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Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)
体内使用DNA结合分子介导染色质免疫沉淀法(enChIP)对于与目的基因组相互作用的蛋白质进行识别   

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参见作者原研究论文

本实验方案简略版
Scientific Reports
Nov 2013

Abstract

Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) for isolation of target genomic regions (Hoshino and Fujii, 2009; Fujita and Fujii, 2011; Fujita and Fujii, 2012; Fujita and Fujii, 2013a; Fujita and Fujii, 2013b; Fujita et al., 2013). Identification and characterization of molecules interacting with the isolated genomic regions facilitates understanding of molecular mechanisms of functions of the target genome regions.

Here, we describe enChIP, in which engineered DNA-binding molecules, such as zinc-finger proteins, transcription activator-like (TAL) proteins, and a catalytically inactive Cas9 (dCas9) plus small guide RNA (gRNA), are utilized for affinity purification of target genomic regions. The scheme of enChIP is as follows:
1. A zinc-finger protein, TAL or dCas9 plus gRNA is generated to recognize DNA sequence in a genomic region of interest.
2. The engineered DNA-binding molecule is fused with a tag(s) and the nuclear localization signal (NLS), and expressed in the cell to be analyzed.
3. The resultant cell is crosslinked, if necessary, and lysed, and DNA is fragmented.
4. The complexes including the engineered DNA-binding molecule are subjected to affinity purification such as mmunoprecipitation. The isolated complexes retain molecules interacting with the genomic region of interest.
5. Reverse crosslinking and subsequent purification of DNA, RNA, or proteins allow identification and characterization of these molecules.
In this protocol, we describe enChIP with a TAL protein to isolate a genomic region of interest and analyze the interacting proteins by mass spectrometry (Fujita et al., 2013).

Keywords: Chromatin immunoprecipitation (染色质免疫沉淀), ChIP (炸薯条), Locus-specific ChIP (位点特异的芯片), EnChIP (enchip), Chromatin (染色质)

Materials and Reagents

  1. Target cells
  2. 37% formaldehyde (Nacalai Tesque, catalog number: 16223-55 )
  3. Glycine (Sigma-Aldrich, catalog number: G7126 )
  4. NaCl (Sigma-Aldrich, catalog number: S9625 )
  5. Agarose S (Wako Pure Chemical Industries, catalog number: 318-01195 )
  6. 1 M Tris (pH 6.8) (AppliChem GmbH, catalog number: A4987 )
  7. 1 M Tris (pH 7.5) (AppliChem GmbH, catalog number: A4263 )
  8. 1 M Tris (pH 8.0) (AppliChem GmbH, catalog number: A4577 )
  9. 0.5 M EDTA (pH 8.0) (Nacalai Tesque, catalog number: 14362-95 )
  10. 0.1 M EGTA (Nacalai Tesque, catalog number: 08947-35 )
  11. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
  12. Triton X-100 (Nacalai Tesque, catalog number: 25987-85 )
  13. 10% SDS solution (Nacalai Tesque, catalog number: 30562-04 )
  14. 8 M LiCl solution (Nacalai Tesque, catalog number: 20077-84 )
  15. 2-mercaptoethanol (Nacalai Tesque, catalog number: 21438-82 )
  16. Sucrose (Nacalai Tesque, catalog number: 30404-45 )
  17. Bromophenol blue (Nacalai Tesque, catalog number: 05808-61 )
  18. Tween-20 (Sigma-Aldrich, catalog number: P5927 )
  19. Deoxycholic acid sodium salt monohydrate (Nacalai Tesque, catalog number: 10712-12 )
  20. 30% lauroylsarcosine (Nacalai Tesque, catalog number: 20135-14 )
  21. Dynabeads-protein G (Life Technologies, catalog number: DB10004 )
  22. Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F1804 )
  23. Normal mouse IgG (Santa Cruz, catalog number: sc-2025 )
  24. 10x phosphate buffered saline (PBS) (pH7.4) (Nacalai Tesque, catalog number: 27575-31 )
  25. PBS (10x dilution of 10x PBS with distilled water)
  26. BSA fraction V (7.5%) (Life Technologies, catalog number: 15260 )
  27. Complete, mini, EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 4693159 )
  28. 10 mg/ml RNase A (Sigma-Aldrich, catalog number: R6513 )
  29. 20 mg/ml Proteinase K (Roche Diagnostics, catalog number: 3115828 )
  30. 3x FLAG peptide (Sigma-Aldrich, catalog number: F4799 )
  31. 2-propanol (Nacalai Tesque, catalog number: 29113-95 )
  32. 3 M sodium acetate buffer solution (pH 5.2) (Nacalai Tesque, catalog number: 06893-24 )
  33. 20 mg/ml glycogen (Roche Diagnostics, catalog number: 901393 )
  34. 70% ethanol
  35. 4-20% mini-PROTEAN TGX gel (10 well, 50 µl) (Bio-Rad Laboratories, catalog number: 456-1094 )
  36. 10x running buffer solution for SDS-PAGE (Tris-Glycine) (Nacalai Tesque, catalog number: 30329-74 )
  37. Coomassie brilliant blue R-250 staining solution (Bio-Rad Laboratories, catalog number: 161-0436 )
  38. In-Gel Tryptic Digestion Kit (Thermo Fisher Scientific, catalog number: 89871 )
  39. 1.25 M glycine solution (200 ml) (see Recipes)
  40. 10% sodium deoxycholate (10 ml) (see Recipes)
  41. 10% Tween-20 (10 ml) (see Recipes)
  42. Cell lysis buffer (CLB) (40 ml) (see Recipes)
  43. Nuclear lysis buffer (NLB) (40 ml) (see Recipes)
  44. Modified lysis buffer 3 (MLB3) (10 ml) (see Recipes)
  45. PBS-T (10 ml) (see Recipes)
  46. PBS-T-BSA (10 ml) (see Recipes)
  47. 5% Triton X-100 (in MLB3) (5 ml) (see Recipes)
  48. Low salt buffer (LSB) (10 ml) (see Recipes)
  49. High salt buffer (HSB) (10 ml) (see Recipes)
  50. LiCl buffer (20 ml) (see Recipes)
  51. TBS-IGEPAL CA-630 (10 ml) (see Recipes)
  52. Elution buffer (500 µl) (see Recipes)
  53. 2x sample buffer (50 ml) (see Recipes)

Equipment

  1. Magnetic stand (magical trapper) (TOYOBO, catalog number: MGS-101 )
  2. Centrifuge
  3. 1.5 ml centrifuge tube (Sarstedt AG, catalog number: 72.690.001 )
  4. 2 ml centrifuge tube (Eppendorf, catalog number: 0030.120.094 )
  5. 50 ml centrifuge tube (BD Biosciences, catalog number: 352070 )
  6. Vortex mixer
  7. Rotator
  8. Vaccum pump connected to a side-arm flask
  9. Sonicator (ultrasonic disruptor UD-201 ) (TOMY SEIKO, catalog number: UD-201)
  10. Shaker
  11. Mass spectrometry facility: a nanoLC-MS/MS system, composed of LTQ Orbitrap Velos (Thermo Fisher Scientific) coupled with nanoLC (Advance CaptiveSpray SOURCE, Michrom BioResources) and HTC-PAL autosampler (CTC Analytics)

Procedure

  1. Preparation of cells
    1. Design the DNA-binding modules of a TAL protein recognizing a specific sequence (approximately 20 bases) in your target genomic region.
    2. Express the TAL protein fused with 3x FLAG tags and the NLS in the cell to be analyzed. An example of the tagged TAL proteins for enChIP is shown in supplemental Figure 1 in the reference (Fujita et al., 2013).

  2. Formaldehyde crosslinking of cells
    1. Culture target cells. Use 2 x 107 cells x 2 (total 4 x 107 cells) (e.g. Ba/F3) for chromatin preparation. Collect cells suspended in culture medium (2 x 107 cells/30 ml) in a 50 ml centrifuge tube (if necessary, increase cell number). Prepare 2 tubes (total 4 x 107 cells).
    2. Add 810 µl of 37% formaldehyde to 1% final concentration into 30 ml of the cell suspension. Formaldehyde solution is harmful, so handle it wearing gloves in a chemical hood. Incubate at 37 °C for 5-10 min (usually 5 min). Crosslinking makes color of cell suspension slightly yellowish.
    3. Stop crosslinking by adding 3.1 ml of 1.25 M glycine solution to 127 mM final concentration. Incubate at room temperature for 10 min.
    4. Collect cells by centrifugation (300 x g, 4 °C, 5 min). Discard carefully the supernatant including formaldehyde by decantation, and store it in a waste bottle.
    5. Resuspend the fixed cells (pellet) in 30 ml of PBS by vortexing. Collect cells by centrifugation (300 x g, 4 °C, 5 min). Discard carefully the supernatant including formaldehyde by decantation, and store it in a waste bottle.
    6. Repeat the step B5 (total twice). The formaldehyde wastes should be handled according to the chemical safety guideline of your laboratory.
    7. Proceed to the step C1, or the centrifuge tube with the fixed cells (pellet) can be frozen and stored at -80 °C.

  3. Preparation of chromatin (per 2 x 107 cells)
    1. Resuspend the fixed cells in 10 ml of CLB. If the fixed cells are frozen in the step B6, it should be thawed before addition of CLB. Incubate on ice for 10 min.
    2. Centrifuge at 930 x g at 4 °C for 8 min. Discard carefully the supernatant by vacuum suction.
    3. Resuspend the pellet in 10 ml of NLB. Incubate on ice for 10 min. Vortex at maximum setting for 2-3 sec every 2-3 min.
    4. Centrifuge at 930 x g at 4 °C for 8 min. Discard carefully the supernatant by vacuum suction.
    5. Resuspend the pellet in 10 ml of PBS. Centrifuge at 930 x g at 4 °C for 10 min. Discard carefully the supernatant by vacuum suction. The pellet is used as the chromatin fraction.
    6. Proceed to the step D1 or the centrifuge tube with the chromatin fraction can be stored at -80 °C after immediate freezing in liquid nitrogen.

  4. Sonication of chromatin (per 2 x 107 cells)
    1. Resuspend the collected chromatin fraction in 800 µl of MLB3. Transfer the suspension into a 1.5 ml microtube.
    2. Sonicate the chromatin by using ultrasonic disruptor UD-201. Conditions are as follows:
      Output: 3
      Duty: 100% (continuous)
      Time: Free
      10-18 cycles of sonication for 10 sec and cooling on ice for 20 sec
      2 min on ice after 5-6 cycles to avoid excessive heating
      Keep the position of the tip of the sonication bar approximately 0.5 cm away from the tube bottom using a clamp or hands to avoid foaming.
    3. Centrifuge at 16,000 x g at 4 °C for 10 min. Transfer the supernatant (800 µl) into a 1.5 ml tube. The 1.5 ml tube with the supernatant can be stored at -80 °C after immediate freezing in liquid nitrogen.

  5. Evaluation of fragmentation of chromatin
    1. Resuspend 10 µl of the fragmented chromatin in 85 µl of distilled water.
    2. Add 4 µl of 5 M NaCl. Incubate at 65 °C overnight.
    3. Add 1 µl of 10 mg/ml RNase A. Incubate at 37 °C for 45 min.
    4. Add 2 µl of 0.5 M EDTA (pH 8.0), 4 µl of 1 M Tris (pH 6.8), and 1 µl of 20 mg/ml Proteinase K. Incubate at 45 °C for 1.5 h.
    5. Prepare 1% agarose gel without staining dye.
    6. Use 10 µl of the sample prepared in the step E4 for electrophoresis in 1% agarose gel without staining dye. Run the gel at 100 V for 30 min. 1 kbp DNA ladder marker should be loaded to estimate DNA fragmentation.
    7. Stain the gel with staining dye (e.g. ethidium bromide) for 0.5-1 h (Figure 1A). An example of DNA fragmentation is shown in Figure 1B. The acceptable range of DNA fragmentation is dependent on the length of your target genomic region. Usually, the range of DNA fragmentation should be generally 4-0.2 kbp (The average length of DNA fragments should be 0.5-2 kbp). If DNA is poorly or excessively fragmented, adjust output or cycles of sonication in the step D2.


      Figure 1. Gel staining and an example of DNA fragmentation. A. Gel staining with staining dye using a shaker (speed: one turn/1-2 sec). B. An example of DNA fragmentation. M: DNA ladder marker.

  6. Preparation of Dynabeads conjugated with antibody (anti-FLAG antibody or normal mouse IgG)
    1. Prepare two 2 ml tubes; one is for anti-FLAG antibody and the other is for normal mouse IgG. Add 300 µl of Dynabeads-Protein G in each tube (300 µl x 2 tubes). In this protocol, we describe preparation of one sample for analysis by mass spectrometry. In this regard, normal mouse IgG is used for pre-clear in the step G3 and anti-FLAG antibody is used for affinity purification in the step G5. If you analyze another sample as a negative control (e.g. affinity purification with normal mouse IgG instead of anti-FLAG antibody), you may prepare chromatin and Dynabeads for it separately.
    2. Put the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting. Handling of a magnet stand is shown in Figure 2.


      Figure 2. Handling of a magnet stand in wash steps. A. Suspension of Dynabeads in a 2 ml tube. B. Setting of the tube on a magnet stand. C. 3 min after the setting. D. Removal of the supernatant by pipetting.

    3. Resuspend the pellet in 1 ml of PBS-T. Put the tubes on a magnet stand and wait for 2 min. Discard the supernatant by pipetting.
    4. Repeat the step F3 (total twice).
    5. Resuspend the pellet in 1.5 ml of PBS-T-BSA.
    6. Add 30 µg of antibody. One tube is for 30 µg of anti-FLAG antibody, and the other is for 30 µg of normal mouse IgG. Rotate at 4 °C overnight.
    7. Centrifuge briefly (400 x g for 2-3 sec). Put the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.
    8. Resuspend the pellet in 1.5 ml of PBS-T. Invert several times and centrifuge briefly (400 x g for 2-3 sec). Put the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.
    9. Repeat the step F8, twice (total three times). The Dynabeads are ready for the next step.

  7. Chromatin immunoprecipitation
    1. Transfer 1.6 ml of the fragmented chromatin, which corresponds to chromatin extracted from 4 x 107 cells, into a 2 ml tube.
    2. Add 400 µl of 5% Triton X-100 (in MLB3) to 1% final concentration.
    3. Add chromatin solution prepared in the step G2 (2 ml) in the tube, in which the Dynabeads conjugated with normal mouse IgG were prepared. Rotate at 4 °C for 1 h.
    4. Put the tube on a magnet stand and wait for 3 min.
    5. Transfer the supernatant into the tube, in which the Dynabeads conjugated with anti-FLAG antibody were prepared. Rotate at 4 °C overnight.
    6. Put the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.
    7. Wash 1: Add 1.8 ml of LSB. Rotate at 4 °C for 5 min. Put the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.
    8. Repeat wash 1 (total twice).
    9. Wash 2: Wash with HSB as described in the step G7. Repeat this wash (total twice).
    10. Wash 3: Wash with LiCl buffer as described in the step G7. Repeat this wash (total twice).
    11. Add 1.8 ml of TBS-IGEPAL CA-630. Rotate at 4 °C for 5 min. Put the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.
    12. Elution: Resuspend the pellet in 200 µl of elution buffer. Incubate at 37 °C for 20 min. Put the tube on a magnet stand and wait for 3 min.
    13. Add the supernatant (200 µl) in the 1.5 ml tube with 0.5 ml of 2-propanol, 25 µl of 3 M Sodium acetate buffer solution (pH 5.2), and 5 µl of 20 mg/ml glycogen. Precipitate proteins at -20 °C overnight.
    14. Centrifuge at 16,000 x g at 4 °C for 30 min. Discard the supernatant.
    15. Rinse with 1 ml of 70% ethanol. Centrifuge at 16,000 x g at 4 °C for 10 min. Discard the supernatant completely by pipetting.
    16. Add 40 µl of 2x sample buffer. Vortex at maximum setting for 5 min to completely dissolve the precipitate. Incubate at 100 °C for 30 min (protein denaturing and reverse-crosslinking).

  8. SDS-PAGE, staining, mass analysis
    1. SDS-PAGE. Run the eluate on polyacrylamide gel until the dye reaches 1 cm from the well.
    2. Coomassie brilliant blue (CBB) staining or silver staining. An example of gel images is shown in Figure 3.


      Figure 3. An example of gel images after CBB staining. The original image is shown in Supplemental Figure 5 in the reference (Fujita et al., 2013).

    3. Cut the gel into 5 pieces x 2 mm.
    4. In gel digestion and mass analysis. You can use In-Gel Tryptic Digestion Kit. Our current system of mass analysis is a nanoLC-MS/MS system, composed of LTQ Orbitrap Velos coupled with nanoLC and HTC-PAL autosampler.

Recipes

  1. 1.25 M glycine solution (200 ml)
    Glycine (MW: 75.07)
    18.8 g
    H2O
    to 200 ml
  2. 10% sodium deoxycholate (10 ml)
    Deoxycholic acid sodium salt monohydrate
    1 g
    H2O
    to 10 ml
  3. Cell lysis buffer (CLB) (40 ml)
    10 mM Tris (pH 8.0), 1 mM EDTA, 0.5% IGEPAL CA-630, 1x protease inhibitors
    1 M Tris (pH 8.0)
    400 µl
    0.5 M EDTA
    80 µl
    IGEPAL CA-630
    200 µl
    Complete, mini, EDTA-free
    4 tablets
    Double distilled water (DDW)
    39.32 ml
  4. Nuclear lysis buffer (NLB) (40 ml)
    10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% lauroylsarcosine, 1x protease inhibitors
    1 M Tris (pH 8.0)
    400 µl
    0.5 M EDTA
    80 µl
    5 M NaCl
    4 ml
    Triton X-100
    400 µl
    10% sodium deoxycholate
    2 ml
    30% lauroylsarcosine
    666 µl
    Complete, mini, EDTA-free
    4 tablets
    DDW
    32.46 ml
  5. Modified lysis buffer 3 (MLB3) (10 ml)
    10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1x protease inhibitors
    1 M Tris (pH 8.0)
    100 µl
    0.5 M EDTA
    20 µl
    0.1 M EGTA
    50 µl
    5 M NaCl
    300 µl
    10% sodium deoxycholate
    100 µl
    10% SDS
    100 µl
    Complete, mini, EDTA-free
    1 tablet
    DDW
    9.33 ml
  6. 10% Tween-20 (10 ml)
    Tween-20
    1 ml
    DDW
    9 ml
  7. PBS-T (10 ml)
    PBS (pH 7.4), 0.01% Tween-20
    PBS
    10 ml
    10% Tween-20
    10 µl
  8. PBS-T-BSA (10 ml)
    PBS (pH 7.4), 0.01% Tween-20, 0.1% BSA
    PBS
    10 ml
    10% Tween-20
    10 µl
    7.5% BSA fraction V
    133 µl
  9. 5% Triton X-100 (in MLB3) (5 ml)
    MLB3
    4.75 ml
    Triton X-100
    250 µl
  10. Low salt buffer (LSB) (10 ml)
    20 mM Tris (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS
    1 M Tris (pH 8.0)
    200 µl
    0.5 M EDTA
    40 µl
    5 M NaCl
    300 µl
    Triton X-100
    100 µl
    10% SDS
    100 µl
    DDW
    9.26 ml
  11. High salt buffer (HSB) (10 ml)
    20 mM Tris (pH 8.0), 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS
    1 M Tris (pH 8.0)
    200 µl
    0.5 M EDTA
    40 µl
    5 M NaCl
    1 ml
    Triton X-100
    100 µl
    10% SDS
    100 µl
    DDW
    8.56 ml
  12. LiCl buffer (20 ml)
    10 mM Tris (pH 8.0), 1 mM EDTA, 0.25 M LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate
    1 M Tris (pH 8.0)
    200 µl
    0.5 M EDTA
    40 µl
    8 M LiCl
    625 µl
    IGEPAL CA-630
    100 µl
    10% sodium deoxycholate
    1 ml
    DDW
    18.035 ml
  13. TBS-IGEPAL CA-630 (10 ml)
    50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% IGEPAL CA-630
    1 M Tris (pH 7.5)
    500 µl
    5 M NaCl
    300 µl
    IGEPAL CA-630
    10 µl
    DDW
    9.19 ml
  14. Elution buffer (500 µl)
    50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% IGEPAL CA-630, 500 µg/ml 3x FLAG peptide
    5 mg/ml 3x FLAG peptide in TBS
    50 µl
    TBS-IGEPAL CA-630
    450 µl
  15. 2x sample buffer (50 ml)
    125 mM Tris (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue
    1 M Tris-HCl (pH 6.8)
    6.25 ml
    2-mercaptoethanol
    5 ml
    SDS
    2 g
    Sucrose
    5 g
    Bromophenol blue
    2 mg
    DDW
    to 50 ml

Acknowledgments

This protocol was adapted from the previously published paper (Fujita et al., 2013). We thank F. Kitaura for technical assistance. This work was supported by Research Institute for Microbial Diseases (T.F. and H.F.), Program for Combined Research Fields from Immunology Frontier Research Center, Osaka University (H.F.), the Takeda Science Foundation (H.F. and T.F.), the Asahi Glass Foundation (H.F.), A-STEP from Japan Science and Technology Agency (H.F.), Grant-in-Aid for Young Scientists (B) (#22710185) (T.F.), Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Cell Fate’’ (#23118516) (T.F.), ‘‘The Genofield’’ (#23114707) (H.F.), ‘‘Transcription Cycle’’ (25118512) (H.F.).

References

  1. Fujita, T. and Fujii, H. (2011). Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One 6(10): e26109.
  2. Fujita, T. and Fujii, H. (2012). Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Advances Biosci Biotechnol 3(5): 626-629.
  3. Fujita, T. and Fujii, H. (2013a). Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun 439(1): 132-136.
  4. Fujita, T. and Fujii, H. (2013b). Locus-specific biochemical epigenetics/chromatin biochemistry by insertional chromatin immunoprecipitation. ISRN Biochem 2013, Article ID 913273.
  5. Fujita, T., Asano, Y., Ohtsuka, J., Takada, Y., Saito, K., Ohki, R. and Fujii, H. (2013). Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci Rep 3: 3171.
  6. Hoshino, A. and Fujii, H. (2009). Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J Biosci Bioeng 108(5): 446-449. 

简介

阐明基因组功能的分子机制需要在体内鉴定与感兴趣的基因组区域相互作用的分子。为此,分离保持分子相互作用的靶区是有用的。我们建立由插入ChIP(iChIP)和工程化的DNA结合分子介导的ChIP(enChIP)组成的基因组特异性染色质免疫沉淀(ChIP)技术用于靶基因组区域的分离(Hoshino和Fujii,2009; Fujita和Fujii,和Fujii,2012; Fujita和Fujii,2013a; Fujita和Fujii,2013b; Fujita等人,2013)。与分离的基因组区域相互作用的分子的鉴定和表征有助于理解靶基因组区域的功能的分子机制。在这里,我们描述enChIP,其中工程化的DNA结合分子,如锌指蛋白,转录激活样(TAL)蛋白和催化失活的Cas9(dCas9)加上小指南RNA(gRNA),被用于亲和纯化靶基因组区。 enChIP的方案如下所示:
1。产生锌指蛋白,TAL或dCas9加gRNA以识别感兴趣的基因组区域中的DNA序列。
2。工程化的DNA结合分子与标签和核定位信号(NLS)融合,并在待分析的细胞中表达。如果需要,所得细胞被交联,并裂解,DNA被片段化。将包括工程化DNA结合分子的复合物进行亲和纯化,例如免疫沉淀。分离的复合物保留分子与感兴趣的基因组区域相互作用。 DNA,RNA或蛋白质的反向交联和随后的纯化允许这些分子的鉴定和表征。在本方案中,我们描述了具有TAL蛋白的enChIP,以分离感兴趣的基因组区域并通过质谱分析相互作用蛋白(Fujita et al。,2013)。

关键字:染色质免疫沉淀, 炸薯条, 位点特异的芯片, enchip, 染色质

材料和试剂

  1. 目标单元格
  2. 37%甲醛(Nacalai Tesque,目录号:16223-55)
  3. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  4. NaCl(Sigma-Aldrich,目录号:S9625)
  5. Agarose S(Wako Pure Chemical Industries,目录号:318-01195)
  6. 1M Tris(pH6.8)(AppliChem GmbH,目录号:A4987)
  7. 1M Tris(pH7.5)(AppliChem GmbH,目录号:A4263)
  8. 1M Tris(pH 8.0)(AppliChem GmbH,目录号:A4577)
  9. 0.5M EDTA(pH8.0)(Nacalai Tesque,目录号:14362-95)
  10. 0.1M EGTA(Nacalai Tesque,目录号:08947-35)
  11. IGEPAL CA-630(Sigma-Aldrich,目录号:I8896)
  12. Triton X-100(Nacalai Tesque,目录号:25987-85)
  13. 10%SDS溶液(Nacalai Tesque,目录号:30562-04)
  14. 8 M LiCl溶液(Nacalai Tesque,目录号:20077-84)
  15. 2-巯基乙醇(Nacalai Tesque,目录号:21438-82)
  16. 蔗糖(Nacalai Tesque,目录号:30404-45)
  17. 溴酚蓝(Nacalai Tesque,目录号:05808-61)
  18. Tween-20(Sigma-Aldrich,目录号:P5927)
  19. 脱氧胆酸钠盐一水合物(Nacalai Tesque,目录号:10712-12)
  20. 30%月桂酰肌氨酸(Nacalai Tesque,目录号:20135-14)
  21. Dynabeads-protein G(Life Technologies,目录号:DB10004)
  22. 抗FLAG M2抗体(Sigma-Aldrich,目录号:F1804)
  23. 正常小鼠IgG(Santa Cruz,目录号:sc-2025)
  24. 10x磷酸盐缓冲盐水(PBS)(pH7.4)(Nacalai Tesque,目录号:27575-31)
  25. PBS(10×PBS用蒸馏水稀释10倍)
  26. BSA级分V(7.5%)(Life Technologies,目录号:15260)
  27. Complete,mini,无EDTA蛋白酶抑制剂(Roche Diagnostics,目录号:4693159)
  28. 10mg/ml RNA酶A(Sigma-Aldrich,目录号:R6513)
  29. 20mg/ml蛋白酶K(Roche Diagnostics,目录号:3115828)
  30. 3x FLAG肽(Sigma-Aldrich,目录号:F4799)
  31. 2-丙醇(Nacalai Tesque,目录号:29113-95)
  32. 3 M乙酸钠缓冲液(pH 5.2)(Nacalai Tesque,目录号:06893-24)
  33. 20mg/ml糖原(Roche Diagnostics,目录号:901393)
  34. 70%乙醇
  35. 4-20%微型PROTEAN TGX凝胶(10孔,50μl)(Bio-Rad Laboratories,目录号:456-1094)
  36. 用于SDS-PAGE(Tris-甘氨酸)(Nacalai Tesque,目录号:30329-74)的10x运行缓冲液溶液
  37. 考马斯亮蓝R-250染色溶液(Bio-Rad Laboratories,目录号:161-0436)
  38. 凝胶胰蛋白酶消化试剂盒(Thermo Fisher Scientific,目录号:89871)
  39. 1.25M甘氨酸溶液(200ml)(参见配方)
  40. 10%脱氧胆酸钠(10ml)(参见配方)
  41. 10%Tween-20(10ml)(参见配方)
  42. 细胞裂解缓冲液(CLB)(40ml)(参见配方)
  43. 核裂解缓冲液(NLB)(40ml)(参见配方)
  44. 改性裂解缓冲液3(MLB3)(10ml)(参见配方)
  45. PBS-T(10ml)(参见配方)
  46. PBS-T-BSA(10ml)(参见配方)
  47. 5%Triton X-100(在MLB3中)(5ml)(参见配方)
  48. 低盐缓冲液(LSB)(10ml)(见配方)
  49. 高盐缓冲液(HSB)(10ml)(见配方)
  50. LiCl缓冲液(20ml)(见配方)
  51. TBS-IGEPAL CA-630(10ml)(参见配方)
  52. 洗脱缓冲液(500μl)(参见配方)
  53. 2x样品缓冲液(50ml)(见配方)

设备

  1. 磁性架(魔法捕集器)(TOYOBO,目录号:MGS-101)
  2. 离心机
  3. 1.5ml离心管(Sarstedt AG,目录号:72.690.001)
  4. 2ml离心管(Eppendorf,目录号:0030.120.094)
  5. 50ml离心管(BD Biosciences,目录号:352070)
  6. 涡流搅拌器
  7. 旋转器
  8. 真空泵连接到侧臂瓶
  9. 超声波破碎机(超声破碎机UD-201)(TOMY SEIKO,目录号:UD-201)
  10. 振动器
  11. 质谱设备:由与nanoLC(Advance CaptiveSpray SOURCE,Michrom BioResources)和HTC-PAL自动进样器(CTC Analytics)结合的LTQ Orbitrap Velos(Thermo Fisher Scientific)构成的nanoLC-MS/MS系统

程序

  1. 细胞的制备
    1. 设计识别目标基因组区域中特定序列(约20个碱基)的TAL蛋白的DNA结合模块。
    2. 在待分析的细胞中表达与3x FLAG标签和NLS融合的TAL蛋白。 enChIP的标记TAL蛋白的实例在参考文献(Fujita等人,2013)中的补充图1中示出。

  2. 甲醛交联的细胞
    1. 培养靶细胞。 使用2×10 7个细胞×2(总共4×10 7个细胞)(例如Ba/F3)用于染色质制备。 收集悬浮在培养基(2×10 7个细胞/30ml)中的细胞在50ml离心管中(如果需要,增加细胞数)。 准备2个管(总共4×10 7个细胞)
    2. 加入810微升37%甲醛至1%终浓度至30毫升的细胞悬浮液。甲醛溶液有害,因此在化学罩中戴手套处理。在37℃孵育5-10分钟(通常为5分钟)。交联使细胞悬浮液的颜色略微发黄。
    3. 通过加入3.1ml 1.25M甘氨酸溶液至终浓度127mM来终止交联。在室温下孵育10分钟。
    4. 通过离心(300×g,4℃,5分钟)收集细胞。通过倾析小心地弃去上清液,包括甲醛,并将其存储在废液瓶中
    5. 通过涡旋将固​​定的细胞(沉淀)重悬在30ml PBS中。通过离心(300×g,4℃,5分钟)收集细胞。通过倾析小心地弃去上清液,包括甲醛,并将其存储在废物瓶中。
    6. 重复步骤B5(总共两次)。甲醛废物应根据实验室的化学安全指南进行处理。
    7. 继续步骤C1,或者可以将具有固定的细胞(沉淀)的离心管冷冻并储存在-80℃。

  3. 染色质的制备(每2×10 7个细胞)
    1. 将固定的细胞重悬在10ml CLB中。 如果在步骤B6中固定的细胞被冷冻,则在添加CLB之前应该将其解冻。 在冰上孵育10分钟。
    2. 在4℃下以930×g离心8分钟。 通过真空抽吸小心弃去上清液。
    3. 将沉淀重悬于10ml NLB中。 在冰上孵育10分钟。 每2-3分钟在最大设置涡旋2-3秒。
    4. 在4℃下以930×g离心8分钟。 通过真空抽吸小心弃去上清液。
    5. 将沉淀重悬在10ml PBS中。 在4℃下以930×g离心10分钟。 通过真空抽吸小心地弃去上清液。 将沉淀用作染色质级分。
    6. 进入步骤D1或带有染色质部分的离心管在液氮中立即冷冻后可储存在-80℃。

  4. 染色质的超声处理(每2×10 7个细胞)
    1. 重新悬浮收集的染色质部分在800μl的MLB3。 将悬浮液转移到1.5 ml微管中
    2. 使用超声波破碎机UD-201超声处理染色质。 条件如下:
      输出:3
      税率:100%(连续)
      时间:免费
      10-18个循环的超声处理10秒,并在冰上冷却20秒 在冰上2分钟,5-6个循环后,以避免过度加热
      使用夹子或手保持超声处理棒尖端的位置离管底约0.5cm,以避免起泡。
    3. 在4℃下以16,000×g离心10分钟。 转移上清(800微升)到1.5毫升管。 具有上清液的1.5ml管可以在液氮中立即冷冻后储存在-80℃
  5. 染色质断裂的评价
    1. 重悬10微升碎片染色质在85微升蒸馏水。
    2. 加入4μl的5 M NaCl。 在65℃孵育过夜。
    3. 加入1微升10毫克/毫升核糖核酸酶A.在37℃下孵育45分钟
    4. 加入2μl0.5M EDTA(pH8.0),4μl1M Tris(pH6.8)和1μl20mg/ml蛋白酶K.在45℃下孵育1.5小时。
    5. 准备1%琼脂糖凝胶,不染色染料
    6. 使用10μl在步骤E4中制备的样品在不染色染料的1%琼脂糖凝胶中电泳。在100 V下运行凝胶30分钟。应加载1 kbp DNA梯形图标记以估计DNA断裂
    7. 用染色染料(例如溴化乙锭)染色凝胶0.5-1小时(图1A)。 DNA断裂的一个例子如图1B所示。 DNA片段化的可接受范围取决于靶基因组区域的长度。通常,DNA断裂的范围通常应为4-0.2kbp(DNA片段的平均长度应为0.5-2kbp)。如果DNA差或过分碎片,在步骤D2中调整超声处理的输出或循环

      图1.凝胶染色和DNA断裂的实施例 A.使用振荡器(染色染料)进行凝胶染色(速度:1圈/1-2秒)。 B.DNA断裂的实例。 M:DNA梯形标记。

  6. 制备与抗体缀合的Dynabeads(抗FLAG抗体或正常小鼠IgG)
    1. 准备两个2毫升管;一个用于抗FLAG抗体,另一个用于正常小鼠IgG。在每个管中加入300μlDynabeads-Protein G(300μl×2管)。在该协议中,我们描述了用于通过质谱法分析的一个样品的制备。在这点上,在步骤G3中使用正常小鼠IgG用于预清除,在步骤G5中将抗FLAG抗体用于亲和纯化。如果您分析另一个样品作为阴性对照(例如使用正常小鼠IgG而不是抗FLAG抗体进行亲和纯化),您可以单独制备染色质和Dynabeads。
    2. 将管放在磁力架上,等待3分钟。通过吸移弃上清。磁铁架的处理如图2所示。


      图2.在洗涤步骤中处理磁体架。 A.将Dynabeads悬浮在2ml管中。 B.在磁体座上设置管。 C.设置后3分钟。 D.通过移液除去上清液。

    3. 将沉淀重悬于1ml PBS-T中。 将管放在磁力架上,等待2分钟。 通过移液除去上清液。
    4. 重复步骤F3(总共两次)。
    5. 将沉淀重悬于1.5ml PBS-T-BSA中。
    6. 加入30μg抗体。 一管用于30μg抗FLAG抗体,另一管用于30μg正常小鼠IgG。 在4℃下旋转过夜。
    7. 短暂离心(400×g/2-3秒)。 将管放在磁力架上,等待3分钟。 通过移液除去上清液。
    8. 将沉淀重悬于1.5ml PBS-T中。 倒置几次并短暂离心(400×g/2-3秒)。 将管放在磁力架上,等待3分钟。 弃去上清液 移液。
    9. 重复步骤F8,两次(总共三次)。 Dynabeads准备好下一步。

  7. 染色质免疫沉淀
    1. 将1.6ml碎片染色质(其对应于从4×10 7个细胞提取的染色质)转移到2ml管中。
    2. 加入400μl5%Triton X-100(在MLB3中)至1%终浓度
    3. 将在步骤G2中制备的染色质溶液(2ml)加入试管中,其中制备与正常小鼠IgG缀合的Dynabeads。 在4℃下旋转1小时。
    4. 将管放在磁力架上,等待3分钟。
    5. 将上清液转移到管中,其中制备与抗FLAG抗体缀合的Dynabeads。 在4℃下旋转过夜。
    6. 将管放在磁力架上,等待3分钟。 通过移液除去上清液。
    7. 洗涤1:加入1.8ml LSB。 在4℃下旋转5分钟。 将管放在磁力架上,等待3分钟。 通过吸移弃上清。
    8. 重复清洗1(总共两次)。
    9. 洗涤2:如步骤G7中所述用HSB洗涤。 重复此洗涤(共两次)。
    10. 洗涤3:如步骤G7中所述用LiCl缓冲液洗涤。 重复此洗涤(共两次)。
    11. 加入1.8ml TBS-IGEPAL CA-630。 在4℃下旋转5分钟。 将管放在磁力架上,等待3分钟。 通过移液除去上清液。
    12. 洗脱:将沉淀重悬在200μl洗脱缓冲液中。 在37℃孵育20分钟。 将管放在磁力架上,等待3分钟。
    13. 将上清液(200μl)加入1.5ml管中,加入0.5ml 2-丙醇,25μl3M乙酸钠缓冲液(pH5.2)和5μl20mg/ml糖原。 在-20℃下沉淀蛋白过夜
    14. 在4℃下以16,000×g离心30分钟。 弃去上清液。
    15. 用1ml 70%乙醇冲洗。 在4℃下以16,000×g离心10分钟。 通过移液完全弃去上清液。
    16. 加入40μl的2x样品缓冲液。 在最大设置下涡旋5分钟以完全溶解沉淀。 在100℃孵育30分钟(蛋白质变性和逆交联)
  8. SDS-PAGE,染色,质量分析
    1. SDS-PAGE。 在聚丙烯酰胺凝胶上运行洗脱液,直到染料从孔中达到1cm。
    2. 考马斯亮蓝(CBB)染色或银染色。 凝胶图像的一个例子如图3所示。


      图3. CBB染色后的凝胶图像的实例。原始图像显示在参考文献中的补充图5中(Fujita等人,2013)。

    3. 将凝胶切成5片×2毫米。
    4. 在凝胶消化和质量分析。 您可以使用凝胶胰蛋白酶消化试剂盒。 我们目前的质谱分析系统是一个nanoLC-MS/MS系统,由LTQ Orbitrap Velos与nanoLC和HTC-PAL自动进样器组成。

食谱

  1. 1.25M甘氨酸溶液(200ml)
    甘氨酸(MW:75.07)
    18.8克
    H sub 2 O
    到200ml
  2. 10%脱氧胆酸钠(10ml)
    脱氧胆酸钠盐一水合物 1克
    H sub 2 O
    到10 ml
  3. 细胞裂解缓冲液(CLB)(40ml) 10mM Tris(pH 8.0),1mM EDTA,0.5%IGEPAL CA-630,1×蛋白酶抑制剂
    1 M Tris(pH 8.0)
    400μl
    0.5 M EDTA
    80μl
    IGEPAL CA-630
    200μl
    完整,迷你,无EDTA 4粒
    双蒸水(DDW)
    39.32毫升
  4. 核裂解缓冲液(NLB)(40ml) 10mM Tris(pH8.0),1mM EDTA,0.5M NaCl,1%Triton X-100,0.5%脱氧胆酸钠,0.5%月桂酰肌氨酸,1×蛋白酶抑制剂
    1 M Tris(pH 8.0)
    400μl
    0.5 M EDTA
    80μl
    5 M NaCl
    4 ml
    Triton X-100
    400μl
    10%脱氧胆酸钠 2 ml
    30%月桂酰肌氨酸 666微升
    完整,迷你,无EDTA 4粒
    DDW
    32.46 ml
  5. 改性裂解缓冲液3(MLB3)(10ml) 10mM Tris(pH8.0),1mM EDTA,0.5mM EGTA,150mM NaCl,0.1%脱氧胆酸钠,0.1%SDS,1×蛋白酶抑制剂
    1 M Tris(pH 8.0)
    100微升
    0.5 M EDTA
    20微升
    0.1 M EGTA
    50微升
    5 M NaCl
    300微升
    10%脱氧胆酸钠 100微升
    10%SDS
    100微升
    完整,迷你,无EDTA 1个平板电脑
    DDW
    9.33 ml
  6. 10%吐温-20(10ml)
    吐温-20
    1 ml
    DDW
    9 ml
  7. PBS-T(10ml) PBS(pH 7.4),0.01%Tween-20
    PBS
    10 ml
    10%Tween-20
    10微升
  8. PBS-T-BSA(10ml) PBS(pH 7.4),0.01%Tween-20,0.1%BSA
    PBS
    10 ml
    10%Tween-20
    10微升
    7.5%BSA馏分V/ 133微升
  9. 5%Triton X-100(在MLB3中)(5ml)
    MLB3
    4.75 ml
    Triton X-100
    250微升
  10. 低盐缓冲液(LSB)(10ml)
    20mM Tris(pH 8.0),2mM EDTA,150mM NaCl,1%Triton X-100,0.1%SDS
    1 M Tris(pH 8.0)
    200μl
    0.5 M EDTA
    40微升
    5 M NaCl
    300微升
    Triton X-100
    100微升
    10%SDS
    100微升
    DDW
    9.26 ml
  11. 高盐缓冲液(HSB)(10ml) 20mM Tris(pH 8.0),2mM EDTA,500mM NaCl,1%Triton X-100,0.1%SDS
    1 M Tris(pH 8.0)
    200μl
    0.5 M EDTA
    40微升
    5 M NaCl
    1 ml
    Triton X-100
    100微升
    10%SDS
    100微升
    DDW
    8.56 ml
  12. LiCl缓冲液(20ml) 10mM Tris(pH8.0),1mM EDTA,0.25M LiCl,0.5%IGEPAL CA-630,0.5%脱氧胆酸钠
    1 M Tris(pH 8.0)
    200μl
    0.5 M EDTA
    40微升
    8 M LiCl
    625μl
    IGEPAL CA-630
    100微升
    10%脱氧胆酸钠 1 ml
    DDW
    18.035 ml
  13. TBS-IGEPAL CA-630(10ml) 50mM Tris(pH7.5),150mM NaCl,0.1%IGEPAL CA-630
    1 M Tris(pH 7.5)
    500微升
    5 M NaCl
    300微升
    IGEPAL CA-630
    10微升
    DDW
    9.19毫升
  14. 洗脱缓冲液(500μl)
    50mM Tris(pH7.5),150mM NaCl,0.1%IGEPAL CA-630,500μg/ml 3x FLAG肽
    5mg/ml在TBS中的3x FLAG肽
    50微升
    TBS-IGEPAL CA-630
    450微升
  15. 2x样品缓冲液(50ml)
    125mM Tris(pH6.8),10%2-巯基乙醇,4%SDS,10%蔗糖,0.004%溴酚蓝
    1 M Tris-HCl(pH 6.8)
    6.25 ml
    2-巯基乙醇 5 ml
    ...
    1 M Tris-HCl(pH 6.8)
    6.25 ml
    2-巯基乙醇 5 ml
    2 mg
    DDW
    to 50 ml

Acknowledgments

This protocol was adapted from the previously published paper (Fujita et al., 2013). We thank F. Kitaura for technical assistance. This work was supported by Research Institute for Microbial Diseases (T.F. and H.F.), Program for Combined Research Fields from Immunology Frontier Research Center, Osaka University (H.F.), the Takeda Science Foundation (H.F. and T.F.), the Asahi Glass Foundation (H.F.), A-STEP from Japan Science and Technology Agency (H.F.), Grant-in-Aid for Young Scientists (B) (#22710185) (T.F.), Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Cell Fate’’ (#23118516) (T.F.), ‘‘The Genofield’’ (#23114707) (H.F.), ‘‘Transcription Cycle’’ (25118512) (H.F.).

References

  1. Fujita, T. and Fujii, H. (2011). Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One 6(10): e26109.
  2. Fujita, T. and Fujii, H. (2012). Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Advances Biosci Biotechnol 3(5): 626-629.
  3. Fujita,T。和Fujii,H。(2013a)。 有效分离特定基因组区域并通过工程化DNA结合分子介导的染色质免疫沉淀来鉴定相关蛋白(enChIP)使用CRISPR。 Biochem Biophys Res Commun 439(1):132-136。
  4. 藤田,和富士,(2013b)。 基因座特异性生化表观遗传学/通过插入染色质免疫沉淀的染色质生物化学。 ISRN Biochem 2013,文章编号913273.
  5. Fujita,T.,Asano,Y.,Ohtsuka,J.,Takada,Y.,Saito,K.,Ohki,R.and Fujii,H。 通过改造的DNA结合分子介导的染色质鉴定端粒相关分子immunocrecipitation(enChIP)。 Sci Rep 3:3171.
  6. Hoshino,A。和Fujii,H。(2009)。 插入染色质免疫沉淀:分离特异性基因组区域的方法 J Biosci Bioeng 108(5):446-449。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fujita, T. and Fujii, H. (2014). Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP). Bio-protocol 4(10): e1124. DOI: 10.21769/BioProtoc.1124.
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