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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

Toshitsugu Fujita Toshitsugu Fujita
Hodaka Fujii Hodaka Fujii

发布: 2014年05月20日第4卷第10期 DOI: 10.21769/BioProtoc.1124 浏览次数: 15617

评审: Sabine Le SauxAnonymous reviewer(s)

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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) search ChIP (炸薯条) search Locus-specific ChIP (位点特异的芯片) search EnChIP (enchip) search Chromatin (染色质) search

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

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

分子生物学 > DNA > DNA-蛋白质相互作用

生物化学 > 蛋白质 > 免疫检测 > 染色质免疫共沉淀（ChIP）

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