Advances in Bioscience and Biotechnology, 2012, 3, 626-629 ABB
http://dx.doi.org/10.4236/abb.2012.35081 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
Efficient isolation of specific genomic regions by insertional
chromatin immunoprecipitation (iChIP) with a
second-generation tagged LexA DNA-binding domain
Toshitsugu Fujita, Hodaka Fujii*
Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
Received 18 June 2012; revised 20 July 2012; accepted 2 August 2012
ABSTRACT
Comprehensive understanding of mechanisms of epi-
genetic regulation requires identification of molecules
bound to genomic regions of interest in vivo. We have
developed a novel method, insertional chromatin im-
munoprecipitatin (iChIP), to isolate specific genomic
regions retain ing in v ivo mol ecular inte raction in order
to perform non-biased identification of interacting
molecules. Here, we developed a second-generation
tagged LexA DNA-binding domain, 3×FNLDD, for the
iChIP analysis. 3×FNLDD consists of 3 × FLAG tags, a
nuclear localization signal (NLS), the DNA-binding
domain (DB) and the dimerization domain of the LexA
protein. Expression of 3×FNLDD can be detected by
immunoblot analysis as well as flowcytometry. We
showed that iChIP using 3×FNLDD is able to consis-
tently isolate more than 10% of input genomic DNA,
several-fold more efficient compared to the first-gen-
eration tagged LexA DB. 3×FNLDD would be a useful
tool to perform the iChIP analysis for locus-specific
biochemical epigenetics.
Keywords: Insertional Chromatin Immunoprecipitation;
iChIP; LexA; FLAG Tag
1. INTRODUCTION
Epigenetic regulation of eukaryotic genomic regions is
mediated by molecular complexes in the context of
chromatin [1]. We recently developed insertional chro-
matin immunoprecipitation (iChIP), which is a method to
biochemically isolate a genomic region of interest which
retains molecular interaction [2]. The scheme of iChIP is
as follows: 1) A repeat of the recognition sequence of an
exogenous DNA-binding protein such as LexA is in-
serted into the genomic region of interest in the cell to be
analyzed. 2) The DNA-binding domain (DB) of the ex-
ogenous DNA-binding protein is fused with a tag(s) and
a nuclear localization signal (NLS)(s) and expressed into
the cell to be analyzed. 3) The resultant cell is stimu-
lated, if necessary, and crosslinked with formaldehyde or
other crosslinkers. 4) The cell is lysed, and the crosslinked
DNA is fragmented by sonication. 5) The complexes
including the exogenous DB are immunoprecipitated
with an antibody against the tag. 6) The isolated com-
plexes retain molecules interacting with the genomic
region of interest. Reverse crosslinking and subsequent
purification of DNA, RNA, proteins, or other molecules
allows their identification and characterization. By using
iChIP, we succeeded in directly identifying protein and
RNA components of an insulator, which functions as
boundaries of chromatin domains [3], showing that iChIP
is a powerful tool for elucidation of molecular mecha-
nisms of epigenetic regulation. In order to easily identify
molecular interaction in vivo, however, more efficient
isolation of specific genomic regions of interest is nec-
essary.
To this end, here, we developed a second-generation
tagged LexA DNA-binding domain, 3×FNLDD, to per-
form the iChIP analysis more efficiently. 3×FNLDD con-
sists of 3 × FLAG tags, an NLS, DB and the dimerization
domain of the LexA protein. Expression of 3×FNLDD can
be detected by immunoblot analysis as well as flowcy-
tometry. We showed that iChIP using 3×FNLDD is able to
consistently isolate more than 10% of input of specific
genomic regions, several-fold more efficient compared to
the first-generation tagged LexA DB. 3×FNLDD would
be useful for the iChIP analysis of specific genomic re-
gions to perform their biochemical analysis.
2. MATERIALS AND METHODS
2.1. Plasmid Construction
To construct 3×FNLDD/pCMV-7.1, the DNA sequence
encoding NLS-LexA DB was cleaved from FCNLD/
pMIR [2] with BamH I and Not I, blunted, and inserted
*Corresponding author.

T. Fujita, H. Fujii / Advances in Bioscience and Biotechnology 3 (2012) 626-629 627
into p3XFLAG-CMV-7.1 (Sigma-Aldrich) that was
cleaved with Bgl II and blunted to generate 3×FNLD/
pCMV-7.1. Subsequently, the DNA sequence encoding
DB and the dimerization domain of LexA was amplified
with the LexA-N (26081) (5'-ccctttCCTGAGGGAATG
AAAGCGTTAACG-3') and LexA-C w/D (26628) (5'-aa
atgtcgaCTACAGCCAGTCGCCGTTGCG-3') primers us-
ing pBTM116 [4] as template. The PCR product was
cleaved with Mlu I and Sal I and inserted into Mlu I- and
Sal I-cleaved 3×FNLD/pCMV-7.1 to generate 3×FNLDD/
pCMV-7.1.
To construct pMXs-I2, the coding sequence of en-
hanced green fluorescent protein (EGFP) of pMXs-IG [5]
was replaced with that of human CD2 antigen (hCD2) [6].
To construct 3×FNLDD/pMXs-I2, the DNA sequence
encoding 3×FNLDD was cleaved with Sac I and Sal I,
blunted, and inserted into the pMXs-I2 vector that was
cleaved with EcoR I and Not I and blunted.
All PCR-derived DNA sequences were verified by
DNA sequencing.
2.2. Cell Lines
293T was maintained in DMEM supplemented with 10%
fetal calf serum (FCS). Ba/F3 [7]-derived cells were
maintained in RPMI-1640 supplemented with 10% FCS,
10 mM Hepes (pH 7.2), 1 × non-essential amino acid, 1
mM sodium pyruvate, 5 nM 2-mercaptoethanol, 1 ng/ml
interleukin-3.
1 × 107 of Ba/F3 were transfected with Mlu I-digested
pGL3C-Neo-cHS4c × 24-LexA × 2 [3] (100 µg) together
with the hygromycin resistance gene (3 µg) by electro-
poration using Gene Pulser II (Bio-Rad) at 250 V, 975
μF. The transfected cells were selected in the presence of
hygromycin (1 mg/ml) to establish the cHS4-core-1.2k
cell line. Subsequently, 1 × 107 of cHS4-core-1.2k were
transfected with Sca I-digested 3×FNLDD/pCMV-7.1
(100 µg) or Hind III-digested FCNLD/pEF (100 µg)
together with the puromycin resistance gene (3 µg) by
electroporation. The transfected cells were selected in the
presence of hygromycin (1 mg/ml) and puromycin (0.6
µg/ml) to establish the 3×FNLDD/cHS4-core-1.2k or
FCNLD/cHS4-core-1.2k cell line.
2.3. Immunoblot Analysis
Nuclear extracts were prepared with NE-PER Nuclear and
Cytoplasmic Extraction Reagents (Pierce). Immunoblot
analysis was performed as described before [6]. Anti-
FLAG M2 Ab was purchased from Sigma-Aldrich.
2.4. Flowcytometry
2 µg of pMXs-I2 or 3×FNLDD/pMXs-I2 was transfected
into 1 × 106 of 293T cells with Lipofectamine 2000 (In-
vitrogen). Two days after transfection, cells were har-
vested and stained with phycoerythrin (PE)-conjugated
anti-hCD2 Ab (BD Biosciences). Subsequently, cells
were intracellularly stained with fluorescein isothiocy-
anate (FITC)-conjugated anti-FLAG M2 (Sigma-Aldrich)
using the Fixation/Permeabilization and Permeabilization
buffer set (eBioscicence). Flowcytometric analysis was
performed on FACS Calibur (BD Biosciences), and data
was analyzed with FlowJo software (TreeStar).
2.5. Chromatin Preparation and iChIP
Cells (4 × 106) were fixed with 1% formaldehyde at 37˚C
for 5 min. The chromatin fraction was extracted and
fragmented (2 kbp-long on average) by sonication and
subjected to iChIP as described previously [3]. The DNA
purified by phenol-chloroform extraction and ethanol
precipitation was used as a template for real-time PCR
with Power SYBR Green PCR Master Mix (Applied
Biosystems) using the Applied Biosystems 7900HT Fast
Real-Time PCR System. PCR cycles were as follows:
heating at 50˚C for 2 min followed by 95˚C for 10 min;
40 cycles of 95˚C for 15 sec and 60˚C for 1 min. The
primers used in this experiment are LexA-N2 (26572)
(5'-ttctctatcgataggtacctcg-3') and LexA-C (26573) (5'-tct
attcagcggatctcgagcg-3').
3. RESULTS AND DISCUSSION
3.1. Generation of the Expression System of
3×FNLDD
For iChIP analysis, we have used the FCNLD protein [2]
consisting of 2 × FLAG tags, a tobacco etch virus (TEV)
protease cleavage site, calmodulin-binidng peptide, the
NLS of SV40-T-antigen, and LexA DB. To generate
more efficient tagged LexA proteins for iChIP analysis,
we constructed a plasmid expressing the 3×FNLDD pro-
tein consisting of 3 × FLAG tags, the NLS of SV40-
T-antigen, and DB as well as the dimerization domain of
LexA (Figure 1(a)). To increase efficiency of immuno-
precipitation, 3 × FLAG tags were used instead of 2 ×
FLAG tags. In addition, we removed the TEV protease
cleavage site and calmodulin-binding peptide because
efficiency of cleavage by TEV was low in crosslinked
chromatin (data not shown).
The pMXs-I2 vector expressing hCD2 or 3×FNLDD/
pMXs-I2 bicistronically expressing 3×FNLDD and hCD2
was transfected into 293T cells. Nuclear extracts were
prepared and subjected to immunoblot analysis using
anti-FLAG Ab. As shown in Figure 1(b), expression of
3×FNLDD was detected.
Next, we examined expression of 3×FNLDD in indi-
vidual cells by flowcytometry. 293T cells transfected
with pMXs-I2 or 3×FNLDD/pMXs-I2 were stained with
PE-conjugated anti-hCD2 Ab and subsequently intracel-

T. Fujita, H. Fujii / Advances in Bioscience and Biotechnology 3 (2012) 626-629
628
lularly stained with FITC-conjugated anti-FLAG Ab. As
shown in Figure 1(c), expression of 3×FNLDD was
clearly detected on hCD2 (+) cells.
3.2. Efficient Isolation of Specific Genomic
Regions by iChIP with 3×FNLDD
Next, we examined efficiency of iChIP with 3×FNLDD.
To this end, we first established a cell line, cHS4-core-
1.2k, by transfection of the pGL3C-Neo-cHS4c × 24-
LexA × 2 plasmid possessing 2 copies of the cHS4c ×
12-LexA cassette, in which 8 × repeats of the LexA
DNA-binding sequence was flanked at each side by six
copies of the core sequence of chicken HS4 insulator
(cHS4-core) (Figure 2(a)). The cHS4-core-1.2k cell line
retained about 100 copies of the cHS4c × 12-LexA cas-
sette in the genome (data not shown), thus about 1200
copies of the cHS4-core sequence are integrated in the
genome. We subsequently transfected the FCNLD/pEF
or the 3×FNLDD/pCMV-7.1 plasmid into the cHS4-
core-1.2l cell line to express FCNLD or 3×FNLDD, re-
spectively. The chromatin fraction was prepared from the
stable cell lines expressing FCNLD or 3×FNLDD and
subjected to iChIP for isolation of cHS4-core as a target
region. Isolation efficiency of cHS4-core was evaluated
by detection of the LexA-binding elements in real-time
PCR (Figure 2(a)). Multiple clones expressing FCNLD
or 3×FNLDD were analyzed to obtain clones showing
high iChIP efficiency. Figure 2(b) shows % input of the
LexA-binding elements purified by iChIP using a
representative FCNLD- or 3×FNLDD-expressing clone.
iChIP with anti-FLAG Ab using the FCNLD-expressing
clone showed 2.9% of the input DNA, which is consistent
with the data we reported previously [2]. In contrast, the
3×FNLDD-expressing clone showed 11.3% of the input,
which is several-fold higher than the FCNLD-expressing
clone. We observed consistent results using clones
established independently (data not shown). In contrast,
% input of the promoter region of the glyceraldehyde
3-phosphate dehydrogenase (GAPDH) gene, a negative
control genomic region, was less than 0.1% in 3×FNLDD-
expressing clones (data not shown), showing that back-
grounds of iChIP with 3×FNLDD are low. These data
indicate that 3×FNLDD would immunoprecipitate the
target sequence more efficiently than FCNLD and be
useful for efficient isolation of specific genomic regions
by iChIP. Increase in the number of the FLAG tag
sequence may contribute to increase in efficiency of
immunoprecipitation. In addition, addition of the dimeri-
zation domain of LexA would help form stable dimers to
increase binding affinity/avidity to the LexA elements.
4. CONCLUSIONS
In this study, we generated a second generation tagged
(a)
(b)
(c)
Figure 1. Scheme of 3×FNLDD. (a) 3×FNLDD consists
of 3 × FLAG tags, a nuclear localization signal (NLS),
the DNA-binding domain (DB) and the dimerization
domain of the LexA protein; (b) Expression of 3×FNLDD
in 293T cells. Nuclear extracts were subjected to im-
munoblot analysis with anti-FLAG Ab. The Coomassie
Brilliant Blue staining (CBB) is shown as a protein
loading control; (c) Flowcytometric detection of 3×FNLDD.
pMXs-I2 or 3×FNLDD/pMXs-I2 was transfected into
293T cells with Lipofectamine 2000. Two days after
transfection, cells were harvested and stained with PE-
conjugated anti-hCD2 Ab. Subsequently, cells were in-
tracellularly stained with FITC-conjugated anti-FLAG
M2. hCD2 (+) cells were gated to quantify expression
levels of 3×FNLDD. Open: pMXs-I2-trans-fected cells;
solid: 3×FNLDD/pMXs-I2-transfected cells. Mean fluo-
rescent intensity of FLAG: pMXs-I2, 8.48; 3×FNLDD/
pMXs-IG, 122. Mean fluorescent intensity of hCD2:
pMXs-IG, 623; 3×FNLDD/pMXs-IG, 636.

T. Fujita, H. Fujii / Advances in Bioscience and Biotechnology 3 (2012) 626-629 629
LexA, 3×FNLDD, for more efficient iChIP analysis. By
using 3×FNLDD, we were able to isolate target genomic
regions much more efficiently than by using FCNLD. %
input by using 3×FNLDD reached more than 10%, which
would enable biochemical analysis of specific genomic
regions much easier.
In addition, we showed that expression of 3×FNLDD
can be detected and quantified by flowcytometry. Flow-
cytometric detection of 3×FNLDD would be useful in
easy quantification of its expression levels in individual
cells. Taken together, 3×FNLDD would be a useful tool
for iChIP analysis of specific genomic regions.
5. ACKNOWLEDGEMENTS
This work was supported by Nakatani Foundation for Advancement of
Measuring Technologies in Biomedical Engineering (T.F.), Japan Sci-
ence and Technology Agency (JST) (H.F.), Grant-in-Aid for Young
Scientists (B) (#22710185) (T.F.), Grant-in-Aid for Scientific Research
on Innovative Areas (#23118516) (T.F.), (#23114707) (H.F.) from the
Ministry of Education, Culture, Sports, Science and Technology of
Japan.
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Figure 2. Efficient isolation of specific genomic regions
by iChIP with 3×FNLDD. (a) Scheme of iChIP in this
study. The cHS4c × 12-LexA regions integrated into the
genome of Ba/F3 cell line were isolated by iChIP with
anti-FLAG Ab and analyzed by real-time PCR with
LexA primers; (b) Quantification of the amounts of the
immunoprecipitated regions by real-time PCR.