Insertional chromatin immunoprecipitation

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Apr 20, 2009 - protein, LexA, was fused with FLAG tag, tobacco etch virus (TEV) protease .... precipitation with anti-FLAG beads, chromatin complexes were.
Journal of Bioscience and Bioengineering VOL. 108 No. 5, 446 – 449, 2009 www.elsevier.com/locate/jbiosc

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Insertional chromatin immunoprecipitation: A method for isolating specific genomic regions Akemi Hoshino,1 and Hodaka Fujii1,2,⁎ Department of Pathology, New York University School of Medicine, New York, NY 10016, USA 1 and Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan 2 Received 20 April 2009; accepted 11 May 2009

We established a novel method, insertional chromatin immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting chromatin structure of genomic region of interest. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chromatin structure; Purification; iChIP; Epigenetics; Transcriptional regulation]

Detailed biochemical and molecular biological analysis of chromatin domains is critical for understanding mechanisms of genetic and epigenetic regulation of gene expression, hetero- and euchromatinization, X-chromosome inactivation, genomic imprinting, and other important biological phenomena (1). However, biochemical nature of chromatin domains is poorly understood. This is mainly because methods for performing biochemical and molecular biological analysis of chromatin structure are limited (2–8). There are several existing methodologies to analyze molecules that interact with specific genomic regions. For example, if interacting proteins of a genomic region of interest are known, it is possible to isolate the region by the ChIP method. However, if interacting proteins are not known, ChIP cannot be used. In addition, since a DNA-binding protein generally binds to many different sites of genomic DNA, complexes immunoprecipitated with Ab against the DNA-binding protein are mixtures of many different genomic regions, causing biochemical analysis to be problematic. For identification of interacting genomic regions, chromosome conformation capture (3C) and its derivatives can be used (9–11). Although non-bias screening is possible with these methods, they include enzymatic reactions such as digestion with restriction enzymes and ligation, which are performed in non-optimal conditions such as under crosslinking, causing artifactual results. In addition, since digestion with restriction enzymes in non-optimal conditions gives rise to incomplete digestion, PCR amplification of neighboring regions of the target genomic region occurs, inhibiting amplification of regions that interact with the target region. Fluorescence microscopy including fluorescence in situ hybridization (FISH) can be used to show colocalization of specific ⁎ Corresponding author. Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan. Tel./fax: +81 6 6879 8358. E-mail address: [email protected] (H. Fujii).

genomic regions and proteins or RNA. However, this technique cannot be used for non-bias screening. Recently, a novel method, proteomics of isolated chromatin segments (PICh), was developed to purify proteins associated with specific genomic loci (8). This method utilizes a specific nucleic acid probe to isolate genomic DNA with its associated proteins, and it was shown that PICh can successfully isolate telomeres that exist in each chromosome and have multiple repeats corresponding to the probe. It is of interest if PICh can be applied to isolation of specific genomic regions in the low copy number genes that contain a single or a few repeats corresponding to the probe. To perform biochemical and molecular biological analysis of specific genomic regions, it is essential to purify those regions. To achieve this goal, we developed the iChIP technology to purify the genomic regions of interest. The scheme of this system is as follows (Fig. 1): (i) A repeat of the recognition sequence of LexA is inserted into the genomic region of interest in the cell to be analyzed (Fig. 1A). This can be achieved by knock-in of the LexA elements into the genomic region of interest. Alternatively, transgenes containing the LexA elements in the genomic region of interest can be transfected into the cell to be analyzed. (ii) The DNA-binding domain (DNA DB) of bacterial DNA-binding protein, LexA, was fused with FLAG tag, tobacco etch virus (TEV) protease cleavage site, calmodulin-binding peptide, and the nuclear localization signal (NLS) of SV40 T-antigen (FCNLD) (Fig. 1B), and expressed into the cell to be analyzed. The TEV cleavage site and calmodulin-binding peptide allow us to perform tandem immunoprecipitation. (iii) The resultant cell is stimulated, if necessary, and crosslinked with formaldehyde. This process crosslinks proteins, RNA, DNA

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to establish FCNLD-BLG. For generation of the FCNLD/pEF plasmid, the cDNA encoding the LexA DB was amplified by PCR with 5′-ccctttcctgagggaatgaaagcgttaacg-3′ and 5′-tgcggccgcttagggttcaccggcagccac-3′ as primers using the pLG plasmid (12) as template. The PCR product was digested with Bsu36 I and Not I and ligated with the oligonucleotide encoding the NLS of SV40 T-antigen (12) into the pBluescript plasmid. After verification of the insert by DNA sequencing, the resultant construct was digested with BamH I and Not I and subcloned into the pMIR-DFT vector, which expresses a protein fused with two FLAG tags, a recognition site of TEV proteinase, and the calmodulin-binding peptide at its N-terminus. The cDNA encoding FCNLD was cleaved from the resultant construct and subcloned into pEF (13) to generate the FCNLD/pEF plasmid. Expression of FCNLD in FCNLD-BLG was confirmed by immunoblot analysis with anti-LexA Ab (06-719, Millipore) (Fig. 2B) as previously described (14). The parental BLG cells or FCNLD-BLG cells were subjected to iChIP analysis. iChIP were performed according to the protocol provided

FIG. 1. Scheme of insertional chromatin immunoprecipitation (iChIP). The system consists of a promoter/enhancer element of a gene of interest linked to LexA-binding sites (the LexA-tagged promoter) (A), and FLAG-tagged, nuclear localization signal (NLS)-fused LexA DNA-binding domain (FCNLD) (B). A TEV protease cleavage site and calmodulin-binding peptide sequence are fused to allow tandem purification scheme. Cells expressing FCNLD are transiently or stably transfected with the LexA-tagged promoter. Alternatively, LexA-binding sites are knocked-in in the promoter/enhancer element of the gene of interest in cells expressing FCNLD. These cells are stimulated with ligand of interest, crosslinked with formaldehyde, and lysed. Then, crosslinked DNA is digested with a restriction enzyme or fragmented by sonication. Subsequently, the LexA-tagged promoter is immunoprecipitated with anti-FLAG antibody, and crosslink is reversed. Molecules (DNA, RNA, proteins, and others) associated with the LexA-tagged promoter are isolated and characterized (C).

and other molecules that interact with the genomic region of interest. This process also crosslinks FCNLD bound to the inserted LexA elements. (iv) The cell is lysed, and the crosslinked DNA is digested with nucleases such as restriction enzymes or fragmented by sonication. (v) The complexes including LexA DB is immunoprecipitated with anti-FLAG antibody (Ab). (vi) The isolated complexes retain molecules interacting with the genomic region of interest. Reverse crosslinking and subsequent purification of DNA, RNA, proteins, or other molecules allow identification and characterization of these molecules. First, we used the BLG cell line (12) to show that this system can enrich specific genomic regions. BLG is a Ba/F3-derived cell line that contains LexA-d1EGFP reporter gene consisting of LexA elements and destabilized GFP gene (Fig. 2A). FCNLD/pEF was transfected into BLG

FIG. 2. Isolation of the genomic region containing LexA-binding sites by iChIP. (A) The LexA-GFP reporter gene. Positions of PCR primers used for detection of the immunoprecipitated DNA are indicated by colored arrowheads. (B) Expression of FCNLD in the FCNLD-BLG cell line. 3 μg of nuclear extracts of the parental BLG and FCNLD-BLG were subjected to immunoblot analysis with anti-LexA Ab. (C) FCNLD was transfected into BLG cells containing the LexA-GFP reporter gene. Cells expressing FCNLD were identified by immunoblot analysis and expanded. 2 × 106 of the parental BLG cells or FCNLD-expressing BLG cells were crosslinked with formaldehyde, and lysed. Then, crosslinked DNA was fragmented by sonication. Subsequently, the LexAGFP reporter gene was immunoprecipitated with control IgG or anti-FLAG Ab, and crosslink is reversed. After RNase and Proteinase K treatment, DNA was purified and subjected to PCR amplification with primers detecting the LexA-GFP reporter gene. The LexA-GFP reporter gene was detected only in FCNLD-expressing BLG cells when antiFLAG Ab but not control IgG was used for immunoprecipitation. PC (positive control): PCR amplification using purified DNA of the LexA-GFP reporter as a template. (D) Quantification of the amounts of the immunoprecipitated LexA-GFP reporter gene by real-time PCR.

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FIG. 3. Detection of IRF-1 promoter in the chromatin complexes isolated by iChIP. (A) The LexA-element IRF-1 promoter GFP (LIPG) reporter. (B) IFNγ-induced GFP expression in FCNLD-expressing Ba/F3 containing the LIPG reporter (FCNLD-BLIPG). (C) IFNγ-induced binding of Stat1 to LIPG. 2 × 107 of FCNLD-BLIPG cells were mockstimulated or stimulated with IFNγ (10 ng/ml) for the indicated time intervals and subjected to ChIP analysis using 5 μg of anti-Stat1 Ab. (D) Detection of Stat1-binding sites adjacent to the LexA-binding elements by iChIP. (E) Detection of Stat1 in the chromatin complexes isolated by iChIP. 1 × 108 cells were subjected to iChIP using antiFLAG beads. Chromatin complexes were washed and digested with AcTEV protease at 30 °C for 3 h. Second immunoprecipitation was performed with normal mouse IgG or anti-Stat1 Ab and Protein G-Sepharose. Immunoprecipitants were washed extensively, and DNA purification and PCR were performed as described in the text. Left panel: input and TEV protease-treated sample. Right panel: results of second ChIP using control IgG and anti-Stat1 Ab.

by Upstate Biotechnology (EZ ChIP kit) with some modifications. 2 × 106 cells in 10 ml of RPMI complete medium were crosslinked with 270 μl of 37% formaldehyde for 10 min at room temperature (RT) and neutralized with 1 ml of 1.25 M glycine for 5 min at RT. After centrifugation (1 krpm) for 5 min at RT, cell pellets were washed with ice-cold PBS twice and suspended with ice-cold PBS containing protease inhibitor cocktail (Complete, Mini, EDTA-free, Roche). After centrifugation (700 ×g) for 5 min at 4 °C, the pellets were suspended in 400 μl of SDS lysis buffer (50 mM Tris (pH 8.1), 10 mM EDTA, 1% SDS, protease inhibitor cocktail). Crosslinked DNA was fragmented by sonication (sonicator: Cole Parmer, Ultra Sonic Processor, Model CP130; probe: CV18, 4273, amplitude 30, 10 s × 4 with 10 s intervals). After centrifugation (10 krpm) for 10 min at 4 °C, supernatant was recovered and diluted with dilution buffer 1 (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl (pH 8.0), 167 mM NaCl, Complete Mini, EDTA-free). Subsequently, the reporter gene was immunoprecipitated with control IgG+Protein G-Sepharose (GE Healthcare) or anti-FLAG M2 affinity gel (Sigma-Aldrich). After washing, immunoprecipitated complexes were eluted with 200 μl of

J. BIOSCI. BIOENG., elution buffer (100 mM NaHCO3, 1% SDS). Crosslink was reversed by adding 8 μl of 5 M NaCl and incubation at 65 °C overnight. After RNase A and Proteinase K treatment, DNA was purified and subjected to PCR amplification. Primers used for detecting the LexA-GFP reporter gene are: 5′-ccccagtgcaagtgcaggtgcc-3′ and 5′-cgtcgccgtccagctcgaccag-3′. As shown in Fig. 2C, the LexA-GFP reporter gene was detected only in the presence of FCNLD when anti-FLAG Ab but not control IgG was used for immunoprecipitation. Real-time PCR analysis showed that 2.5% of the input LexA-GFP reporter was recovered by iChIP with antiFLAG Ab (Fig. 2D). These results showed that iChIP can enrich specific genomic regions. Next, we examined whether we can purify promoter regions adjacent to the LexA-binding sites by iChIP. LexA-binding sites were inserted into upstream of Stat-binding sites of human IRF-1 promoter fused to GFP gene to generate the LexA-element IRF-1 promoter GFP reporter (LIPG). Stat-binding sites of human IRF-1 promoter were shown to be essential for interferon (IFN) γ-induced transcription of IRF-1 gene (15). In addition, Stat1 was shown to be necessary for IFNγ-induced transcription of IRF-1 gene (16, 17). For construction of LIPG, the 1.1 kbp Sac I–Sac II fragment of human IRF-1 promoter (15) was blunted and inserted into Kpn I-digested LexA-d1EGFP (12) after the ends were blunted. The resultant plasmid was digested with Xho I, and after its ends were blunted, the blunted 220 bp Sac I–Sac II fragment of human IRF-1 promoter was inserted. LIPG was stably transfected into Ba/F3 to generate the BLIPG cell line. Subsequently, FCNLD was stably transfected into BLIPG to generate the FCNLD-BLIPG cell line. Expression of FCNLD was confirmed by immunoblot analysis with anti-LexA Ab (data not shown). IFNγ-induced expression of GFP reporter was confirmed by flowcytometry (Fig. 3B). Flowcytometric analysis of GFP expression was performed as previously described (18). We also detected IFNγ-induced Stat1-binding to LIPG by ChIP using anti-Stat1 Ab (06-501, Santa Cruz Biotechnology) (Fig. 3C). The FCNLD-BLIPG cells were mock-stimulated or stimulated with IFNγ for 30 min and subjected to iChIP analysis. 1 × 108 cells per condition were used. After immunoprecipitation with anti-FLAG beads, chromatin complexes were washed and digested with 1000 units of AcTEV protease (Invitrogen) at 30 °C for 3 h. Supernatants (ca. 3 ml) were collected by centrifugation and diluted by adding 12 ml of dilution buffer 2 (0.39% SDS, 1.0% Triton X-100, 4.75 mM EDTA, 21.1 mM Tris–HCl (pH 8.0), 144.5 mM NaCl, Complete Mini, EDTA-free). Second immunoprecipitation was performed with 5 μg of normal mouse IgG (Millipore) or anti-Stat1 Ab and 40 μl of Protein G-Sepharose. Immunoprecipitants were washed extensively, and DNA purification and PCR were performed as described above. Primers used for detecting the LIPG are: 5′-tgtacttccccttcgccgctagct-3′ and 5′-gcaatccaaacacttagcgggatt-3′. As shown in Fig. 3D, the Statbinding sites were detected both in mock- and IFNγ-stimulated cells when anti-FLAG Ab but not control IgG was used for immunoprecipitation. These results showed that iChIP system can be used to purify promoter regions adjacent to the LexA-binding sites. Next, to examine if the iChIP-isolated IRF-1 promoter region contains Stat1, we performed sequential ChIP assay. After immunoprecipitation with anti-FLAG beads, chromatin complexes were released by TEV cleavage. Then, second ChIP was performed with control IgG or anti-Stat1 Ab. As shown in Fig. 3E, a band was specifically detected in the sample precipitated with anti-Stat1 Ab in an IFNγ-dependent manner. These results showed that the isolated chromatin complexes by iChIP contain transcription factors activated by extracellular signal and can be subjected to biochemical and molecular biological analysis. The iChIP system enables us to purify specific genomic DNA regions. Biochemical and molecular biological analysis of the purified complexes can identify their constituents including DNA, RNA, proteins, and others. In other words, this method enables us to identify unknown interaction of specific genomic DNA regions with

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DNA, RNA, proteins and others. Non-bias screening of interacting molecules is possible using this system. In addition, unknown modifications of chromatin constituents such as histone proteins can be revealed. Interacting proteins can be detected by enzymelinked immunosorbent assay (ELISA), immunoblot analysis, or mass spectrometry. Interacting DNA can be detected by region-specific PCR, DNA microarray analysis, or sequencing. Interacting RNA can be detected by microarray, Northern blot analysis, or RT-PCR. Other developing technologies for detection of protein, DNA, RNA, and other types of molecules can be flexibly combined with this technology. In summary, we developed the iChIP technology, which can be applied for (i) identification of transcription factors involved in transcriptional regulation of a gene of interest, (ii) identification of distant enhancers, (iii) identification of intra- and interchromosomal interaction, (iv) biochemical analysis of chromatin structure of specific genomic regions, (v) biochemistry of euchromatin/heterochromatin, and (vi) biochemical analysis of inactive X-chromosome. We used transgenes to show the feasibility of the iChIP technology. We recognize the possibility that chromosomal condition of genomic transgene may be different from that of endogenous wild-type locus. The knocking-in of the LexA-binding elements in the genome by gene targeting would enable us to utilize the iChIP technique in more physiological contexts. Knock-in technology can be applied to cell lines and primary cells from any organisms to which the technology has been shown to be applicable. Although cultured cell lines are much easier to handle, primary cells with targeted modification can be obtained from individuals derived from targeted embryonic stem cells. Recent development of induced pluripotent stem (iPS) cells (19) enables us to apply the iChIP technology to primary human cells. However, insertion of the LexA elements could change the physiological chromatin structure. Therefore, it is necessary to validate results obtained from the iChIP analysis using wild-type cells with conventional techniques. The iChIP technique will be a useful tool for dissecting chromatin structure. ACKNOWLEDGMENTS We thank Dr. L. Zhou for the pMIR-DFTC vector. We also thank Drs. S. Saint Fleur and T. Fujita for critical reading of the manuscript. References 1. Kornberg, R. D. and Lorch, Y.: Chromatin rules, Nat. Struct. Mol. Biol., 14, 986–988 (2007).

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