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In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease
体外组蛋白H3裂解分析法测定酵母和鸡肝H3蛋白酶的活性   

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本实验方案简略版
Yeast
Jun 2016

Abstract

Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.

Keywords: Histone H3 (组蛋白H3), Chicken liver H3 protease (鸡肝H3蛋白酶), Yeast H3 protease (酵母H3蛋白酶), Histone clipping (组蛋白剪切), Chromatin (染色质)

Background

Histone H3 clipping is the least understood mechanism of chromatin modification and regulation. It is expected that H3 clipping will permanently erase PTMs from the nucleosomes that might affect chromatin related events. Moreover, the fate of cleaved histones is still under investigation and it has been suggested that the cleaved histones might be recycled at specific regions of chromatin or they are targeted for degradation. There are various reports that describe in vivo clipping of histone H3 in different organisms, while in vitro assays for histone H3-specific clipping are limited. We need an efficient and robust in vitro assay for characterizing histone specific proteases. To this end, we present a protocol that can be used to examine the in vitro histone H3 clipping activity of yeast and chicken liver histone H3 proteases. We have optimized temperature and pH conditions for the assay. Under our optimized conditions, proteases were found to specifically cleave histone H3 out of all core histones. We have extensively used this protocol in our recent publications (Chauhan et al., 2016; Chauhan and Tomar, 2016; Azad and Tomar, 2016; Mandal et al., 2014; Mandal et al., 2013; Mandal et al., 2012). This protocol can be used to identify and characterize histone H3 specific proteases from different organisms ranging from yeast to mammals.

Materials and Reagents

  1. 1.5 ml centrifuge tubes (Tarsons)
  2. Dialysis tubing cellulose membrane (Sigma-Aldrich, catalog number: D9277 )
  3. Amicon ultra centrifugal filters (EMD Millipore, catalog number: UFC900396 )
  4. Cheese cloth
  5. Freshly extracted or frozen chicken brain tissue
  6. Saccharomyces cerevisiae yeast cells (BY4743 strain)
  7. Freshly extracted liver tissue from chicken
  8. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  9. Sucrose (Sigma-Aldrich, catalog number: S7903 )
  10. Sodium chloride (NaCl) (EMD Millipore, catalog number: 567440 )
  11. Acetone (HiMedia Laboratories, catalog number: AS024 )
  12. Lyticase (Sigma-Aldrich, catalog number: L4025 )
  13. Protein A sepharose bead (GE Healthcare, catalog number: 17-0963-03 )
  14. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
  15. Ammonium sulfate [(NH4)2SO4] (Sigma-Aldrich, catalog number: A2939 )
  16. Coomassie Brilliant Blue R (Sigma-Aldrich, catalog number: B0149 )
  17. Potassium chloride (KCl) (EMD Millipore, catalog number: 104936 )
  18. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  19. Spermine (Sigma-Aldrich, catalog number: 85590 )
  20. EDTA (Sigma-Aldrich, catalog number: EDS )
  21. EGTA (Sigma-Aldrich, catalog number: E3889 )
  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  23. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 7626 )
  24. Disodium hydrogen phosphate (EMD Millipore, catalog number: 106586 )
  25. Sodium dihydrogen phosphate (EMD Millipore, catalog number: 106370 )
  26. HEPES (Sigma-Aldrich, catalog number: H3375 )
  27. Glycerol (EMD Millipore, catalog number: 104093 )
  28. Protease inhibitor cocktail (PIC) (Sigma-Aldrich, catalog number: P2714 )
  29. Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
  30. DTT (Sigma-Aldrich, catalog number: D9779 )
  31. Sorbitol (Sigma-Aldrich, catalog number: S6021 )
  32. Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: 63064 )
  33. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  34. Trizma base (Sigma-Aldrich, catalog number: T6066 )
  35. Hydroxyapatite resin (Sigma-Aldrich, catalog number: 289396 )
  36. Sulfuric acid (H2SO4) (EMD Millipore, catalog number: 112080 )
  37. Acrylamide (Sigma-Aldrich, catalog number: A8887 )
  38. GLUD1 antibody (Sigma-Aldrich, catalog number: SAB2100932-50UG )
    Note: This Product has been discontinued.
  39. Solution 1 (see Recipes)
  40. Hypotonic solution (see Recipes)
  41. Sodium phosphate buffer (1 M), pH 6.8 (see Recipes)
  42. HB buffer (see Recipes)
  43. HAP buffer (see Recipes)
  44. Potassium acetate solution (see Recipes)
  45. Prespheroplasting buffer (see Recipes)
  46. Spheroplasting buffer (see Recipes)
  47. Wash buffer (see Recipes)
  48. Lysis buffer (see Recipes)
  49. Protein A sepharose beads (50% slurry) (see Recipes)
  50. Yeast-protease resuspension buffer (see Recipes)
  51. Protease dialysis buffer (see Recipes)
  52. Reaction buffer
    1. Yeast protease reaction buffer (see Recipes)
    2. Chicken liver protease reaction buffer (see Recipes)
  53. 10% Triton X-100 solution (see Recipes)
  54. 6x SDS-PAGE loading dye (see Recipes)
  55. 10x SDS-PAGE running buffer (see Recipes)

Equipment

  1. Homogenizer (Pro Scientific, model: Bio-Gen PRO200 homogenizer )
  2. Potter-Elvehjem PTFE pestle and glass tube (Sigma-Aldrich, catalog number: P7734 )
  3. Centrifuge (Beckman Coulter, models: Avanti® J-E centrifuge-floor , Microfuge® 22R centrifuge )
  4. Sonicator (Boston Industries, model: Branson Digital Sonifier 250 & 102C Converter Ultrasonic Dismembrator )
    Note: This product has been sold out.
  5. Ultracentrifuge (Beckman Coulter, model: OptimaTM L-100K ultracentrifuge )
  6. Bio-Rad electrophoresis power supply (Bio-Rad Laboratories, model: PowerPacTM Basic Power Supply )
  7. Bio-Rad SDS-PAGE running apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN tetra cell )
  8. Shaker (Eppendorf, model: New BrunswickTM Innova® 44 )
  9. Rotamer (Tarsons, catalog number: 3090 )
  10. Heating block, Thermocell Cooling and heating Block (Bio-Equip, model: HB-202 )
  11. FPLC cold cabinet (UniEquip, model: Unichromat 1500 )
  12. SuperoseTM 6, 10/300 GL chromatography column (GE Healthcare, catalog number: 17-5172-01 )

Procedure

  1. Preparation of core histones
    1. For protease assay, good quality of histones is required. We purified core histones from chicken brain tissue. To isolate histones from tissue requires preparation of nuclei as detailed below.
      1. Homogenize the chicken brain tissue (5 g) via a motor-driven homogenizer in solution 1 (50 ml) for 30 sec to make a 10% homogenate solution.
      2. Add 1.25 ml 10 % Triton X-100 drop-wise with constant shaking (swirling with hand) to the homogenate to a final concentration of 0.25%.
      3. Centrifuge the sample at 4 °C, 3,024 x g for 20 min and discard the supernatant to obtain nuclei pellet.
      4. Wash nuclei pellets (at 4 °C, 3,024 x g for 20 min) 3 times with 50 ml chilled solution 1 containing 0.34 M sucrose.
      5. Suspend the nuclei thoroughly into the 7 ml hypotonic buffer at a concentration of 1 mg DNA/ml. Check DNA concentration by measuring A280.
    2. Purify core histones from isolated nuclei by hydroxyapatite affinity purification (HAP) method as described below:
      1. Sonicate nuclei in 7 ml hypotonic solution to prepare soluble chromatin.
        Note: We have observed that soluble chromatin preparation is fine when made as described above but this step can be improved. To improve soluble chromatin preparation, first dissolve nuclei in 7 ml HB buffer and apply 5 strokes by Potter-Elvehjem homogenizer. Centrifuge (27,216 x g, 4 °C for 5 min) the sample to collect nuclei. Repeat the same step (resuspension in HB buffer, homogenization, centrifugation and discard the supernatant) 6 times in total.
      2. Further, dissolved nuclei in 7 ml HAP buffer containing 0.4 M NaCl and apply 5 strokes by hand homogenizer. Collect nuclei by centrifugation at 27,216 x g, 4 °C for 5 min. Repeat this step 2 times.
      3. Resuspend nuclei pellet in HAP buffer containing 0.6 M NaCl and sonicate (60% amplitude, 4 min-10 sec on, 10 sec at 4 °C) off it. These steps are adapted from Côté et al., 1995; Workman et al., 1991.
      4. Mix 5 g of hydroxyapatite resin (1.5 mg DNA/g hydroxyapatite) with 50 ml of 50 mM sodium phosphate buffer, pH 6.8. Keep it at room temperature for 10 min and centrifuge it (3,024 x g, 5 min). Discard the supernatant. Repeat it for two more times.
        Note: Alternatively, equilibration of hydroxyapatite resin can be done with HAP buffer containing 0.6 M NaCl.
      5. Mix soluble chromatin (1 mg/ml) with hydroxyapatite resin equilibrated in 50 mM sodium phosphate buffer, pH 6.8 (1.5 mg DNA/g hydroxyapatite).
      6. Add NaCl (5 M) to the final concentration of 0.6 M and incubate chromatin-HAP resin for half an hour at 4 °C with 10 rotations per minute on rotator.
      7. Wash chromatin mixed hydroxyapatite with 50 ml 50 mM phosphate buffer containing 0.6 M NaCl. Perform washing through centrifugation (3,024 x g, 5 min). Discard the supernatant. Repeat it 6 times.
      8. Elute total histones with 15 ml phosphate buffer containing 2.0 M NaCl. Perform the elution three times.
        Note: Alternatively, we can elute histones with 15 ml HAP buffer containing 2.5 M NaCl. Perform the elution three times.
      9. Desalt the eluted histones by dialysis against 1,000 ml of 10 mM Tris-Cl (pH 7.5) overnight at 4 °C with 4 changes.
        Note: Alternatively, we can remove salt by spin filter and concentrate core histones, which can be used directly for assays.
      10. Precipitate histones by addition of 3.5 volumes of chilled acetone. After adding acetone, keep the solution for precipitation at -20 °C for 1 h.
        Collect the precipitated histones by centrifugation at 4 °C, 12,096 x g for 15 min and wash 2-3 times with chilled acetone.
        Air-dry the pellet for 15 min and dissolve it in 500 μl 10 mM Tris-Cl (pH 7.5).
        Check purity of core histones by running 18% SDS PAGE as shown in Figure1.


        Figure 1. Purification of core histones. Core histones were purified from chicken brain tissue through hydroxyapatite resin and the purity was checked by running 18% SDS-PAGE. 2 μg core histones were loaded onto a lane.

  2. Purification of H3 protease
    1. Partial purification of H3-protease from Saccharomyces cerevisiae
      1. Make extract from sporulation-phase yeast cells (BY4743 strain) as it yields maximum H3-protease activity.
      2. Harvest 100 ml yeast cells growing in exponential phase (OD600-0.8) by centrifugation (2,300 x g, 5 min).
      3. Discard supernatant. Suspend yeast cell pellet in 2% (w/v) potassium acetate solution (50 ml).
      4. Allow the cells to grow further for 3 h at 30 °C with constant shaking (200 rpm). Under these condition, the yeast cells reach sporulation phase.
      5. Harvest yeast cells grown to sporulation-phase by centrifugation (2,300 x g, 5 min). Transfer the harvested cells into 1.5 ml centrifuge tubes. Wash the cell pellet with 2 ml of prespheroplasting buffer and discard the supernatant.
      6. Incubate the yeast cells in 0.5 ml spheroplasting buffer containing lyticase (250 U) at 37 °C for 15 min. Prepare the spheroplasts through lyticase enzymatic degradation of the yeast cell walls.
      7. Harvest the spheroplasts by centrifugation at low speed (3,300 x g) for 3 min at 4 °C.
      8. Discard the supernatant and wash the pellet once with 1 ml ice-cold wash buffer.
      9. Lyse the spheroplasts with 0.8 ml lysis buffer through incubation on ice for 15 min. Mix the cells by hand tapping at regular intervals.
      10. Centrifuge the cell lysates at 13,400 x g for 15 min at 4 °C to obtain clear lysates and transfer it into a new 1.5 ml centrifuge tube. Discard the pellet.
      11. To acquire H3-specific proteolytic activity, incubate the cell lysates with 100 µl of protein A sepharose beads (50% slurry) for 1 h at 4 °C with constant shaking on a rotator.
      12. Centrifuge (800 x g, 5 min, 4 °C) the bead mixed cell lysate and discard the supernatant.
      13. Similarly, wash the beads two times with 1 ml of lysis buffer (by reversing the microcentrifuge tube by hand, settle beads by centrifugation and throw the supernatant ) followed by washing twice with lysis buffer without Triton X-100.
      14. Resuspend the beads in yeast-protease resuspension buffer (100 µl) and use it for in vitro H3 cleavage assays.
    2. Partial purification of H3-protease from chicken liver
      1. Extract liver tissue from chicken and wash it with saline solution.
      2. Prepare the microsomes as described earlier (Grillo et al., 2002). Briefly, the method to purify microsomes from chicken liver tissue is described below.
      3. Homogenize 5 g chicken liver tissue via a motor-driven homogenizer in 50 ml of chilled solution 1 to make 10% homogenate and filter it through four layered cheese cloth.
      4. Remove nuclei and mitochondrial contaminations from homogenate subsequently by centrifugation at 5,000 x g, 4 °C for 10 min and 27,000 x g, 4 °C for 10 min respectively.
      5. Centrifuge the collected supernatant at 105,000 x g, 4 °C for 1 h to pellet down the microsomes.
      6. Resuspend the collected microsomes in 5 ml of 10 mM Tris-Cl, pH 8.0. Extract the resuspended microsomes with 0.2% Triton X-100 for 1 h at 4 °C by continuous rotation (rotator, 10 rpm).
      7. Centrifuge the extracted microsomes at 105,000 x g, 4 °C for 1 h. This time collect the supernatant and further precipitate it with 30% ammonium sulfate for 30 min at 4 °C.
      8. Collect the precipitates by centrifugation (27,216 x g, 4 °C, 30 min) and dialyze it over-night, against 2,000 ml dialysis buffer at 4 °C to remove ammonium sulfate.
      9. After dialysis, perform the centrifugation (27,216 x g, 4 °C, 15 min) to remove insoluble precipitates.
      10. To further purify H3 protease, perform SuperoseTM 6, 10/300 GL chromatography (Sample volume-500 μl, flow rate-0.3 ml/min, fraction volume-0.3 ml). Buffer for size exclusion chromatography is same as dialysis buffer. Collect positive fractions ranging from 39th to 51st as shown in Figure 2.
      11. Pool fractions (39th to 51st) together and use them directly (no need to concentrate these fractions for experiments) for in vitro histone H3 cleavage assay.


        Figure 2. Western blot of liver microsomal extract with Glud1 antibody. Size exclusion chromatography fractions of chicken liver microsomal extract probed with Glud1 (Chicken liver H3 protease) antibody to confirm the presence of H3 protease in these fractions.

  3. Set up of in vitro histone H3 cleavage assay
    1. Protease source: yeast (BY4743 strain)
      1. Perform in vitro protease assay in reaction buffer (20 µl).
      2. Initiate assay by addition of 10 μl protein A sepharose bead-bound proteins into reaction mix containing 1-2 μg core histones.
      3. Incubate the reaction mix for 1 h at 37 °C, with constant shaking.
      4. Stop the in vitro cleavage reaction by addition of 3.33 μl 6x SDS-PAGE sample loading buffer into the reaction mixture (final concentration of SDS-loading dye in reaction mix: 1x).
    2. Protease source: chicken
      1. Perform the in vitro proteolytic assay in reaction buffer.
      2. Incubate 2 μg core histones with 0.25 μg of partially purified H3 protease extract from chicken liver tissue in 20 μl reaction volume.
      3. Incubate the reaction mix for 1 h at 37 °C.
      4. Stop the reaction by addition of 3.33 μl 6x SDS-PAGE sample loading buffer.

  4. Visualization of core histones after assay
    1. Stop the in vitro cleavage reaction by the addition of SDS-PAGE loading dye as mentioned earlier.
    2. Incubate the samples at 95 °C for 5 min.
    3. Examine the cleavage of histone H3 by running 18% SDS-PAGE as shown in Figure 3.
    4. Visualize the bands of core histones by staining with Coomassie blue staining.


      Figure 3. In vitro histone H3 cleavage assay with core histones. A. In vitro activity assay with yeast protease. B. In vitro activity assay with chicken liver protease. Out of four core histones, only histone H3 get cleaved as protease is specific for histone H3. ‘-’ and ‘+’ indicate absence and presence of H3 protease respectively.
      Note: The in vitro histone H3 cleavage assay works best when proteases are freshly prepared. The core histone should be stored in small aliquots at -80 °C. Repeated freeze-thawing of both histones and proteases should be avoided.

Recipes

  1. Solution 1
    0.34 M sucrose
    15 mM Tris-Cl, pH 7.5
    15 mM NaCl
    60 mM KCl
    0.5 mM spermidine
    0.15 mM spermine
    2 mM EDTA
    0.5 mM EGTA
    15 mM β-mercaptoethanol
    0.2 mM PMSF (added before use)
  2. Hypotonic solution
    10 mM Tris-Cl, pH 7.5
    15 mM β-mercaptoethanol
    2 mM PMSF
  3. Sodium phosphate buffer (1 M), pH 6.8 (100 ml)
    49 ml of 1 M disodium hydrogen phosphate
    51 ml of sodium dihydrogen phosphate
  4. HB buffer
    20 mM HEPES, pH 7.5
    0.4 M NaCl
    1 mM EDTA
    10% glycerol
    0.1% NP-40
    1 mM β-ME
    PIC (Protease inhibitor cocktail) (added before use)
  5. HAP buffer
    50 mM sodium phosphate buffer
    1 mM β-ME
    PIC (Protease inhibitor cocktail) (added before use)
  6. Potassium acetate solution
    2 g potassium acetate dissolved in 100 ml water
  7. Prespheroplasting buffer
    100 mM Tris-Cl, pH 8.8
    10 mM DTT
  8. Spheroplasting buffer
    50 mM Tris-Cl, pH 7.5
    0.6 M sorbitol
    10 mM DTT
  9. Wash buffer
    100 mM KCl
    50 mM HEPES-KOH, pH 7.5
    2.5 mM MgCl2
    0.4 M sorbitol
  10. Lysis buffer
    20 mM Tris-Cl, pH 8.5
    200 mM KCl
    25 mM EDTA
    1% (v/v) Triton X-100
  11. Protein A sepharose beads (50% slurry)
    Take 500 µl protein A sepharose beads in 1.5 ml Eppendorf tube
    Centrifuge it at 4 °C, 800 x g for 5 min
    Discard supernatant
    Add of 1 ml of lysis buffer to beads
    Centrifuge it again and discard the supernatant
    Repeat it two more time
    Finally, resuspend the bead pellet in 500 µl of lysis buffer to obtain 50% slurry
  12. Yeast-protease resuspension buffer
    20 mM Tris-Cl, pH 8.5
    200 mM KCl
    25 mM EDTA
  13. Protease dialysis buffer
    25 mM Tris-Cl, pH 7.5
    100 mM NaCl
    10% glycerol
    1 mM β-mercaptoethanol
    0.2 mM EDTA
  14. Reaction buffer
    1. Yeast protease reaction buffer
      25 mM Tris-Cl, pH 7.5
      150 mM NaCl
      10% glycerol
      2 mM β-mercaptoethanol
      0.1 mM EDTA
    2. Chicken liver protease reaction buffer
      10 mM HEPES, pH 5.5
      100 mM NaCl
      1 mM β-mercaptoethanol
      0.2 mM EDTA
      10% glycerol
  15. 10% Triton X-100 solution
    10 ml of Triton X-100 from stock (100%)
    90 ml water
    Solution mixed well through rotation
    10% solution
    Stored at 4 °C
  16. 6x SDS-PAGE loading dye
    2.5 ml of 4x Tris-Cl, pH 6.8
    3 ml glycerol
    1 g SDS, 0.5 ml
    1.2 mg bromophenol blue
    Add water to make final volume to 10 ml
  17. 10x SDS-PAGE running buffer
    30.0 g of Trizma base
    10.0 g of SDS
    144.0 g of glycine
    Dissolve in 1,000 ml of H2O
    Dilute 10x buffer to 1x before use

Acknowledgments

This work was supported by the Department of Biotechnology (Government of India), Department of Science and Technology (Government of India) to RST. Short version of this protocol was published in Chauhan et al., 2016, Chauhan and Tomar, 2016; Azad and Tomar, 2016; Mandal et al., 2014; Mandal et al., 2013; Mandal et al., 2012. CSIR is acknowledged for providing fellowship support to Sakshi. Authors also acknowledge members of RST lab for their comments on this work.

References

  1. Azad, G. K. and Tomar, R. S. (2014). Proteolytic clipping of histone tails: the emerging role of histone proteases in regulation of various biological processes. Mol Biol Rep 41(5): 2717-2730.
  2. Azad, G. K. and Tomar, R. S. (2016). Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae. Yeast 33(6): 217-226.
  3. Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res 21(3): 381-395.
  4. Chauhan, S., Mandal, P. and Tomar, R. S. (2016). Biochemical analysis reveals the multifactorial mechanism of histone H3 clipping by chicken liver histone H3 protease. Biochemistry 55(38): 5464-5482.
  5. Chauhan, S. and Tomar, R. S. (2016). Efficient expression and purification of biologically active human cystatin proteins. Protein Expr Purif 118: 10-17.
  6. Côté, J., Utley, R. T., and Workman, J. L. (1995). Basic analysis of transcription factor binding to nucleosomes. Methods Mol Genet 6:108-128.
  7. Grillo, C., Coppari, S., Turano, C. and Altieri, F. (2002). The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain. Biochem Biophys Res Commun 295(1): 67-73.
  8. Mandal, P., Azad, G. K. and Tomar, R. S. (2012). Identification of a novel histone H3 specific protease activity in nuclei of chicken liver. Biochem Biophys Res Commun 421(2): 261-267.
  9. Mandal, P., Chauhan, S. and Tomar, R. S. (2014). H3 clipping activity of glutamate dehydrogenase is regulated by stefin B and chromatin structure. FEBS J 281(23): 5292-5308.
  10. Mandal, P., Verma, N., Chauhan, S. and Tomar, R. S. (2013). Unexpected histone H3 tail-clipping activity of glutamate dehydrogenase. J Biol Chem 288(26): 18743-18757.
  11. Workman, J. L., Taylor, I. C., Kingston, R. E. and Roeder, R. G. (1991). Control of class II gene transcription during in vitro nucleosome assembly. Methods Cell Biol 35: 419-447.

简介

组蛋白受到广泛的可逆和不可逆的翻译后修饰(PTM)(Bannister和Kouzarides,2011; Azad和Tomar,2014)。已知组蛋白上的PTM调节染色质结构和功能。组蛋白不可逆地修饰其尾部结构域的蛋白水解剪切。组蛋白尾巴的蛋白水解剪切不断吸引研究人员在染色质生物学领域的兴趣。我们可以通过在体外实施H3切割测定来概括H3-剪切。在这里,我们提供了详细的方案来进行体外实验。

背景 组蛋白H3剪切是染色质修饰和调节最不了解的机制。预期H3剪切将永久性消除可能影响染色质相关事件的核小体的PTM。此外,切割的组蛋白的命运仍在研究之中,并且已经表明,切割的组蛋白可能在染色质的特定区域被再循环,或者它们被靶向降解。有各种各样的报告描述了不同生物中组蛋白H3的体内剪切,而组蛋白H3特异性剪切的体外测定是有限的。我们需要一种有效和稳健的体外实验来鉴定组蛋白特异性蛋白酶。为此,我们提出了一个可用于检查酵母和鸡肝组织蛋白H3蛋白酶的体外组蛋白H3剪切活性的方案。我们已经优化了测定的温度和pH条件。在我们优化的条件下,发现蛋白酶在所有核心组蛋白中特异性切割组蛋白H3。我们在最近的出版物(Chauhan等人,2016年; Chauhan和Tomar,2016年; Azad和Tomar,2016; Mandal等人,2014年)中广泛使用了这一协议。 ; Mandal等人,2013; Mandal等人,2012)。该方案可用于鉴定和表征来自酵母和哺乳动物的不同生物体的组蛋白H3特异性蛋白酶。

关键字:组蛋白H3, 鸡肝H3蛋白酶, 酵母H3蛋白酶, 组蛋白剪切, 染色质

材料和试剂

  1. 1.5ml离心管(Tarsons)
  2. 透析管纤维素膜(Sigma-Aldrich,目录号:D9277)
  3. Amicon超离心过滤器(EMD Millipore,目录号:UFC900396)
  4. 奶酪布
  5. 新鲜提取或冷冻鸡脑组织
  6. 酿酒酵母酵母细胞(BY4743菌株)
  7. 来自鸡肉的新鲜提取的肝组织
  8. Triton X-100(Sigma-Aldrich,目录号:T8787)
  9. 蔗糖(Sigma-Aldrich,目录号:S7903)
  10. 氯化钠(NaCl)(EMD Millipore,目录号:567440)
  11. 丙酮(HiMedia Laboratories,目录号:AS024)
  12. 溶胶酶(Sigma-Aldrich,目录号:L4025)
  13. 蛋白A琼脂糖珠(GE Healthcare,目录号:17-0963-03)
  14. 每硫酸铵(Sigma-Aldrich,目录号:A3678)
  15. 硫酸铵[(NH 4)2 SO 4](Sigma-Aldrich,目录号:A2939)
  16. 考马斯亮蓝R(Sigma-Aldrich,目录号:B0149)
  17. 氯化钾(KCl)(EMD Millipore,目录号:104936)
  18. 亚精胺(Sigma-Aldrich,目录号:S2626)
  19. 精胺(Sigma-Aldrich,目录号:85590)
  20. EDTA(Sigma-Aldrich,目录号:EDS)
  21. EGTA(Sigma-Aldrich,目录号:E3889)
  22. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  23. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:7626)
  24. 磷酸氢二钠(EMD Millipore,目录号:106586)
  25. 磷酸二氢钠(EMD Millipore,目录号:106370)
  26. HEPES(Sigma-Aldrich,目录号:H3375)
  27. 甘油(EMD Millipore,目录编号:104093)
  28. 蛋白酶抑制剂混合物(PIC)(Sigma-Aldrich,目录号:P2714)
  29. 醋酸钾(Sigma-Aldrich,目录号:P1190)
  30. DTT(Sigma-Aldrich,目录号:D9779)
  31. 山梨醇(Sigma-Aldrich,目录号:S6021)
  32. 氯化镁六水合物(MgCl 2)(Sigma-Aldrich,目录号:63064)
  33. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  34. Trizma碱(Sigma-Aldrich,目录号:T6066)
  35. 羟基磷灰石树脂(Sigma-Aldrich,目录号:289396)
  36. 硫酸(H 2 SO 3 SO 4)(EMD Millipore,目录号:112080)
  37. 丙烯酰胺(Sigma-Aldrich,目录号:A8887)
  38. GLUD1抗体(Sigma-Aldrich,目录号:SAB2100932-50UG)
    注意:本产品已停产。
  39. 解决方案1(见配方)
  40. 低音解决方案(请参阅食谱)
  41. 磷酸钠缓冲液(1M),pH 6.8(参见食谱)
  42. HB缓冲(见配方)
  43. HAP缓冲区(见配方)
  44. 醋酸钾溶液(见配方)
  45. 前置放电缓冲液(参见食谱)
  46. 旋转缓冲液(见配方)
  47. 洗涤缓冲液(见配方)
  48. 裂解缓冲液(见配方)
  49. 蛋白A琼脂糖珠(50%浆)(参见食谱)
  50. 酵母蛋白酶再悬浮缓冲液(参见食谱)
  51. 蛋白酶透析缓冲液(见配方)
  52. 反应缓冲液
    1. 酵母蛋白酶反应缓冲液(参见食谱)
    2. 鸡肝蛋白酶反应缓冲液(参见食谱)
  53. 10%Triton X-100溶液(参见食谱)
  54. 6x SDS-PAGE加载染料(见配方)
  55. 10x SDS-PAGE运行缓冲液(参见食谱)

设备

  1. 均质器(Pro Scientific,型号:Bio-Gen PRO200均质器)
  2. Potter-Elvehjem PTFE杵和玻璃管(Sigma-Aldrich,目录号:P7734)
  3. 离心机(Beckman Coulter,型号:Avanti J-E离心机底盘,Microfuge ®/22R离心机)
  4. 超声波发生器(Boston Industries,型号:Branson Digital Sonifier 250& 102C Converter Ultrasonic Dismemberator)
    注意:本产品已售完。
  5. 超速离心机(Beckman Coulter,型号:Optima TM L/100K超速离心机)
  6. Bio-Rad电泳电源(Bio-Rad Laboratories,型号:PowerPac TM 基本电源)
  7. Bio-Rad SDS-PAGE运行装置(Bio-Rad Laboratories,型号:Mini-PROTEAN tetra cell)
  8. 振荡器(Eppendorf,型号:New Brunswick TM Innova ® 44)
  9. Rotamer(Tarsons,目录号:3090)
  10. 加热块,Thermocell冷却和加热块(Bio-Equip,型号:HB-202)
  11. FPLC冷柜(UniEquip,型号:Unichromat 1500)
  12. Superose TM 6/10/300 GL色谱柱(GE Healthcare,目录号:17-5172-01)

程序

  1. 核心组蛋白的制备
    1. 对于蛋白酶测定,需要良好的组蛋白质量。我们从鸡脑组织中纯化了核心组蛋白。要从组织中分离组蛋白需要准备如下所述的细胞核。
      1. 通过马达驱动的均化器在溶液1(50ml)中将鸡脑组织(5g)均化30秒,以制备10%匀浆溶液。
      2. 将恒定摇动(用手旋动)逐滴加入1.25ml 10%Triton X-100至匀浆至0.25%的终浓度。
      3. 将样品在4℃,3024×g离心20分钟,弃去上清液,得到核沉淀。
      4. 用含有0.34M蔗糖的50ml冷冻溶液1洗涤细胞核(4℃,3024×g×20分钟)3次。
      5. 以1毫克DNA /毫升的浓度将细胞核充分悬浮于7毫升低渗缓冲液中。通过测量A280检查DNA浓度。
    2. 通过如下所述的羟基磷灰石亲和纯化(HAP)方法从分离的核中纯化核心组蛋白:
      1. 在7ml低渗溶液中超声处理细胞核以制备可溶性染色质。
        注意:我们已经观察到,如上所述制备可溶性染色质制剂时可以很好,但可以改善这一步骤。为了改善可溶性染色质制备,首先将细胞核溶解在7 ml HB缓冲液中,并用Potter-Elvehjem匀浆器涂5次。离心机(27,216×g,4℃,5分钟)样品收集细胞核。重复相同的步骤(HB缓冲液中的再悬浮液,匀浆,离心并丢弃上清液)总共6次。
      2. 此外,将溶解的核溶解在含有0.4M NaCl的7ml HAP缓冲液中,并用手均化器施加5次。通过在27,216×g,4℃离心5分钟收集细胞核。重复此步骤2次。
      3. 将核沉淀重悬于含有0.6M NaCl和超声波(60%振幅,4分-10秒,4℃10秒)的HAP缓冲液中。这些步骤是从Côté等人改编的,1995; Workman等人,1991。
      4. 将5g羟基磷灰石树脂(1.5mg DNA/g羟基磷灰石)与50ml 50mM磷酸钠缓冲液(pH6.8)混合。将其在室温下保持10分钟并离心(3,024×g,5分钟)。丢弃上清液。再重复两次。
        注意:或者,羟磷灰石树脂的平衡可以用含有0.6M NaCl的HAP缓冲液进行。
      5. 将可溶性染色质(1mg/ml)与在50mM磷酸钠缓冲液(pH6.8)(1.5mg DNA/g羟基磷灰石)中平衡的羟基磷灰石树脂混合。
      6. 加入NaCl(5 M)至最终浓度为0.6 M,并在4℃下将染色质-HAP树脂在旋转器上以每分钟10转的速度孵育半小时。
      7. 用含有0.6M NaCl的50ml 50mM磷酸盐缓冲液洗涤染色质混合羟基磷灰石。通过离心(3,024×g,5分钟)进行洗涤。丢弃上清液。重复6次。
      8. 用含有2.0M NaCl的15ml磷酸盐缓冲液洗脱总组蛋白。进行三次洗脱。
        注意:或者,我们可以用含有2.5M NaCl的15ml HAP缓冲液洗脱组蛋白。进行三次洗脱。
      9. 通过透析对1000毫升10mM Tris-Cl(pH7.5)进行透析,在4℃下4次变化,使洗脱的组蛋白脱盐。
        注意:或者,我们可以通过旋转过滤器去除盐,并浓缩核心组蛋白,可直接用于测定。
      10. 通过加入3.5体积的冷冻丙酮沉淀组蛋白。加入丙酮后,保持溶液在-20℃下沉淀1小时 通过在4℃,12,096×g离心15分钟收集沉淀的组蛋白,并用冷却的丙酮洗涤2-3次。
        将沉淀物干燥15分钟,并将其溶于500μl10mM Tris-Cl(pH7.5)中。
        通过运行18%SDS PAGE检查核心组蛋白的纯度,如图1所示

        图1.核心组蛋白的纯化。核酸组蛋白通过羟基磷灰石树脂从鸡脑组织中纯化,并通过运行18%SDS-PAGE检查纯度。将2μg核心组蛋白加载到泳道上
  2. H3蛋白酶的纯化
    1. 从酿酒酵母中部分纯化H3-蛋白酶
      1. 从孢子形成酵母细胞(BY4743菌株)中提取,因为它产生最大的H3-蛋白酶活性。
      2. 通过离心(2,300×g,5分钟)收获以指数期生长的100ml酵母细胞(OD 600,-0.8)。
      3. 丢弃上清液。将酵母细胞沉淀悬浮于2%(w/v)乙酸钾溶液(50ml)中。
      4. 允许细胞在30℃下持续摇动(200rpm)进一步生长3小时。在这些条件下,酵母细胞达到孢子形成阶段。
      5. 通过离心收获酵母细胞生长至孢子形成阶段(2,300×g,5分钟)。将收获的细胞转移到1.5ml离心管中。用2ml预先加工的缓冲液洗涤细胞沉淀,弃去上清液。
      6. 将酵母细胞在含有溶解酶(250U)的0.5ml球形沉淀缓冲液中于37℃孵育15分钟。通过酵解细胞壁的溶解酶酶降解来制备原生质球。
      7. 通过在4℃下低速离心(3,300×g )3分钟收获原生质球。
      8. 弃去上清液,并用1ml冰冷的洗涤缓冲液洗涤沉淀一次。
      9. 通过在冰上孵育15分钟,将0.8ml裂解缓冲液裂解成原生质球。用手敲定细胞,定期混合细胞。
      10. 在4℃下以13,400×g离心细胞裂解物15分钟以获得澄清的裂解物并将其转移到新的1.5ml离心管中。丢弃颗粒。
      11. 为了获得H3特异性的蛋白水解活性,将细胞裂解物与100μl蛋白A琼脂糖凝胶珠(50%浆液)一起在4℃下恒温摇动在旋转器上孵育1小时。
      12. 离心机(800xg,5分钟,4℃)珠混合细胞裂解物并丢弃上清液。
      13. 类似地,用1ml裂解缓冲液洗涤珠子两次(通过手动倒置微量离心管,通过离心沉淀珠子并抛出上清液),然后用无Triton X-100的裂解缓冲液洗涤两次。
      14. 将珠重悬于酵母 - 蛋白酶再悬浮缓冲液(100μl)中,并用于体外 H3切割测定。
    2. 从鸡肝部分纯化H3蛋白酶
      1. 从鸡中提取肝组织,并用盐水溶液洗涤。
      2. 如前所述准备微粒体(Grillo et al。,2002)。简言之,下面描述从鸡肝组织中纯化微粒体的方法。
      3. 通过马达驱动的均化器在50ml冷冻溶液1中均匀化5g鸡肝组织以制备10%匀浆并通过四层干酪布过滤。
      4. 从匀浆中去除细胞核和线粒体污染物,然后分别以5,000 x g,4℃离心10分钟和27,000 x g,4℃离心10分钟。
      5. 将收集的上清液以10万5千克,4℃离心1小时以使微粒体沉淀。
      6. 将收集的微粒体重悬于5ml的10mM Tris-Cl,pH8.0中。通过连续旋转(旋转器,10rpm)在4℃下用0.2%Triton X-100将悬浮的微粒体提取1小时。
      7. 将提取的微粒体以105,000 x g,4℃离心1小时。此时收集上清液,并在4℃下用30%硫酸铵进一步沉淀30分钟。
      8. 通过离心(27,216×g,4℃,30分钟)收集沉淀物,并在4℃下对2000ml透析缓冲液进行透析,以除去硫酸铵。
      9. 透析后,进行离心(27,216×g,4℃,15分钟)以除去不溶性沉淀物。
      10. 为了进一步纯化H3蛋白酶,执行Superose TM,6/10/300GL色谱(样品体积-500μl,流速-0.3ml/min,级分体积-0.3ml)。用于大小排阻色谱的缓冲液与透析缓冲液相同。收集从39 th 到51 st 的正分数,如图2所示。
      11. 中将池分数(39 th 至51 st )一起直接使用(不需要将这些部分用于实验)组织蛋白H3切割试验。


        图2.含有Glud1抗体的肝微粒体提取物的Western印迹用Glud1(鸡肝H3蛋白酶)抗体检测的鸡肝微粒体提取物的大小排阻色谱部分,以证实这些级分中存在H3蛋白酶。

  3. 组织体外组蛋白H3切割分析
    1. 蛋白酶来源:酵母(BY4743菌株)
      1. 在反应缓冲液(20μl)中进行体外蛋白酶测定。
      2. 通过将10μl蛋白A琼脂糖珠结合蛋白加入含有1-2μg核心组蛋白的反应混合物中来启动测定。
      3. 将反应混合物在37℃下恒温摇动孵育1小时。
      4. 通过向反应混合物中加入3.33μl6x SDS-PAGE样品加样缓冲液(反应混合物中的SDS-加载染料的最终浓度:1×),停止体外裂解反应。
    2. 蛋白酶来源:鸡
      1. 在反应缓冲液中进行体外蛋白水解测定。
      2. 孵育2微克核心组蛋白与0.25微克部分纯化的H3蛋白酶提取物从鸡肝组织在20微升的反应体积。
      3. 将反应混合物在37℃下孵育1小时。
      4. 加入3.33μl6x SDS-PAGE样品加载缓冲液停止反应
  4. 测定后核心组蛋白的可视化
    1. 如前所述,通过添加SDS-PAGE加载染料来停止体外的裂解反应。
    2. 在95℃下孵育样品5分钟
    3. 通过运行18%SDS-PAGE检查组蛋白H3的切割,如图3所示
    4. 通过用考马斯蓝色染色染色可视化核心组蛋白条带

      图3。 体外 组织蛋白H3切割测定与核心组蛋白。 em>活性测定用酵母蛋白酶。 B.用鸡肝蛋白酶进行体外活性测定。在四个核心组蛋白中,只有组蛋白H3被切割,因为蛋白酶特异于组蛋白H3。 " - "和"+"分别表示H3蛋白酶的不存在和存在 注意:当蛋白酶新鲜制备时,体外组蛋白H3切割测定法最有效。核心组蛋白应储存在-80℃的小等分试样中。应避免重复冻融组蛋白和蛋白酶。

食谱

  1. 解决方案1
    0.34 M蔗糖
    15mM Tris-Cl,pH7.5
    15 mM NaCl
    60 mM KCl
    0.5mM亚精胺
    0.15mM精胺
    2 mM EDTA
    0.5 mM EGTA
    15mMβ-巯基乙醇
    0.2 mM PMSF(使用前加入)
  2. 低调解决方案
    10mM Tris-Cl,pH7.5
    15mMβ-巯基乙醇
    2 mM PMSF
  3. 磷酸钠缓冲液(1M),pH 6.8(100ml) 49毫升1 M磷酸氢二钠 51毫升磷酸二氢钠
  4. HB缓冲区
    20 mM HEPES,pH 7.5
    0.4 M NaCl
    1 mM EDTA
    10%甘油
    0.1%NP-40
    1 mMβ-ME
    PIC(蛋白酶抑制剂鸡尾酒)(使用前加入)
  5. HAP缓冲区
    50 mM磷酸钠缓冲液
    1 mMβ-ME
    PIC(蛋白酶抑制剂鸡尾酒)(使用前加入)
  6. 醋酸钾溶液
    2克醋酸钾溶于100ml水中
  7. 前置放电缓冲器
    100mM Tris-Cl,pH8.8
    10 mM DTT
  8. 球磨缓冲器
    50mM Tris-Cl,pH7.5
    0.6 M山梨醇
    10 mM DTT
  9. 洗涤缓冲液
    100 mM KCl
    50mM HEPES-KOH,pH7.5
    2.5mM MgCl 2
    0.4 M山梨醇
  10. 裂解缓冲液
    20mM Tris-Cl,pH8.5
    200 mM KCl
    25 mM EDTA
    1%(v/v)Triton X-100
  11. 蛋白A琼脂糖珠(50%浆)
    在1.5 ml Eppendorf管中取500μl蛋白A琼脂糖珠 在4℃,800 x g离心5分钟
    弃上清
    加入1 ml裂解缓冲液至珠粒上 再次离心并丢弃上清液 再次重复两次
    最后,将珠粒沉淀在500μl裂解缓冲液中,以获得50%浆液
  12. 酵母蛋白酶再悬浮缓冲液
    20mM Tris-Cl,pH8.5
    200 mM KCl
    25 mM EDTA
  13. 蛋白酶透析缓冲液
    25mM Tris-Cl,pH7.5
    100 mM NaCl
    10%甘油
    1mMβ-巯基乙醇
    0.2 mM EDTA
  14. 反应缓冲液
    1. 酵母蛋白酶反应缓冲液
      25mM Tris-Cl,pH7.5
      150 mM NaCl
      10%甘油
      2mMβ-巯基乙醇
      0.1mM EDTA
    2. 鸡肝蛋白酶反应缓冲液
      10 mM HEPES,pH 5.5
      100 mM NaCl
      1mMβ-巯基乙醇
      0.2 mM EDTA
      10%甘油
  15. 10%Triton X-100溶液
    10%Triton X-100(100%)
    90 ml水
    解决方案通过旋转混合很好
    10%解决方案
    储存于4°C
  16. 6x SDS-PAGE加载染料
    2.5ml 4x Tris-Cl,pH6.8
    3毫升甘油
    1g SDS,0.5ml
    1.2毫克溴酚蓝
    加水至最终体积至10 ml
  17. 10倍SDS-PAGE运行缓冲液
    30.0克Trizma底座
    10.0克SDS
    144.0g甘氨酸
    溶解于1000ml H 2 O 3/
    中 使用前稀释10倍缓冲液至1x

致谢

这项工作得到了生物科技部(印度政府),科学技术部(印度政府)向RST提供的支持。该协议的简短版本在2016年Chauhan等人,2016年Chauhan和Tomar发表。 Azad和Tomar,2016; Mandal 等,2014年; Mandal 等人,2013;曼德勒等人,2012年。CSIR被公认为向Sakshi提供奖学金支持。作者还承认了RST实验室的成员对这项工作的意见。

参考文献

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  3. Bannister,AJ和Kouzarides,T.(2011)。  通过组蛋白修饰来调节染色质。 Cell Res 21(3):381-395。
  4. Chauhan,S.,Mandal,P.和Tomar,RS(2016)。  生化分析揭示鸡肝组蛋白H3蛋白酶组蛋白H3剪切的多因素机制。生物化学 55(38):5464-5482。
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引用:Chauhan, S., Azad, G. K. and Tomar, R. S. (2017). In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease. Bio-protocol 7(1): e2085. DOI: 10.21769/BioProtoc.2085.
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