Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain

doi:10.4236/abb.2012.35081

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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

Email: *[email protected]

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.

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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.

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