分钟。
    2. 加入5μl20%SDS和2μl蛋白酶K(20mg / ml)。
      在65°C孵育3小时。
    3. 加入200μl7.5M NH 4 Ac和300μlddH 2 O.
    4. 加600μl的PCI和涡旋。
      以最快速度在微型离心机中旋转5分钟
    5. 取水相,用1倍体积的2-丙醇沉淀。

    6. 在异丙醇沉淀后,在室温下以最大速度旋转20分钟
    7. 用1毫升70%乙醇洗沉淀,旋转5分钟,空气干燥1小时。

    8. 用10μlTE重悬沉淀
    9. 加入2μl6x上样缓冲液,并在含有0.5μg/ ml溴化乙锭的1.5%TAE琼脂糖凝胶上以5V / cm运行50分钟。

  4. 链霉抗生物素蛋白 - 下拉
    1. 用E缓冲液在4℃平衡CL2B琼脂糖珠。为了阻断珠子,每个样品添加10μg胰岛素/40μl浆液,在4℃下旋转30分钟。将MNase消化的样品(步骤B4中所述)加入到40μl浆液预封闭的CL2B琼脂糖珠中,在4℃下旋转30-60分钟。
    2. 以8,000×g g离心1分钟,4℃。取30μl上清液+ 30μl2x SB作为'输入'样品。在-20°C储存。
    3. 转移上清到一个新的1.5毫升管。加入预先用胰岛素预先封闭的30μl浆液链霉抗生物素蛋白 - 琼脂糖珠(每个样品使用10微克胰岛素/30μl浆液,如上述方法),在4℃下旋转2小时。
    4. 以8,000×g g离心1分钟,4℃。
      以30μl上清液+ 30μl2x SB作为'Unbound'样本
    5. 在4°C用1 ml W缓冲液洗珠3次,每次4°C旋转5分钟。在4℃下旋转8,000×gg 1分钟,然后丢弃上清液。
    6. 最后一次清洗后,用26号针在真空下除去所有上清液。在50μl2x SB中重悬珠。在-20°C储存。

  5. 蛋白质印迹
    1. 将样品在100°C煮10分钟。
    2. 加载10μl0分钟和60分钟样品,15μl输入样品和8μl结合样品。在恒定8mA的1x SDS-运行缓冲液中的17%SDS-PAGE凝胶上运行,而溴酚蓝通过积层凝胶,当其通过分离凝胶时为18mA。溴酚蓝从底部流出后继续运行15分钟以改善组蛋白的分辨率。总运行时间约为2小时。

    3. 转移到印迹缓冲液中的硝酸纤维素膜恒温0.5分钟。
    4. 在室温下将膜在5%牛奶/ TBST中封闭1小时。
    5. 用抗V5抗体(在5%牛奶/ TBST中1:10,000稀释)在4℃孵育膜过夜。

    6. 每次用5%乳/ TBST洗膜三次,每次10分钟
    7. 用二抗孵育膜(在5%乳/ TBST中抗 - 小鼠IgG 1:15,000稀释)1小时。

    8. 用TBST每次清洗膜三次10分钟
    9. 去除TBST并用1 ml ECL(增强型化学发光)溶液孵育膜10分钟。通过使用ChemiDoc触摸成像系统(Chemiluminescent Blot模式,以最高分辨率)进行检测。

数据分析

用Bio-Rad'Image Lab'软件使用'Volume Tools'量化H3-H3交联物质。乐队的区域是用一个矩形框包围它的(图3)。使用相同的体积面积来测量从带强度中减去的背景信号。用以下公式计算沉淀的H3二聚体的百分比:
总输入='输入线上H3二聚体的带强度'x'输入部分的总体积'/'输入部分的加载体积'
总绑定='绑定车道上H3二聚体的绑定强度'x'绑定分数的总容量'/'绑定分数的装载量'
沉淀的H3二聚体的百分比=(总结合量/总投入量)×100
平均值和标准偏差由3次独立重复实验的值计算。


图3.使用Bio-Rad图像实验室进行数据分析使用'Volume Tools'将H3-H3二聚体带和空白区域用矩形框包围。

笔记

  1. 使用新鲜细胞(在交联之前不要冷冻细胞)。
  2. 使用新鲜的DMS库存。
  3. 请注意,在珠粒打浆过程中,出现可见的聚集体,其中许多残留在DMS交联部分的步骤A7中的带有废弃玻璃珠的螺旋盖管中。这是正常的,并不表示有问题。
  4. 在步骤A11的DMS交联反应期间温育60分钟足以使我们获得交联的H3二聚体。但是,您可以根据您的目的和样品条件更改孵化时间。
  5. PVDF膜可用于蛋白质印迹,但根据我们的经验,硝酸纤维素膜更敏感地检测组蛋白。
  6. 丙烯酰胺是有毒的。
    在制作SDS-PAGE凝胶时戴上检查手套。

食谱

  1. 合成媒体
    没有氨基酸的0.67%酵母氮碱
    2%葡萄糖
    注意:没有调整pH值
  2. MNase
    在10 mM Tris-HCl pH 7.4中20 U /μl
  3. 提取(E)缓冲区
    20 mM硼酸钠
    0.35M NaCl
    2 mM EDTA
    用HCl调节pH至9.00 1毫米PMSF(新鲜加入)
  4. 2x SDS样品缓冲液(SB)
    0.1M Tris-HCl pH 6.8
    2%SDS
    20%甘油
    0.02%溴酚蓝
    1/50体积的2-巯基乙醇
  5. 洗(W)缓冲液
    10 mM Tris-HCl pH 8.0
    1 mM EDTA
    2 M NaCl
    0.2%吐温20
  6. 17%SDS-PAGE凝胶(0.75mm厚,14孔梳)
    2.13毫升40%丙烯酰胺
    0.18毫升2%双丙烯酰胺
    1.875ml 1.0M Tris-HCl pH8.8
    25μl20%SDS
    0.78毫升的H 2 O 20μl10%过硫酸铵
    5μlTEMED
  7. 5%浓缩胶
    0.31毫升40%丙烯酰胺
    0.18毫升2%双丙烯酰胺
    0.31ml 1.0M Tris-HCl pH 6.8
    12.5μl的20%SDS
    1.69ml的H 2 O 2 10μl10%过硫酸铵
    5μlTEMED
  8. 1x SDS运行缓冲区
    25 mM Tris
    192 mM甘氨酸
    0.1%SDS
  9. 40倍碳酸钠缓冲液
    251 mM NaHCO 3 3 173 mM Na 2 CO 3 3/2 用NaOH调节pH至9.5
  10. 印迹缓冲液
    1x碳酸钠缓冲液
    20%甲醇
  11. TBST
    25 mM Tris-HCl pH 8.0
    137mM NaCl
    2.68mM KCl
    0.1%吐温20

致谢

这项工作得到了美国国立卫生研究院拨款R01GM100164的支持。作者没有利益冲突或利益冲突。

参考

  1. Bartley,J.A。和Chalkley,R。(1972)。 脱氧核糖核酸与组蛋白在天然核蛋白组合中的结合 J Biol Chem 247(11):3647-3655。
  2. Beckett,D.,Kovaleva,E。和Schatz,P.J。(1999)。 生物素全酶合成酶催化生物素化中的最小肽底物。 Protein Sci 8(4):921-929。
  3. Ichikawa,Y.,Connelly,CF,Appleboim,A.,Miller,TC,Jacobi,H.,Abshiru,NA,Chou,HJ,Chen,Y.,Sharma,U.,Zheng,Y.,Thomas,PM, Chen,HV,Bajaj,V.,Muller,CW,Kelleher,NL,Friedman,N.,Bolon,DN,Rando,OJ和Kaufman,PD(2017)。 探索核小体对称性的合成生物学方法 Elife 6.
  4. Kornberg,R.D。和Thomas,J.O。(1974)。 染色质结构;组蛋白的低聚物。 184(4139):865-868。
  5. Shema,E.,Jones,D.,Shoresh,N.,Donohue,L.,Ram,O。和Bernstein,B.E。(2016)。 单分子解码组合修饰核小体 科学 352(6286):717-721。
  6. Thomas,J.O.(1989)。 组蛋白的化学交联方法Enzymol 170 :549-571。
  7. Voigt,P.,LeRoy,G.,Drury,W.J.,3rd,Zee,B.M.,Son,J.,Beck,D.B。,Young,N.L.,Garcia,B.A.and Reinberg,D。(2012)。 不对称修饰的核小体。 Cell 151(1): 181-193。
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Copyright Ichikawa and Kaufman. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ichikawa, Y. and Kaufman, P. D. (2018). Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes. Bio-protocol 8(6): e2770. DOI: 10.21769/BioProtoc.2770.
  2. Ichikawa, Y., Connelly, C. F., Appleboim, A., Miller, T. C., Jacobi, H., Abshiru, N. A., Chou, H. J., Chen, Y., Sharma, U., Zheng, Y., Thomas, P. M., Chen, H. V., Bajaj, V., Muller, C. W., Kelleher, N. L., Friedman, N., Bolon, D. N., Rando, O. J. and Kaufman, P. D. (2017). A synthetic biology approach to probing nucleosome symmetry. Elife 6.
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