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; Hodaka  Fujii

doi:10.21769/BioProtoc.1124

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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

Hodaka Fujii email

发布: 2014年05月20日第4卷第10期 DOI: 10.21769/BioProtoc.1124 浏览次数: 14651

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)

ChIP (炸薯条)

Locus-specific ChIP (位点特异的芯片)

EnChIP (enchip)

Chromatin (染色质)

Materials and Reagents

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

Equipment

Magnetic stand (magical trapper) (TOYOBO, catalog number: MGS-101 )
Centrifuge
1.5 ml centrifuge tube (Sarstedt AG, catalog number: 72.690.001 )
2 ml centrifuge tube (Eppendorf, catalog number: 0030.120.094 )
50 ml centrifuge tube (BD Biosciences, catalog number: 352070 )
Vortex mixer
Rotator
Vaccum pump connected to a side-arm flask
Sonicator (ultrasonic disruptor UD-201 ) (TOMY SEIKO, catalog number: UD-201)
Shaker
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

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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.