Insertional chromatin immunoprecipitation: A method for isolating
specific genomic regions
Akemi Hoshino,1and Hodaka Fujii1,2,⁎
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA1and Combined Program on Microbiology and Immunology,
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan2
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 chro-
matin domains is critical for understanding mechanisms of genetic
and epigenetic regulation of gene expression, hetero- and euchroma-
tinization, X-chromosomeinactivation,genomic imprinting,andother
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 areseveralexistingmethodologies 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 interact-
ing 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
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
(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
Journal of Bioscience and Bioengineering
VOL. 108 No. 5, 446–449, 2009
⁎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: firstname.lastname@example.org (H. Fujii).
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved.
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
(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
to establish FCNLD-BLG. For generation of the FCNLD/pEF plasmid, the
cDNA encoding the LexA DB was amplified by PCR with 5′-ccctttcct-
gagggaatgaaagcgttaacg-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 sequen-
cing, 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).
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. 2×106of the parental
BLG cells or FCNLD-expressing BLG cells were crosslinked with formaldehyde, and
lysed. Then, crosslinked DNA was fragmented by sonication. Subsequently, the LexA-
GFP 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 anti-
FLAG 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
TECHNICAL NOTE447VOL. 108, 2009
by Upstate Biotechnology (EZ ChIP kit) with some modifications.
2×106cells in 10 ml of RPMI complete mediumwere 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
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 anti-
FLAG Ab (Fig. 2D). These results showed that iChIP can enrich specific
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. 1×108cells 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. Super-
natants (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 Stat-
binding sites were detected both in mock- and IFNγ-stimulated cells
when anti-FLAG Ab but not control IgG was used for immunopreci-
pitation. 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 immuno-
precipitation 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
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. 2×107of FCNLD-BLIPG cells were mock-
stimulated 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.1×108cells were subjected to iChIP using anti-
FLAG 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 wereperformed 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.
448HOSHINO AND FUJIIJ. BIOSCI. BIOENG.,
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 enzyme-
linked 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
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 conven-
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.
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