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Apr 2020
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Reconstitution of Chromatin by Stepwise Salt Dialysis
逐步盐透析重组染色质   

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Abstract

Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for a wide range of applications. Here, we describe simple and straightforward protocols for the reconstitution of chromatin by stepwise salt dialysis and the analysis of the chromatin by the micrococcal nuclease (MNase) digestion assay. Chromatin that is reconstituted with this method can be used for efficient homology-directed repair (HDR)-mediated gene edited with the CRISPR-Cas9 system as well as for biochemical studies of chromatin dynamics and function.

Keywords: Core histones (核心组蛋白), DNA (DNA), Chromatin (染色质), Chromatin reconstitution (染色质重建), Salt dialysis (盐透析), Micrococcal nuclease (微球菌核酸酶)

Background

DNA in the eukaryotic nucleus is organized into chromatin. Therefore, the analysis of DNA-directed processes, such as replication, recombination, repair, and transcription, would ideally be performed with chromatin templates rather than with plain DNA (Kadonaga, 2019). To this end, chromatin can be reconstituted in vitro from purified components by using either ATP-dependent or ATP-independent approaches (for a review, see Lusser and Kadonaga, 2004).


Here, we describe the specific method of chromatin reconstitution that we employed in our studies of HDR-mediated gene editing with the CRISPR-Cas9 system in cells (Cruz-Becerra and Kadonaga, 2020). In this work, we found that precise HDR-mediated insertion of DNA is enhanced by the use of a chromatin donor template relative to a plain (naked) DNA donor template. We have also used this method for the biochemical analysis of chromatin, which includes the characterization of high mobility group N (HMGN) proteins (Rattner et al., 2009), chromatin dynamics (Torigoe et al., 2013), prenucleosomes (Fei et al., 2015), and nucleosome-destabilizing factor (NDF) (Fei et al., 2018).


The nucleosome, which is the repeating unit of chromatin, consists of an octamer of core histones that is associated with about 147 bp of DNA. A simple and reliable technique for reconstituting chromatin in vitro involves the ATP-independent formation of nucleosomes from purified DNA and core histones by stepwise salt dialysis (Figure 1). In this method, the core histones and the DNA are combined at high ionic strength (1 M NaCl), and nucleosomes are formed by gradually decreasing the salt concentration (Stein, 1989; Jeong et al., 1991). Some important considerations for the successful reconstitution of chromatin are as follows.


  1. Core histones. For chromatin reconstitution, we recommend using core histones that were purified as octamers (which exist as octamers at high salt concentrations and as H2A-H2B dimers and H3-H4 tetramers at low salt concentrations) rather than individual core histones because the separate histones need to be combined in the correct stoichiometry and then purified as octamers before use (Khuong et al., 2017). Protocols for the purification of core histone octamers from different sources are described elsewhere (Laybourn and Kadonaga, 1991; Bulger and Kadonaga, 1994; Fyodorov and Levenstein, 2002; Peterson and Hansen, 2008; Khuong et al., 2017). Histones prepared by these methods have been extensively used for the efficient assembly of chromatin in vitro. We also tested a sample of commercially available native human histone octamers (catalog number 52065; BPS Bioscience, San Diego, CA) and found that they are suitable for reconstituting chromatin by the salt dialysis method.


  2. DNA. To achieve the efficient reconstitution of chromatin, the quality of the DNA is extremely important. For the protocol described here, the DNA can be easily prepared by using the HiSpeed Plasmid Maxi Kit (catalog number 12662; QIAGEN, Hilden, Germany) or by carrying out two successive CsCl density gradient centrifugation steps. We typically use plasmid DNA prepared by these methods for reconstituting chromatin donor templates for HDR-mediated gene editing (Cruz-Becerra and Kadonaga, 2020). In addition, for mononucleosome gel shift assays, we use this salt dialysis protocol to reconstitute mononucleosomes with DNA fragments (≥ 147 bp) that are prepared by PCR amplification followed by purification with the QIAquick Gel Extraction Kit (catalog number 28704; QIAGEN, Hilden, Germany) (Chavez et al., 2019).


  3. Histone:DNA ratio. It is essential to determine experimentally the optimal histone:DNA ratio for the desired level of nucleosome occupancy with each new preparation of DNA or histone stock. To this end, we recommend performing a titration with variations of about 10% in the histone:DNA mass ratio in a series of chromatin reconstitution reactions. For instance, in HDR-mediated gene editing with the CRISPR-Cas9 system and chromatin donor templates, we first reconstituted chromatin with histone:DNA mass ratios of 0.80:1.0, 0.90:1.0, 1.0:1.0, 1.1:1.0, and 1.2:1.0. We then evaluated the quality of the reconstituted chromatin by MNase digestion analysis and used the chromatin with the most extensive arrays of nucleosomes (Cruz-Becerra and Kadonaga, 2020).


  4. MNase digestion assay. In this assay, the partial digestion of chromatin with different concentrations of MNase (which cleaves the linker DNA between nucleosomes) reveals the formation of arrays of nucleosomes on the DNA template. After deproteinization, the resulting DNA fragments that are derived from the oligonucleosomes show a repeating ladder pattern on an agarose gel. The detection of a periodic array of oligonucleosomes, such as tetra- and pentanucleosomes, is an indication of high quality chromatin.




Figure 1. Schematic outline of the workflow in the reconstitution of chromatin by stepwise salt dialysis. The DNA and the histone octamers are combined in TE Buffer containing 1.0 M NaCl. Then, the histone-DNA mixture is dialyzed sequentially against TE Buffer containing 0.80 M, 0.60 M and 0.05 M NaCl. After dialysis, the quality of the resulting chromatin is assessed by subjecting a portion of the sample to micrococcal nuclease digestion analysis.


In summary, we describe a protocol for the reconstitution of chromatin by simple stepwise salt dialysis. This protocol can be completed in about 12 h (total time for chromatin reconstitution and analysis), and can be used with core histones from different sources and DNA prepared by standard laboratory procedures. In addition to the reconstitution of nucleosomes onto circular plasmid DNA, this method can be used for the preparation of mononucleosomes or polynucleosomes with linear DNA of different lengths (Chavez et al., 2019).


Materials and Reagents

  1. 200 µl barrier tips (Denville Scientific, catalog number: P1122 )

  2. 20 µl barrier tips (Denville Scientific, catalog number: P1121 )

  3. 10 µl barrier tips (Denville Scientific, catalog number: P1096-FR )

  4. 1.7 ml low binding microcentrifuge tubes (Sorenson Bioscience, catalog number: 39640T )

  5. 0.65 ml safeseal low binding microcentrifuge tubes (PGC Scientific, catalog number: 505-195 )
    Note: This product has been discontinued and should be substituted by other 0.65 ml low binding microcentrifuge tubes (e.g., Sorenson Bioscience, catalog number: 11300 ).

  6. Microcentrifuge tube lid locks (VWR, catalog number: 14229-941 )

  7. Dialysis membrane, 3.5 kDa molecular weight cutoff (Spectrum, catalog number: 132592 ) or Slide-A-Lyzer MINI dialysis device (Thermo Fisher Scientific, catalog number: 69550 )

  8. Liquid nitrogen

  9. Micrococcal nuclease (Sigma, catalog number: N5386 )

  10. Proteinase K (Roche, catalog number: 03115879001)

  11. 123 bp DNA ladder (Sigma, catalog number: D5042 )

  12. Glycogen (Sigma, catalog number: G-0885 )

  13. Sodium chloride (Fisher Scientific, catalog number: S671-3 )

  14. Orange G sodium salt (Sigma, catalog number: O-3756 )

  15. Calcium chloride dihydrate (Fisher Scientific, catalog number: C79-500 )

  16. HEPES (Fisher Scientific, catalog number: BP-310-1 )

  17. Potassium chloride (Fisher Scientific, catalog number: P-217-500 )

  18. Magnesium chloride hexahydrate (Fisher Scientific, catalog number: M33-500 )

  19. EGTA (Sigma, catalog number: E-4378 )

  20. Ammonium acetate (Fisher Scientific, catalog number: A637-500 )

  21. Glycerol (Fisher Scientific, catalog number: G33-20 )

  22. Sodium dodecyl sulfate (Bio-Rad, catalog number: 161-0302 )

  23. Tris base (Fisher Scientific, catalog number: BP152-5 )

  24. EDTA disodium salt dihydrate (Macron Fine Chemicals, catalog number: 4931-04 )

  25. Phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) (Thermo Fisher Scientific, catalog number: J75831-AN )

  26. Ethanol (Koptec, catalog number: V1016 )

  27. Agarose (Promega, catalog number: V3125 )

  28. Boric acid (Fisher Scientific, catalog number: BP-168-500 )

  29. Ethidium bromide (Sigma, catalog number: E-8751 )

  30. Glass distilled water

  31. Plasmid DNA prepared with the HiSpeed Plasmid Maxi Kit (QIAGEN, catalog number: 12662 )

  32. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )

    Note: This product is required only when the DNA is prepared by PCR amplification.

  33. Histone octamer stock (BPS Bioscences, catalog number: 52065 ; or purified by the methods cited above)

  34. TE Buffer, pH 8.0 (see Recipes)

  35. 10× TE Buffer, pH 8.0 (see Recipes)

  36. TBE Buffer (see Recipes)

  37. 10× TBE Buffer (see Recipes)

  38. Orange G Loading Buffer (see Recipes)

  39. 200 U/ml MNase stock (see Recipes)

  40. MNase Buffer R (see Recipes)

  41. Stop Buffer (see Recipes)

  42. HEG Buffer (see Recipes)

  43. 10 mg/ml Ethidium bromide stock solution (see Recipes)

Equipment

  1. Ice bucket

  2. Liquid nitrogen Dewar flask

  3. Magnetic stir bars

  4. 1 L cylinders (Pyrex, catalog number: 3022 )

  5. 1 L beakers (Pyrex, catalog number: 1000 )

  6. Fine tip transfer pipettes (Samco Scientific, catalog number: 231 )

  7. 10 μl Micropipette (Rainin, model: SL 10-XLS)

  8. 20 μl Micropipette (Rainin, model: SL 20)

  9. 200 μl Micropipette (Rainin, model: SL 200)

  10. Magnetic stirrer (Fisher Scientific, catalog number: 11-500-75 )

  11. Centrifuge (Eppendorf, model: 5424 )

  12. Minicentrifuge (RPI, model: PMC-060 )

  13. Vortex mixer (Fisher, catalog number: 12-812 )

  14. Horizontal gel electrophoresis apparatus (Gibco, model: H5 )

  15. Electrophoresis power supply (Bio-Rad, catalog number: Power Pac 200)

  16. UV transilluminator (Protein Simple, model: Alpha Imager HP)

Procedure

  1. Reconstitution of chromatin

Here we describe the protocol for a standard chromatin reconstitution experiment. In this protocol, it is assumed that the optimal amounts of histones and DNA have been previously established.

To determine the optimal histone:DNA ratio with new preparations of histones and/or DNA, this protocol can be performed at 0.5× scale (that is, each reaction has 25 µg of DNA in 75 µl final volume instead of 50 µg of DNA in 150 µl final volume) in a series of reactions with different histone:DNA ratios. For this purpose, we typically carry out reactions with histone:DNA mass ratios of 0.80:1.0, 0.90:1.0, 1.0:1.0, 1.1:1.0 and 1.2:1.0.

  1. For each reconstitution reaction, prepare a 150 µl histone-DNA mix that contains 50 µg of DNA and the desired mass of histone octamers in TE Buffer (pH 8.0) containing a final concentration of 1.0 M NaCl. Mix gently. Then, incubate for 15 min on ice.

    It is important to consider the salt contribution from the histone stock in the calculations of the final concentration of NaCl in the histone-DNA mixture. For example, a reconstitution reaction with a desired final histone:DNA ratio of 1.0:1.0 could be prepared from 42 µl of a 1.2 mg/ml DNA stock (50 µg DNA), 42 µl of a 1.2 mg/ml histone stock containing 2.5 M NaCl (50 µg histones), 9.0 µl of 5.0 M NaCl (to give 1.0 M NaCl final concentration), 15 µl of 10× TE Buffer (i.e., 1× TE final), and 42 µl of water in a final volume of 150 µl.

  1. Load the histone-DNA mix from Step A1 into a dialysis chamber.

    The dialysis chamber consists of the sample reservoir, the dialysis membrane, and a sealing ring (Figure 2). Assemble the dialysis chamber as follows:

    1. Cut a microcentrifuge tube (please see Note 8) such that the lid of the tube (i.e., the sample reservoir) is surrounded by a ring (i.e., the sealing ring).

    2. Detach the sample reservoir from the sealing ring by cutting the linker between them. Then pull the two pieces apart.

    3. Cut a square piece (about 1.5 cm × 1.5 cm) of dialysis membrane and rinse it with water to remove the storage solution. Then, immerse the membrane in TE Buffer containing 0.80 M NaCl.

    4. Load the sample into the reservoir with a 200 µl pipette.

    5. Drain the excess buffer from the membrane by gently tapping the edge over a paper towel.

    6. Cover the sample with the membrane.

    7. Close the chamber with the sealing ring (i.e., place the ring on top of the membrane, then push it down over the sample reservoir piece).

    8. Fasten the dialysis chamber with a lid lock, and immediately immerse it into TE Buffer containing 0.80 M NaCl. The dialysis membrane should be in direct contact with the dialysis buffer (i.e., the membrane side of the dialysis chamber faces down).



    Figure 2. Diagram of the procedure for the assembly of a dialysis chamber. The dialysis chamber is prepared from a microcentrifuge tube and a small piece of dialysis membrane as follows. A. Cut the top part of a microcentrifuge tube. B. Separate the lid and the portion of the tube that remained attached. The lid serves as the sample reservoir and the cropped tube as the sealing ring. C. Pipet 150 μl of sample into the reservoir. Cover the sample with a small square of dialysis membrane (denoted in blue). Then, place the sealing ring on top of the membrane. Close the dialysis chamber by pushing the sealing ring down over the reservoir. D. Fully assembled dialysis chamber. E. Secure the dialysis chamber with a lid lock.


  2. Dialyze at room temperature (22 °C) in a stepwise manner against decreasing salt concentrations as follows:

    1. 1 L of TE Buffer containing 0.80 M NaCl for 2 h.

    2. 1 L of TE Buffer containing 0.60 M NaCl for 2 h.

    3. 1 L of TE Buffer containing 0.05 M NaCl for 2 h.

      Notes:

      1. When transferring the sealed dialysis chamber between the different buffers, it is not necessary to remove the small amount of excess buffer that is associated with the outside of the dialysis chamber.

      2. Several reconstitution reactions can be dialyzed simultaneously in a beaker containing 1 L of dialysis buffer at medium speed on a magnetic stirrer. The dialysis chambers should be gently swirling around the top of the buffer during dialysis.

  3. Remove the dialysis chamber from the TE Buffer containing 0.050 M NaCl and place it over a paper towel with the membrane side facing up.

  4. Remove the remaining excess TE Buffer on the outside of the membrane with a 200 µl pipette. Then, with a new tip, carefully perforate the membrane and transfer the dialysate (i.e., reconstituted chromatin) to a microcentrifuge tube.

  5. Store the chromatin at 4 °C. The chromatin is stable at 4 °C for several months. Do not freeze the chromatin.


  1. Quantification of the DNA concentration in the salt dialyzed chromatin

  2. With the chromatin reconstitution procedure described in Section A, we generally recover greater than 90% of the starting amount of DNA (Figure 3). For many applications, the nucleosomal DNA concentrations can be estimated (within 10% of the method described below and shown in Figure 3) by the A260nm readings (using the extinction coefficient for pure double-stranded DNA) on a NanoDrop OneC spectrophotometer (model ND-ONEC-W; Thermo Fisher Scientific, Waltham, MA). This is probably due to the low A260nm absorbance of the histones because of their low content of aromatic amino acid residues. Alternatively, the nucleosomal DNA concentrations can be determined by removal of the histones and quantification of the resulting DNA samples, as described below. In contrast, the use of a Qubit Fluorometer is not recommended because it does not provide accurate quantification of the nucleosomal DNA in the chromatin sample, probably because the binding of fluorescent dye molecules to nucleosomal DNA is less efficient than their binding to free DNA.



    Figure 3. Quantification of the DNA in chromatin reconstituted by salt dialysis. A sample of salt dialyzed chromatin was diluted 8-, 16-, and 32- fold in TE Buffer containing 0.05 M NaCl. After deproteinization of a 2.0 μl aliquot of each of the three chromatin dilutions, the resulting DNA samples, along with DNA standards of known concentration (20, 40 and 80 ng naked DNA), were subjected to agarose gel electrophoresis analysis and visualized by staining with ethidium bromide (EB). The numbers under Nucleosomal DNA correspond to the calculated amounts of DNA in 2.0 µl of the 32-, 16-, and 8- fold dilutions (from left to right) of the chromatin sample after quantification by using the Gel Analysis function of the ImageJ software (NIH, Bethesda, MD). In this experiment, the reconstituted chromatin contained about 0.30 µg/µl DNA in 160 µl final volume (i.e., 48 µg of DNA, which corresponds to about 96% of the starting amount in the chromatin reconstitution reaction).

    1. Measure the volume of the sample after dialysis by using a 200 μl pipette. With this method, the volume increases by about 7% of the original reaction volume during the salt dialysis (i.e., in chromatin reconstitution reactions performed with initial volumes of 150 μl and 75 μl, we typically recover about 160 μl and 80 μl, respectively).

    2. Quantify the DNA by agarose gel electrophoresis followed by analysis with ImageJ as follows.

      1. Prepare 40, 20, and 10 ng/μl standards (in TE Buffer containing 0.05 M NaCl) of the DNA stock that was used for reconstituting the chromatin. These standards will be used to make a DNA standard curve to determine the amount of DNA in the chromatin sample (Step B2q).

      2. Dilute 2.0 μl of the reconstituted chromatin in 14 μl of TE Buffer containing 0.050 M NaCl (i.e., 8-fold dilution). Then perform two 2-fold serial dilutions in the same buffer to give 16- and 32-fold dilutions of the reconstituted chromatin.

      3. To 2.0 μl of the 40, 20, and 10 ng/μl DNA standards (from Step B2a), and 2.0 μl of the 8-, 16-, and 32-fold dilutions of the chromatin (from Step B2b), add 48 μl of HEG Buffer to give a final volume of 50 μl of each sample.

      4. To each 50 μl sample, add 100 μl of Stop Buffer and 5.0 μl of 2.5 mg/ml proteinase K. Mix gently. Incubate 30 min at 37 °C.

      5. Add 155 μl of phenol:chloroform:isoamyl alcohol and vortex vigorously for 30 s.

      6. Centrifuge at 16,000 × g for 5 min.

      7. Transfer 140 μl of the aqueous phase to a new microcentrifuge tube.

      8. To the aqueous phase, add 25 μl of 2.5 M ammonium acetate and 600 μl of 100% ethanol. Mix by inverting the tube several times.

      9. Centrifuge at 16,000 × g for 20 min.

      10. Remove the supernatant by using a transfer pipette. Then, spin the tubes briefly in a minicentrifuge and remove any leftover liquid with a 10 μl pipette tip.

      11. Air dry the pellet for 5 min.

      12. To the pellet, add 5.0 μl of Orange G Loading Buffer. Incubate for 5 min. Vortex gently, then spin briefly.

      13. Subject the DNA to electrophoresis on a 1.0% agarose-TBE gel at 3.7 V/cm until the Orange G dye reaches the bottom of the gel. The running time will vary according to the gel dimensions. As an example, a gel that is 5 cm × 6 cm × 0.5 cm (width × length × height) will take about 40 min at 100 V in a horizontal electrophoresis chamber in which the anode and the cathode are separated by 27 cm. To estimate the total voltage, multiply the distance between the electrodes in the electrophoresis chamber by the desired voltage (V) per centimeter (cm). In this example, 27 cm × 3.7 V/cm = 100 V.

      14. Stain the gel for 10 min in EB staining solution.

      15. Rinse the gel with water for 10 min.

      16. Visualize the DNA with a UV transilluminator and record an image of the DNA in TIF format for quantitative analysis.

      17. Quantify the DNA on the gel by using the Gel Analysis function of the ImageJ software (National Institutes of Health, Bethesda, MD) as described elsewhere (https://imagej.nih.gov/ij/docs/menus/analyze.html#gels).

        Note: Adjust the calculated DNA amount on the gel according to the dilution factor that corresponds to each sample.

      18. To obtain the DNA concentration in the chromatin sample, divide the calculated total amount of DNA by the volume of sample recovered after the dialysis steps (from Step B1).


  1. MNase digestion analysis of the reconstituted chromatin

The quality of the reconstituted chromatin is assessed by using the MNase digestion assay (Figures 4 and 5).



Figure 4. Determination of the optimal histone:DNA ratio for efficient chromatin reconstitution by salt dialysis. Chromatin was reconstituted with histone:DNA mass ratios of 0.80:1.0, 0.90:1.0, 1.0:1.0, and 1.1:1.0 (i.e., 25 µg of DNA with 20 µg, 22.5 µg, 25 µg, and 27.5 µg of histone octamers, respectively) by using the salt dialysis methodology outlined in Figure 1. To evaluate the quality of the reconstituted chromatin, the samples were subjected to partial digestion with four different concentrations of MNase. After deproteinization, the resulting DNA fragments were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide (EB). The arrows indicate the DNA bands that correspond to mono-, di-, tri-, tetra-, and pentanucleosomes. In this experiment, there are higher proportions of tetra- and pentanucleosomal DNA fragments at histone:DNA ratios of 0.90:1.0 (yellow dots) and 1.0:1.0 (green dots) than at 0.80:1.0 (pink dots) or 1.1:1.0 (blue dots). For many applications, such as HDR-mediated gene editing (Cruz-Becerra and Kadonaga, 2020), it is best to use chromatin with the lowest histone:DNA ratio that yields the most extensive nucleosome arrays. In this case, the 0.90:1.0 chromatin and the 1.0:1.0 chromatin are of comparable quality and are better than the 0.80:1.0 chromatin and the 1.1:1.0 chromatin, as assessed by the MNase analysis. Therefore, the recommended histone:DNA ratio is 0.90:1.0. The DNA size markers (M) are the 123 bp ladder (Millipore Sigma), which consists of bands that are integral multiples of 123 bp. The smallest 123 bp band migrates slightly faster than the mononucleosome DNA band.



Figure 5. MNase digestion analysis of salt dialyzed chromatin. Chromatin was reconstituted with three different preparations of core histones and the same plasmid DNA template (pBKS-GAPDH-HDR; 5.6 kbp; Cruz-Becerra and Kadonaga, 2020) by using the salt dialysis methodology outlined in Figure 1. A. Chromatin prepared with core histones from Drosophila melanogaster (Dm) 0-12 h embryos. The histones were purified by using the method of Fyodorov and Levenstein (2002). B. Chromatin reconstituted with HeLa core histones prepared as described in Khuong et al. (2017) (denoted as Sample A). C. Chromatin assembled with HeLa core histones obtained from BPS Bioscience (San Diego, CA) (denoted as Sample B). After partial digestion of the chromatin samples with four different concentrations of MNase, the samples were deproteinized, and the resulting DNA fragments were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide (EB). The arrows indicate the DNA bands that correspond to mono-, di-, tri-, tetra-, and pentanucleosomes. The DNA size markers (M) are the 123 bp ladder (Millipore Sigma), which consists of bands that are integral multiples of 123 bp. The smallest 123 bp band migrates slightly faster than the mononucleosome DNA band.


  1. Perform partial MNase digestion of the reconstituted chromatin (from Step A5) with four different concentrations of enzyme. Each MNase digestion reaction contains 35 µl of chromatin (5.7 ng/µl DNA; 200 ng total DNA), 9.0 µl of 10 mM CaCl2, and 5.0 µl of MNase at various concentrations (e.g., 1.3, 0.67, 0.33, and 0.17 mU/μl; the optimal concentrations will vary with different preparations of MNase) in a final volume of 49 µl. The reactions are set up as follows:

    1. Dilute an aliquot of chromatin to 5.7 ng/μl DNA in HEG Buffer in 175 µl final volume.

    2. Thaw an aliquot of a 200 U/ml MNase stock in a container with water at 4 °C. After thawing, keep the enzyme stock on ice.

    3. Make MNase working stocks that contain 1.3, 0.67, 0.33, and 0.17 mU/μl in MNase Buffer R as follows:

      1. Dilute the 200 U/ml enzyme stock to 13 mU/μl in cold MNase Buffer R (e.g., 2.0 ul of 200 U/ml MNase in 30 μl final volume).

      2. Make a 10-fold dilution of 13 mU/μl MNase to obtain 1.3 mU/μl.

      3. Perform three 2-fold serial dilutions starting with 1.3 mU/μl MNase to obtain 0.67, 0.33, and 0.17 mU/μl.

    4. Immediately before use, combine 5.0 μl of the MNase working stock with 9.0 μl of 10 mM CaCl2 to give a final volume of 14 μl MNase-CaCl2 mix. Note that it is necessary to prepare a different MNase-CaCl2 mix for each of the four MNase working stocks from Step C1c in this section. If several chromatin samples will be analyzed simultaneously, prepare the required volume of each MNase-CaCl2 mix for the total number of samples plus one (e.g., for four chromatin samples, prepare 5 × 14 µl = 70 μl of each MNase-CaCl2 mix).

    5. Add 14 μl of MNase-CaCl2 mix to 35 μl of chromatin (5.7 ng/μl DNA; from Step C1a in this section) to give a final volume of 49 µl. Mix gently, then spin briefly. Incubate exactly 10 min. Repeat this step with each MNase-CaCl2 mix (and each chromatin sample).

  1. Stop the MNase digestion reactions by adding 7.0 μl of 0.50 M EDTA (to 63 mM EDTA final concentration) to each tube. Mix gently.

  2. Recover the DNA as follows.

    1. Add 100 μl of Stop Buffer and 5.0 μl of 2.5 mg/ml proteinase K. Mix gently. Incubate 40 min at 37 °C.

    2. Add 155 μl of phenol:chloroform:isoamyl alcohol and vortex vigorously for 30 s.

    3. Centrifuge at 16,000 × g for 5 min.

    4. Transfer 140 μl of the aqueous phase to a new microcentrifuge tube.

    5. To the aqueous phase, add 25 μl of 2.5 M ammonium acetate and 600 μl of 100% ethanol. Incubate for 10 min.

    6. Centrifuge at 16,000 × g for 20 min.

    7. Remove the supernatant by using a transfer pipette. Then, spin the tubes briefly in a minicentrifuge and remove any leftover liquid with a pipette tip.

    8. Air dry the pellet for 5 min.

  3. Resolve the DNA fragments by agarose gel electrophoresis.

    1. To the pellet, add 5.0 μl of Orange G Loading Buffer. Incubate for 5 min. Vortex gently, then spin briefly.

    2. Subject the DNA to electrophoresis on a 1.3% agarose-TBE gel at 3.3 V/cm until the Orange G dye reaches the bottom of the gel (this will take about 2.5 h with a gel that is 11 cm, 14 cm, and 0.6 cm in width, length, and height, respectively).

  4. Visualize the DNA by EB staining.

    1. Stain the gel for 30 min in EB staining solution.

    2. Remove the excess EB with water (typically, two 10 min rinses in water).

    3. Visualize the DNA with a UV transilluminator.

Notes

  1. The catalog numbers, models, and manufacturers that are given in the Materials and Reagents and Equipment sections are those of specific items that we have used to perform this method. This protocol could be performed with comparable items from other sources.

  2. Before performing chromatin reconstitution experiments, we recommend analyzing the quality of the histone and DNA preparations by SDS-polyacrylamide and agarose gel electrophoresis, respectively. For the histones, an equimolar stoichiometry of all four histones should be observed with no detectable degradation or contamination. The plasmid DNA should be mostly supercoiled DNA and should not contain bacterial genomic DNA.

  3. DNA and histone stocks of ≥ 1 mg/ml concentration are recommended.

  4. Avoid contamination of the materials and reagents with nucleases and proteases. Use nuclease- and protease-free laboratory plasticware and glassware, and wear clean gloves throughout the entire procedure.

  5. Use only low binding microcentrifuge tubes to reduce the adhesion of the histones and DNA to the plastic.

  6. Mix the reaction reagents by flicking the tube, except when vortexing is indicated.

  7. Perform all the steps at room temperature (22 °C) except when stated otherwise.

  8. Chromatin can be reconstituted in reaction volumes of 150 µl and 75 µl with similar results. We use dialysis chambers prepared from 1.7 ml or 0.65 ml low binding tubes for 150 µl or 75 µl reconstitution reactions, respectively. Do not use smaller reaction volumes with these dialysis chambers.

  9. As an alternate option, the Slide-A-Lyzer MINI dialysis units from Thermo Fisher Scientific (catalog number: 69550 , Thermo Fisher Scientific, Waltham, MA) could be used instead of the dialysis chambers described here.

  10. We recommend that the conductivity of the reconstituted chromatin sample is measured to determine the completion of dialysis. The conductivity of the reconstituted chromatin at the end of dialysis should be within 10% of the conductivity of TE Buffer containing 0.05 M NaCl.

Recipes

  1. TE Buffer, pH 8.0

    10 mM Tris

    1.0 mM EDTA

  2. 10× TE Buffer, pH 8.0

    100 mM Tris

    10 mM EDTA

  3. TBE Buffer

    89 mM Tris base

    89 mM Boric acid

    2.0 mM EDTA

  4. 10× TBE Buffer

    0.89 M Tris base

    0.89 M Boric acid

    20 mM EDTA

  5. Orange G Loading Buffer

    6.25% (v/v) glycerol with Orange G (add Orange G dye until the solution is medium-dark orange)

    1× TBE Buffer

  6. 200 U/ml MNase stock

    Resuspend the lyophilized enzyme in 5.0 mM sodium phosphate, pH 7.0 containing 2.5 µM of CaCl2

    Make small aliquots

    Quick-freeze in liquid nitrogen and store at -80 °C

  7. MNase Buffer R

    10 mM K+-HEPES, pH 7.6

    10 mM KCl

    1.5 mM MgCl2

    0.50 mM EGTA

    10% (v/v) glycerol

  8. Stop Buffer

    20 mM EDTA

    0.20 M NaCl

    1.0% (w/v) SDS

    0.25 mg/ml glycogen

  9. HEG Buffer

    25 mM K+-HEPES, pH 7.6

    0.10 mM EDTA

    10% (v/v) glycerol

  10. 10 mg/ml Ethidium bromide stock solution

    Dissolve 100 mg of ethidium bromide in 10 ml of water

    Store at 4 °C; protect from light

  11. 1.0 µg/ml Ethidium bromide staining solution

    Perform a 10,000-fold dilution of 10 mg/ml ethidium bromide stock solution in water

Acknowledgments

We thank all of the previous members of the Kadonaga laboratory whose biochemical work on chromatin assembly has served as the reference for the protocol described here. We are also grateful to Sharon Torigoe, George Kassavetis, Long Vo ngoc, and Claudia Medrano for critical reading of the manuscript, and to Daniel Zamorano for helping with Figure 2. J.T.K is the Amylin Chair in the Life Sciences. G.C.-B. is a recipient of a Pew Latin American Postdoctoral Fellowship, and the University of California at San Diego Molecular Biology Cancer Fellowship. This work was supported by a grant from the National Institutes of Health (R35 GM118060) to J.T.K. The protocol described here was used to reconstitute chromatin donor templates for HDR-mediated gene editing via the CRISPR-Cas9 system in Cruz-Becerra and Kadonaga (2020).

Competing interests

The authors declare no competing financial interests.

References

  1. Bulger, M. and Kadonaga, J. T. (1994). Biochemical reconstitution of chromatin with physiological nucleosome spacing. Methods Mol Gen 5: 241-262.
  2. Chavez, C., Cruz-Becerra, G., Fei, J., Kassavetis, G.A. and Kadonaga, J. T. (2019). The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. Elife 8: e47682.
  3. Cruz-Becerra, G. and Kadonaga, J. T. (2020). Enhancement of homology-directed repair with chromatin donor templates in cells.Elife 9: e55780.
  4. Fei, J., Ishii, H., Hoeksema, M. A., Meitinger, F., Kassavetis, G. A., Glass, C. K., Ren, B. and Kadonaga, J. T. (2018). NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes. Genes Dev 32(9-10): 682-694.
  5. Fei, J., Torigoe, S.E., Brown, C.R., Khuong, M.T., Kassavetis, G.A., Boeger, H. and Kadonaga, J.T. (2015). The prenucleosome, a stable conformational isomer of the nucleosome. Genes Dev 29(24): 2563-2575.
  6. Fyodorov, D. V. and Levenstein, M. E. (2002). Chromatin assembly using Drosophila systems. Curr Protoc Mol Biol Chapter 21: 7.
  7. Jeong, S. W., Lauderdale, J. D. and Stein, A. (1991). Chromatin assembly on plasmid DNA in vitro. Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5 . J Mol Biol 222(4): 1131-1147.
  8. Kadonaga, J. T. (2019). The transformation of the DNA template in RNA polymerase II transcription: a historical perspective.Nat Struct Mol Biol 26(9): 766-770.
  9. Khuong, M. T., Fei, J., Cruz-Becerra, G. and Kadonaga, J. T. (2017). A simple and versatile system for the ATP-dependent assembly of chromatin.J Biol Chem 292(47): 19478-19490.
  10. Laybourn, P. J. and Kadonaga, J. T. (1991). Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II.Science 254(5029): 238-245.
  11. Lusser, A. and Kadonaga, J.T. (2004). Strategies for the reconstitution of chromatin. Nat Methods 1(1): 19-26.
  12. Peterson, C. L. and Hansen, J. C. (2008). Chicken erythrocyte histone octamer preparation. CSH Protoc: pdb.prot 5112.
  13. Rattner, B. P., Yusufzai, T. and Kadonaga, J. T. (2009). HMGN proteins act in opposition to ATP-dependent chromatin remodeling factors to restrict nucleosome mobility. Mol Cell 34(5): 620-626.
  14. Stein, A. (1989). Reconstitution of chromatin from purified components. Methods Enzymol 170: 585-603.
  15. Torigoe, S. E., Patel, A., Khuong, M. T., Bowman, G. D. and Kadonaga, J. T. (2013). ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling.Elife 2: e00863.


简介

[摘要]染色质而不是普通的DNA是介导细胞核中DNA定向过程的分子机器的天然底物。染色质可以重新构建d体外b ÿ使用不同的方法。盐渗析法产生的染色质由纯化的组蛋白和DNA组成。这种生物化学纯的染色质非常适合广泛的应用。在这里,我们描述了通过逐步盐透析和通过微球菌核酸酶(MNase)消化测定法对染色质进行分析的染色质重构的简单明了的协议。该复原用该方法染色质,可用于高效同源定向修复(HDR)介导的基因编辑编 使用CRISPR-Cas9系统,以及用于染色质动力学和功能的生化研究。


[背景]真核细胞中的DNA被组织成染色质。因此,理想情况下,DNA定向过程的分析(例如复制,重组,修复和转录)将使用染色质模板而不是纯DNA进行(Kadonaga,2019)。为此,染色质可通过使用ATP依赖性或ATP依赖性方法在体外从纯化的成分中重建(有关综述,请参见Lusser和Kadonaga,2004年)。

在这里,我们描述了染色质重建的特定方法,该方法在我们利用CRISPR-Cas9系统对细胞进行HDR介导的基因编辑研究中采用了(Cruz-Becerra和Kadonaga,2020年)。在这项工作中,我们发现相对于普通的(裸)DNA供体模板,通过使用染色质供体模板可以增强精确的HDR介导的DNA插入。我们还将这种方法用于染色质的生化分析,包括高迁移性N组(HMGN)蛋白(Rattner等,2009),染色质动力学(Torigoe等,2013),前核小体(Fei)的表征。等人,2015年),以及核小体失稳因子(NDF)(F ei等人,2018年)。

核小体是染色质的重复单元,由核心组蛋白的八聚体组成,该组蛋白与约147 bp的DNA相关。一种用于染色质重构简单且可靠的技术在体外INVO LVES从纯化的DNA,并通过分步透析盐核心组蛋白(图1)的核小体的ATP依赖性形成。在这种方法中,核心组蛋白和DNA在高离子强度(1 M NaCl)下结合,并通过逐渐降低盐浓度形成核小体(Stein,1989; Jeong等,1991)。成功重建染色质的一些重要考虑因素如下。

核心组蛋白。对于染色质重建,我们建议使用纯化的八聚体核心组蛋白(在高盐浓度下以八聚体形式存在,在低盐浓度下以H2A-H2B二聚体和H3-H4四聚体形式存在),而不是单独的核心组蛋白,因为单独的组蛋白需要以正确的化学计量比结合,然后在使用前作为八聚体纯化(Khuong et al。,201 7)。从不同来源纯化核心组蛋白八聚体的方案在其他地方有描述(Laybourn和Kadonaga,1991; Bulger和Kadonaga,1994; Fyodorov和Levenstein,2002; Peterson和Hansen,2008; Khuong等人,2017)。通过这些方法制备的组蛋白已被广泛用于体外染色质的有效组装。我们还测试了市售天然人组蛋白八聚体的样品(产品目录号52065;BPS Bioscience,圣地亚哥,加利福尼亚),发现它们适用于通过盐透析方法重构染色质。
脱氧核糖核酸。为了实现染色质的有效重建,DNA的质量极为重要。对于此处描述的方案,可以使用HiSpeed Plasmid Maxi试剂盒(目录号12662; QIAGEN ,Hilden,德国)或执行两个连续的CsCl密度梯度离心步骤轻松制备DNA 。我们通常使用通过这些方法制备的质粒DNA来重构染色质供体模板,以进行HDR介导的基因编辑(Cruz-Becerra和Kadonaga,2020年)。此外,对于单核小体凝胶位移测定,我们使用这种盐渗析方案来重组具有DNA片段(≥147 bp)的单核小体,这些片段通过PCR扩增,然后用QIAquick凝胶提取试剂盒(目录号28704; QIAGEN,Hilden,德国)(Chavez等人,2019)。
组蛋白:DNA比率。对于每种新制备的DNA或组蛋白原液,必须根据实验确定最佳的组蛋白:DNA比例,以达到所需的核小体占据水平。为此,我们建议在一系列染色质重构反应中进行组蛋白:DNA质量比变化约10%的滴定。例如,在使用CRISPR-Cas9系统和染色质供体模板进行HDR介导的基因编辑中,我们首先以组蛋白:DNA质量比为0.80:1.0、0.90:1.0、1.0:1.0、1.1:1.0和1.2重构染色质: 1.0。然后,我们通过MNase消化分析评估了重组染色质的质量,并将染色质与最广泛的核小体阵列一起使用(Cruz-Becerra和Kadonaga,2020年)。
MNase消化测定。在该测定中,用不同浓度的MNase(切割核小体之间的连接子DNA)对染色质进行部分消化,揭示了DNA模板上核小体阵列的形成。脱蛋白后,源自寡核小体的所得DNA片段在琼脂糖凝胶上显示出重复的梯形图案。所述寡核,如四-和pentanucleosomes的周期性阵列的检测,是高品质的染色质的指示。


图1.通过逐步盐透析重建染色质的工作流程示意图。将DNA和组蛋白八聚体在含有1.0M NaCl的TE缓冲液中合并。然后,将组蛋白-DNA混合物依次用含有0.80M,0.60M和0.05M NaCl的TE缓冲液透析。透析后,通过对一部分样品进行微球菌核酸酶消化分析,评估所得染色质的质量。



总而言之,我们描述了通过简单的逐步盐析法重建染色质的方案。该方案可在约12小时内完成(染色质重建和分析的总时间),并可与来自不同来源的核心组蛋白和通过标准实验室程序制备的DNA一起使用。除了将核小体重建成环状质粒DNA外,该方法还可用于制备具有不同长度的线性DNA的单核小体或多核小体(Chavez等人,2019)。

关键字:核心组蛋白, DNA, 染色质, 染色质重建, 盐透析, 微球菌核酸酶




材料和试剂



200个μ升屏障提示(登维尔科学,目录号:P1122)
20个μ升屏障提示(登维尔科学,目录号:P1121)
10个μ升屏障提示(登维尔科学,目录号:P1096-FR)
1.7 ml低结合微量离心管(Sorenson Bioscience,目录号:39640T)
0.65 ml的Safeseal低结合微量离心管(PGC Scientific,目录号:505-195)注:该产品已停产,应用其他0.65 ml的低结合微量离心管(例如Sorenson Bioscience,目录号:11300)代替。
微量离心管盖锁(VWR,目录号:14229-941 )
透析膜,截留分子量为3.5 kDa(光谱,目录号:132592)或Slide-A-Lyzer MINI透析装置(Thermo Fisher Scientific,目录号:69550)
液氮
微球菌核酸酶(Sigma,目录号:N5386)
蛋白酶K(罗氏(Roche),目录号:03115879001)
123 bp DNA阶梯(Sigma,目录号:D5042)
糖原(Sigma,目录号:G-0885)
氯化钠(Fisher Scientific,目录号:S671-3)
橙色G钠盐(西格玛,目录号:O-3756)
二水合氯化钙(Fisher Scientific,目录号:C79-500)
HEPES(Fisher Scientific,目录号:BP-310-1)
氯化钾(Fisher Scientific,目录号:P-217-500)
六水合氯化镁(Fisher Scientific,目录号:M33-500)
EGTA(Sigma,目录号:E-4378)
醋酸铵(Fisher Scientific,目录号:A637-500)
甘油(Fisher Scientific,目录号:G33-20)
十二烷基硫酸钠(Bio - Rad,目录号:161-0302)
Tris base(Fisher Scientific,目录号:BP152-5)
EDTA二钠二水合物盐(Macron Fine Chemicals,目录号:4931-04)
苯酚:氯仿:异戊醇(25:24:1,v / v / v)(Thermo Fisher Scientific,目录号:J75831-AN)
乙醇(Koptec,目录号:V1016)
琼脂糖(Promega,目录号:V3125)
硼酸(Fisher Scientific,目录号:BP-168-500)
溴化乙锭(Sigma,目录号:E-8751)
玻璃蒸馏水
用HiSpeed Plasmid Maxi Kit(QIAGEN,目录号:12662)制备的质粒DNA
QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
注意:Ť只有当DNA通过PCR扩增制备,需要他的产品。

组蛋白八聚体储备液(BPS Bioscences,目录号:52065;或通过以上引用的方法纯化)
pH 8.0的TE缓冲液(请参见配方)
10×TE Buffer,pH 8.0(请参见配方)
TBE缓冲液(请参阅配方)
10×TBE缓冲液(请参见配方)
橙色G加载缓冲液(请参见配方)
200 U / ml MNase库存(请参阅食谱)
MNase缓冲液R(请参见配方)
停止缓冲区(请参见配方)
HEG缓冲液(请参阅食谱)
10 mg / ml溴化乙锭储备溶液(请参阅食谱)


设备



    冰桶
    液氮杜瓦瓶
    磁力搅拌棒
    1 L气瓶(派热克斯(Pyrex),货号:3022)
    1 L烧杯(派热克斯(Pyrex),货号:1000)
    细尖移液管(Samco Scientific,目录号:231)
    10微升微管(的Rainin,型号:SL 10 XLS)
    20微升微管(的Rainin,型号:SL 20)
    200微升微管(的Rainin,型号:SL 200)
磁力搅拌器(Fisher Scientific,目录号:11-500-75 )
离心机(Epp endorf,型号:5424)
微型离心机(RPI,型号:PMC-060)
涡旋混合器(Fisher,目录号:12-812)
卧式凝胶电泳仪(Gibco,型号:H5)
电泳电源(Bio - Rad,目录号:Power Pac 200)
紫外线透射仪(Protein Simple,型号:Alpha Imager HP)


程序



A.染色质的重建     

在这里,我们描述了标准染色质重构实验的协议。在此协议中,假定事先已确定了最佳量的组蛋白和DNA。

为了用组蛋白和/或DNA的新制剂确定最佳的组蛋白:DNA比例,可以以0.5倍的规模执行该方案(也就是说,每个反应在75 µl的最终体积中有25 µg的DNA而不是在50 µg的DNA中)。最终反应体积为150 µl),以不同的组蛋白:DNA比例进行一系列反应。为此,我们通常以组蛋白:DNA质量比为0.80:1.0、0.90:1.0、1.0:1.0、1.1:1.0和1.2:1.0进行反应。

对于每个重构反应,准备一个150 µl组蛋白-DNA混合物,其中含有50 µg DNA和所需质量的组蛋白八聚物在TE Buffer (pH 8.0 )中,最终浓度为1.0 M NaCl。轻轻混合。然后,孵育用于在冰上15分钟。
在计算组蛋白-DNA混合物中NaCl的最终浓度时,必须考虑组蛋白储备液中盐的贡献。例如,可以从42 µl的1.2 mg / ml DNA储备液(50 µg DNA),42 µl的1.2 mg / ml组蛋白储备液中制备最终组蛋白:DNA比例为1.0:1.0的重组反应。含有2.5 M氯化钠(50微克组蛋白),9.0μ升的5.0 M氯化钠(以得到1.0M的NaCl的最终浓度),15μ升10 × TE缓冲液(即,1 × TE最终),和42μ升的水在150μ的最终体积升。

加载组蛋白DNA混合物˚F ROM小号TEP阿1到透析室。
透析室由样品池,透析膜和密封环组成(图2)。组装透析室如下:

一种。切微量离心管中(请参阅Ñ OTE 8) ,使得所述管的盖(即,样本池)由环(包围即,密封环)。     

b。通过切割样品容器之间的连接器,将其从密封环上拆下。然后将两块分开。     

C。切一块方形(约1.5厘米× 1.5厘米)的透析膜,然后用水冲洗以除去储存液。然后,将膜浸入含有0.80 M NaCl的TE Buffer中。     

d。用200 µl移液器将样品装入储液器。     

e。轻轻拍打纸巾上的边缘,从膜上排走多余的缓冲液。     

F。用膜覆盖样品。       

G。用密封环关闭腔室(即,将密封环放在膜的顶部,然后将其向下推到样品储存器上方)。     

H。用盖锁固定透析室,然后立即将其浸入含有0.80 M NaCl的TE Buffer中。透析膜应与透析缓冲液直接接触(即,透析室的膜侧朝下)。     





图2.透析室组装程序图。透析室由微量离心管和一小片透析膜组成,如下所示。A.切下微量离心管的顶部。B.分离盖子和仍然连接的那部分管子。盖子用作样品容器,短管用作密封环。C.吸取150微升样品到贮存器。用一小块透析膜(蓝色表示)覆盖样品。然后,将密封环放在膜的顶部。通过将密封环向下推到储液器上方来关闭透析室。D.完全组装的透析室。E.用盖锁固定透析室。



在室温(22°C)下分步进行透析以防止盐浓度降低,如下所示:
1 L含0.80 M NaCl的TE缓冲液2小时。
1 L的含0.60 M NaCl的TE缓冲液放置2小时。
1 L含0.05 M NaCl的TE缓冲液2小时。
笔记:

W¯¯母鸡传送不同的缓冲器之间的密封透析室,它是没有必要删除与透析室的外部相关联的过量缓冲液的少量。
小号everal重构反应可以同时在含有以中等速度1升透析缓冲液在磁力搅拌器的烧杯中进行透析。透析过程中,透析室应在缓冲液顶部周围缓慢旋转。
从含有0.050 M NaCl的TE缓冲液中取出透析室,将其放在纸巾上,使膜的一面朝上。
用200 µl移液器除去膜外侧剩余的过量TE缓冲液。然后,用新的尖端小心地在膜上打孔,然后将透析液(即重构的染色质)转移到微量离心管中。
将染色质保存在4°C。染色质在4 °C稳定数月。不要冻结染色质。


B.盐透析染色质中DNA浓度的定量     

随着描述的染色质重构过程小号挠度A,我们一般恢复大于90%的DNA(图3)的起始量的。对于许多应用,在核小体DNA的浓度可以被估计(在10%范围内通过以下描述并在图3中所示的方法)将A 260nm处上的读数(使用纯的双链DNA的消光系数)纳米滴一个Ç分光光度计(型号ND-ONEC-W; Thermo Fisher Scientific,Waltham,MA)。这可能是由于组蛋白的260nm吸光度低,因为它们的芳香族氨基酸残基含量低。或者,如下所述,可以通过除去组蛋白和定量所得的DNA样品来确定核小体DNA的浓度。相反,不建议使用Qubit荧光计,因为它不能提供对染色质样品中核小体DNA的准确定量,这可能是因为荧光染料分子与核小体DNA的结合效率不如其与游离DNA的结合。





图3.通过盐透析重构的染色质中DNA的定量。将盐渗析的染色质样品在含有0.05 M NaCl的TE缓冲液中稀释8倍,16倍和32倍。脱蛋白后的2.0μ升每三个染色质稀释液的等分试样,将得到的DNA样品,用已知浓度(20,40和80纳克裸DNA)的DNA标准一起,被进行琼脂糖凝胶电泳分析和可视化通过用溴化乙锭染色(EB )。使用ImageJ的凝胶分析功能进行定量后,核糖体DNA下的数字对应于2.0 µl染色质样品的32倍,16倍和8倍稀释度(从左到右)中的DNA计算量。软件(NIH,Beth es da,马里兰州)。在该实验中,重构的染色质在160 µl的最终体积中包含约0.30 µg / µl DNA (即48 µg DNA,约占染色质重构反应起始量的96%)。



透析后,使用200μl移液管测量样品的体积。与此方法中,所述盐透析期间由原始反应体积的约7%的体积增大(即,在染色质重构反应用150的初始体积进行微升和75微升,我们通常回收大约160微升和80微升分别)。
通过琼脂糖凝胶电泳对DNA进行定量,然后如下图所示用Image J进行分析。
制备40、20和10 ng / μl标准液(在含有0.05 M NaCl的TE缓冲液中)用于重建染色质的DNA储备液。这些标准品将用于绘制DNA标准曲线,以确定染色质样品中的DNA量(S tep B 2q)。
在含0.050 M NaCl的14μlTE缓冲液中稀释2.0μl重组染色质(即8倍稀释)。然后在相同的缓冲液中进行两次2倍系列稀释,以得到16倍和32倍稀释的染色质稀释液。
到2.0微升的40,20,和10ng / μl的DNA标准品(来自小号TEP乙2a)中,和2.0微升8-,16-,和染色质的32倍稀释液(从小号TEP乙2b)的,加入48μlHEG缓冲液,使每个样品的最终体积为50μl 。
向每个50μl样品中,添加100μl终止缓冲液和5.0μl2.5 mg / ml蛋白酶K。轻轻混合。在37孵育30分钟℃。
添加155微升的苯酚:氯仿:异戊醇并剧烈涡流30秒。
以16,000 × g离心5分钟。
将140μl水相转移至新的微量离心管中。
向水相中加入25μl的2.5 M乙酸铵和600μl的100%乙醇。颠倒管数次进行混合。
以16,000 × g离心20分钟。
用移液管移出上清液。然后,在微量离心机中短暂旋转试管,并用10μl移液器吸头除去所有残留的液体。
风干沉淀5分钟。
向沉淀中加入5.0μl的Orange G Loading Buffer。孵育为5分钟。轻轻涡旋,然后短暂旋转。
使DNA在3.7 V / cm的1.0%琼脂糖TBE凝胶上进行电泳,直到橙色G染料到达凝胶底部。运行时间将根据凝胶的维数而变化。例如,在水平电泳室中,阳极和阴极之间相隔27厘米,在100 V电压下,长5厘米× 6厘米× 0.5厘米(宽×长×高)的凝胶大约需要40分钟。要估算总电压,请将电泳室中电极之间的距离乘以所需的每厘米(cm)的电压(V)。在此示例中,27 cm × 3.7 V / cm = 100 V.
在EB染色溶液中将凝胶染色10分钟。
用水冲洗凝胶10分钟。
用紫外线透射仪观察DNA并以TIF格式记录DNA图像以进行定量分析。
如其他地方所述(https://imagej.nih.gov/ij/docs/menus/analyze.html#凝胶)。
注意:根据与每个样品对应的稀释倍数,调整凝胶上计算的DNA量。

为了获得染色质样品中的DNA浓度,将计算出的DNA总量除以透析步骤后回收的样品体积(来自S tep B 1)。


C.所述重构染色质MNase消化分析     

重组染色质的质量通过使用MNase消化测定法进行评估(图4和5)。





图4.确定通过盐渗析进行有效染色质重构的最佳组蛋白:DNA比率。染色质用组蛋白:DNA的质量比为0.80:1.0、0.90:1.0、1.0:1.0和1.1:1.0重构(即25 µg DNA与20 µg,22.5 µg,25 µg和27.5 µg组蛋白八聚体,分别使用图1中概述的盐渗析方法。为评估重构染色质的质量,将样品用四种不同浓度的MNase进行部分消化。脱蛋白后,通过琼脂糖凝胶电泳分离所得的DNA片段,并通过用溴化乙锭(EB)染色来可视化。箭头指示对应于单核小体,双核小体,三核小体,四核小体和五核小体的DNA条带。在该实验中,组蛋白:DNA比为0.90:1.0(黄点)和1.0:1.0(绿点)的四核和五核小体DNA片段的比例高于0.80:1.0(粉红色点)或1.1:1.0(蓝点)。对于许多应用,例如HDR介导的基因编辑(Cruz-Becerra和Kadonaga,2020年),最好使用组蛋白:DNA比率最低的染色质,产生最广泛的核小体阵列。在这种情况下,按MNase分析评估,0.90:1.0染色质和1.0:1.0染色质的质量相当,并且优于0.80:1.0染色质和1.1:1.0染色质。因此,推荐的组蛋白:DNA比率为0.90:1.0。DNA大小标记(M)是123 bp的阶梯(Millipore Sigma),由123 bp整数倍的条带组成。最小的123 bp带迁移比单核小体DNA带迁移快一些。





图5.盐透析染色质的MNase消化分析。通过使用图1中概述的盐渗析方法,用三种不同的核心组蛋白制剂和相同的质粒DNA模板(pBKS-GAPDH-HDR; 5.6 kbp; Cruz-Becerra和Kadonaga,2020)重构染色质。果蝇(Dm)0-12 h胚胎的核心组蛋白。使用Fyodorov和Levenstein(2002)的方法纯化组蛋白。B.用Khuong等人所述制备的HeLa核心组蛋白重构的染色质。(2017)(表示为样本A)。C.染色质与从BPS Bioscience(加利福尼亚州圣地亚哥)获得的HeLa核心组蛋白组装在一起(表示为样品B)。用四种不同浓度的MNase对染色质样品进行部分消化后,将样品脱蛋白,然后通过琼脂糖凝胶电泳分离所得的DNA片段,并通过溴化乙锭(EB)染色进行可视化。箭头指示对应于单核小体,双核小体,三核小体,四核小体和五核小体的DNA条带。的DNA大小标记(M)是在123 bp梯度(Millipore公司Sigma公司),其由作为123 bp的整数倍频段。最小的123 bp波段迁移的速度比单核小体DNA波段迁移的速度稍快。



用四种不同浓度的酶对重构的染色质(来自S tep A 5)进行部分MNase消化。每个MNase消化反应均包含35 µl染色质(5.7 ng / µl DNA; 200 ng总DNA),9.0 µl的10 mM CaCl 2和5.0 µl的各种浓度的MNase(例如1.3、0.67 , 0.33和0.17 mU / μl ;最佳浓度将随MNase的不同制备而变化,最终体积为49 µl 。反应设置如下:
染色质稀释的等分试样至5.7纳克/微升DNA在HEG缓冲液在175 μ升的最终体积。
在4 °C的水中用容器解冻200 U / ml MNase储备液的等分试样。解冻后,将酶储备置于冰上。
使MNase Buffer R中含有1.3、0.67、0.33和0.17 mU / μl的MNase工作储备液如下:
稀释200 U / ml的酶储备到13 MU / μl的在冷MNase缓冲器R(例如,2.0ü升200U /的毫升在30 MNase微升最终体积)。
使13亩的10倍稀释/微升MNase以获得1.3 MU /微升。
执行三个2倍连续稀释以1.3 MU /开始微升MNase得到0.67,0.33 ,和0.17 MU /微升。
在即将使用前,结合5.0微升的MNase工作原液与9.0微升的10mM的CaCl 2 ,得到的14的最终体积微升MNase-的CaCl 2混合。需要注意的是,有必要准备一个不同MNase-的CaCl 2混合用于每个从四个MNase工作储备的小号TEP Ç在这个部分1c。如果要同时分析几个染色质样品,则为样品总数加一个准备所需体积的每种MNase- CaCl 2混合物(例如,对于四个染色质样品,准备5 × 14 µl = 70μl的每种MNase- CaCl 2混合)。
加入14微升的MNase-的CaCl 2混合物至35微升的染色质(5.7纳克/微升DNA;从小号TEP Ç此部分1a)中,得到49μ的最终体积升。轻轻混合,然后短暂旋转。精确孵育10分钟。对每种MNase- CaCl 2混合物(和每种染色质样品)重复此步骤。
通过添加7.0停止MNase消化反应微升0.50M的EDTA(〜63毫摩尔EDTA终浓度)到每个管中。轻轻混合。
回收DNA,如下所示。
加入100μl终止缓冲液和5.0μl2.5 mg / ml蛋白酶K。轻轻混合。在37 °C下孵育40分钟。
添加155微升的苯酚:氯仿:异戊醇并剧烈涡流30秒。
以16,000 × g离心5分钟。
将140μl水相转移至新的微量离心管中。
向水相中加入25μl的2.5 M乙酸铵和600μl的100%乙醇。孵育10分钟。
以16,000 × g离心20分钟。
用移液管移出上清液。然后,在微型离心机中短暂旋转试管,并用移液器吸头除去所有残留的液体。
风干沉淀5分钟。
通过琼脂糖凝胶电泳分离DNA片段。
向沉淀中加入5.0μl的Orange G Loading Buffer。孵育为5分钟。轻轻涡旋,然后短暂旋转。
使DNA在1.3%琼脂糖TBE凝胶上以3.3 V / cm的速度进行电泳,直到橙色G染料到达凝胶底部为止(对于11 cm,14 cm和0.6 cm的凝胶,这大约需要2.5小时分别在宽度,长度和高度上)。
通过EB染色可视化DNA 。
在EB染色溶液中将凝胶染色30分钟。
用水除去过量的EB (通常在水中漂洗两次10分钟)。
用紫外线透射仪可视化DNA。


笔记



在“材料和试剂与设备”部分中提供的目录号,型号和制造商是我们用于执行此方法的特定项目的目录号,型号和制造商。该协议可以与其他来源的可比较项目一起执行。
在进行染色质重建实验之前,我们建议分别通过SDS-聚丙烯酰胺和琼脂糖凝胶电泳分析组蛋白和DNA制剂的质量。对于组蛋白,应观察到所有四个组蛋白的等摩尔化学计量,且不可检测到降解或污染。质粒DNA应该主要是超螺旋DNA,并且不应包含细菌基因组DNA。
建议使用浓度≥1 mg / ml的DNA和组蛋白原液。
避免核酸酶和蛋白酶污染材料和试剂。使用无核酸酶和蛋白酶的实验室塑料器皿和玻璃器皿,并在整个过程中戴上干净的手套。
仅使用低结合力的微量离心管以减少组蛋白和DNA对塑料的粘附。
轻拂试管将反应试剂混合,除非有涡旋现象。
除非另有说明,否则请在室温(22 °C )下执行所有步骤。
染色质可以在150的反应体积中重构μ升和75 μ升具有类似的结果。我们使用从1.7制备透析室毫升0.65毫升低结合管150 μ升或75 μ升分别重构反应,。这些透析室请勿使用较小的反应体积。
作为替代选择,可以使用Thermo Fisher Scientific的Sli de-A-Lyzer MINI透析装置(产品目录号:69550,Thermo Fisher Scientific,沃尔瑟姆,马萨诸塞州)代替此处所述的透析室。
我们建议对重构的染色质样品的电导率进行测量,以确定透析的完成程度。透析结束时,重组染色质的电导率应在含0.05 M NaCl的TE Buffer的电导率的10%以内。


菜谱



TE缓冲液,pH 8.0
10 mM Tris

1.0毫米EDTA

10 × TE缓冲液,pH 8.0
100 mM Tris

10毫米EDTA

TBE缓冲液
89 mM Tris底座

89毫米硼酸

EDTA 2.0毫米

10 × TBE缓冲器
83万Tris基地

0.89 M硼酸

EDTA 20毫米

橙色G加载缓冲液
含橘子G的6.25%(v / v)甘油(添加橘子G染料,直到溶液为中等深橙色)

1 × TBE缓冲器

200 U / ml MNase库存
将冻干的酶重悬于5.0 mM磷酸钠(pH 7.0)中,其中含有2.5 µM CaCl 2

制作小份

速冻在液氮中,并储存在- 80℃下

MNase缓冲液R
10 mM K + -HEPES,pH 7.6

10毫米氯化钾

1.5毫米MgCl 2

0.50毫米EGTA

10%(v / v)甘油

停止缓冲区
EDTA 20毫米

0.20 M氯化钠

1.0%(w / v)安全数据表

0.25毫克/毫升糖原

HEG缓冲液
25 mM K + -HEPES,pH 7.6

0.10毫米EDTA

10%(v / v)甘油

10 mg / ml溴乙锭储备溶液
将100毫克溴化乙锭溶于10毫升水中

储存在4℃;避光

1.0 µg / ml溴乙锭染色液
执行1 0,的000倍稀释10mg / ml的溴化乙锭的储备溶液在水



致谢



我们感谢Kadonaga实验室的所有以前的成员,他们在染色质组装方面的生化研究已作为此处所述方案的参考。我们也感谢Sharon Torigoe,George Kassavetis,Long Vo ngoc和Claudia Medrano对手稿的批判性阅读,并感谢Daniel Zamorano对图2的帮助。JTK是生命科学领域的Amylin主席。GC-B。是皮尤拉丁美洲博士后研究金的获得者,也是加利福尼亚大学圣地亚哥分校分子生物学癌症研究金的获得者。这项工作得到了美国国立卫生研究院(R35 GM118060)对JTK的资助支持。此处描述的协议用于通过Cruz-Becerra和Kadonaga的CRISPR-Cas9系统重构用于HDR介导的基因编辑的染色质供体模板(2020年)。



利益争夺



作者宣称没有竞争的经济利益小号。



参考



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Copyright Cruz-Becerra and Kadonaga. 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. Cruz-Becerra, G. and Kadonaga, J. T. (2021). Reconstitution of Chromatin by Stepwise Salt Dialysis. Bio-protocol 11(7): e3977. DOI: 10.21769/BioProtoc.3977.
  2. Cruz-Becerra, G. and Kadonaga, J. T. (2020). Enhancement of homology-directed repair with chromatin donor templates in cells.Elife 9: e55780.
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