Sleeping Beauty Transposase Has an Affinity for Heterochromatin Conformation


MOLECULAR AND CELLULAR BIOLOGY, Mar. 2007, p. 1665–1676 Vol. 27, No. 5
0270-7306/07/$08.00�0 doi:10.1128/MCB.01500-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Sleeping Beauty Transposase Has an Affinity for
Heterochromatin Conformation�

Ryuji Ikeda,1 Chikara Kokubu,1,2 Kosuke Yusa,1 Vincent W. Keng,2 Kyoji Horie,1 and Junji Takeda1,2*
Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita,

Osaka 565-0871, Japan,1 and Center for Advanced Science and Innovation, Osaka University,
2-1 Yamada-oka, Suita, Osaka 565-0871, Japan2

Received 11 August 2006/Returned for modification 22 September 2006/Accepted 6 December 2006

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in
mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition
efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of
methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influ-
enced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin
status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into
the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline
repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline
operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the trans-
position event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with
intense DAPI (4�,6�-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation
analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited
to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for
heterochromatin conformation leads to enhancement of SB transposition efficiency.

Transposable elements, constituting a large part of the mam-
malian genome, may have a major impact on the evolution and
integrity of the genome (30). One of the most active trans-
posases in mammalian cells, Sleeping Beauty (SB), was recon-
structed from the salmonid genome and it is categorized within
the Tc1/mariner superfamily of transposons (20). SB-based
gene mobilization has been utilized as a tool for insertional
mutagenesis (3, 4, 7, 14, 19, 24) and several gene therapy
paradigms (32, 39). The SB transposon system has been devel-
oped as a nonautonomous system consisting of two indepen-
dent components: transposon and transposase. The SB trans-
poson is a DNA fragment flanked by the terminal inverted
repeats (IRs) and is mobilized by SB transposase. Each IR
contains two copies of a short (15- to 20-bp) direct repeat
(DR), and the resultant structures are named IR/DRs. SB
transposase can initiate a transposition event by binding to
IR/DRs. The two IR/DRs are then probably paired through
interactions of the transposase subunits (21), thereby forming
a synaptic complex (Fig. 1A), followed by excision and rein-
sertion of the SB transposon into another locus. The excision
events result in a “footprint” containing several additional base
pairs at the excised site.

The epigenetic modification for transposable-element mo-
bility is an important issue for understanding the relationship
between host and exogenous elements, such as transposons.
When the copy number of the retrotransposon is increased in

Arabidopsis, the transposed retrotransposon is inactivated by
DNA methylation. These retrotransposons were found to be
reactivated in a ddm (for decrease in DNA methylation) mu-
tant background (17, 29). Moreover, although expression of
retrovirus was often repressed in the genome by cytosine meth-
ylation, it was transcriptionally activated in mouse embryos
deficient in DNA methyltransferase-1 (38) and by treatment
with 5-azacytidine, which induces genomewide demethylation
(27, 36). Thus, reactivation of silent retroviral or retrotrans-
posable elements due to the lack of DNA methylation suggests
that this may have evolved as a basic defense mechanism to
prevent the harmful activity of mobile elements within the
mammalian genome (37, 40).

On the other hand, we demonstrated previously that CpG
methylation within the transposon sequence enhances the
transposition frequency of the SB transposon (42). However,
that study was performed using cultured cells introduced with
in vitro-methylated DNA. In order to further clarify this result,
we took advantage of a tetracycline-controlled transrepressor
(tTR) protein, in which the tetracycline repressor (TetR) from
Escherichia coli Tn10 is fused to the Kruppel-associated box
(KRAB) domain of human KOX1 (5), which can induce epi-
genetic gene silencing in specific regions of the genome.
KRAB is a 75-amino-acid transcriptional repression domain
found in many zinc finger-containing proteins (2) and can
suppress in an orientation-independent manner both polymer-
ase II- and polymerase III-mediated transcription within a
distance of up to 3 kb from its binding site, presumably by
triggering heterochromatin conformation change (5, 28, 34).
This heterochromatic conformation induced by the KRAB do-
main has been found to be mitotically heritable (1). When
linked to the DNA-binding domain of TetR, KRAB can mod-

* Corresponding author. Mailing address: Department of Social and
Environmental Medicine H3, Graduate School of Medicine, Osaka
University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone:
81-6-6879-3262. Fax: 81-6-6879-3266. E-mail: takeda@mr-envi.med
.osaka-u.ac.jp.

� Published ahead of print on 18 December 2006.

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FIG. 1. Schematic representation of the initial transposition reaction and localization of SB transposase. (A) Summary and schematic representation
of steps occurring in the initial transposition event: step 1 (I), SB transposase binds to IR/DRs; step 2 (II), synaptic-complex formation, followed by
excision and reinsertion of the transposon from the original donor site. IR/DR(R) and IR/DR(L) indicate right and left IR/DRs, respectively.
(B) Colocalization of SB transposase with intense DAPI staining. After SB transposase gene transfection, ES cells were fixed, followed by staining them
with anti-SB antisera (red) and counterstaining with DAPI (blue). Scale bar, 10 �m. (C) Intracellular distribution of SB and HP1 proteins. ES cells
expressing exogenous SB10 or vector control were extracted with an NE-PER kit (see Materials and Methods), and the indicated fractions were examined
by Western blot analysis using the indicated antibodies.

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ulate transcription from an integrated promoter juxtaposed
with tetracycline operator (tetO) sequences (5). In the absence
of doxycycline (DOX), tTR binds specifically to tetO and sup-
presses the activity of any nearby promoter. Conversely, in the
presence of DOX, tTR is sequestered away from tetO, thus
permitting gene expression (5). Using this mechanism, we per-
formed a tTR-based study by inducing heterochromatin for-
mation in inserted tetO sequences within or near the transpo-

son IR/DRs and compared the efficiencies of SB transposition
in vivo.

MATERIALS AND METHODS

Construction of plasmids. A targeting vector to introduce a single-copy SB
transposon into the Pax1 locus was generated as part of a separate project in our
laboratory, and full details will be described elsewhere (C. Kokubu, K. Hories, R.
Ikeda, and J. Takeda, unpublished data). As shown in Fig. 2A, tetO sequences were

FIG. 2. Targeted insertion of a transposon into the Pax1 locus. (A) Introduction of a single SB transposon copy into the Pax1 locus by
insertion-type homologous recombination. The open boxes indicate homologous sequences for recombination. An Hsp68 minimal promoter-LacZ
cassette and tetO were inserted between IR/DRs of the transposon. (B) Southern blot analysis of the targeted ES cells. DNA from targeted and
wild-type (WT) ES cells were double digested with EcoO65I and PacI and analyzed with two different probes. The locations of the probes are
shown in panel A.

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inserted within the SB transposon immediately inside both IR/DRs. An enhancer
detection cassette, consisting of the Hsp68 minimal promoter and the LacZ reporter
gene, was also inserted into the transposon between the tetO elements. The resulting
SB transposon was inserted between the puromycin resistance gene and the phos-
phoglycerate kinase (PGK) promoter. The PGK-Neo cassette, located out-
side the transposon, was used as a positive selection marker for genomic
integration of the entire targeting vector.

To generate the pCAG-IRES-Hygro vector, the fusion PCR product of the
internal ribosome entry site (IRES)-hygromycin resistance gene was inserted
downstream of the CAG promoter. This vector was used for generating control
embryonic stem (ES) cell clones. To generate the tTR expression vector (pCAG-
tTR-IRES-Hygro), the tTR fragment generated from pCMV-TetR(B/E)-KRAB
(12) was cloned into the HpaI site of the pCAG-IRES-Hygro vector.

Cell culture and gene targeting. Mouse ES cells were cultured in Dulbecco
modified Eagle medium containing 20% fetal bovine serum, nonessential amino
acids, sodium pyruvate, and leukemia-inhibitory factor (1,000 U/ml) on mitomy-
cin C-treated (15 �g/ml for 2.5 h) mouse embryonic fibroblasts.

Targeted integration of the SB transposon at the Pax1 locus was performed by
means of insertion-type homologous recombination. Briefly, 25 �g of the target-
ing vector was linearized at the BamHI site located in the homologous region and
introduced into 107 ES cells by electroporation (240 V; 500 �F) with a Gene
Pulser II (Bio-Rad, Hercules, CA). Cells were selected for 7 days with G418 (150
�g/ml), after which resistant clones were picked up, expanded, and analyzed by
Southern blotting.

Generation and characterization of stably transfected cell clones. For the
generation of ES cell lines containing a stably expressed tTR, ES cells with a
targeted integration of the transposon element in the Pax1 locus were transfected
with either the pCAG-IRES-Hygro control or pCAG-tTR-IRES-Hygro expres-
sion vector and cultured in ES cell medium containing 200 �g/ml hygromycin B.
The colonies were isolated, expanded, and tested for G418 (150 �g/ml) resis-
tance and LacZ expression using X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galac-
topyranoside) staining 3 days after the addition of DOX.

Antibodies. The antibodies used and their sources were as folows: anti-
dimethyl H3-K4 (Upstate Biotechnology, Lake Placid, NY), anti-dimethyl
H3-K9 (Abcam, Cambridge, United Kingdom), anti-trimethyl H3-K9 (Abcam,
Cambridge, United Kingdom), anti-acetyl H3 (Upstate Biotechnology), anti-
HP1� (Upstate Biotechnology), anti-HP1� (Chemicon International, Inc., Te-
mecula, CA), anti-HP1� (Upstate Biotechnology), anti-SB antisera (a gift from
Zoltan Ivics), anti-tubulin (Sigma Chemical Co., St. Louis, MO), and anti-Sp1
(Sigma Chemical Co., St. Louis, MO).

ChIP assay. The chromatin immunoprecipitation (ChIP) experiment was per-
formed with a ChIP assay kit using the protocol recommended by the supplier
(Upstate Biotechnology) with some modifications. Briefly, 107 (for histone mod-
ification [see Fig. 4]) and 2 � 106 (for recruitment of SB transposase [see Fig. 8])
ES cells were cross-linked with 1% formaldehyde at 37°C for 10 min. The
cross-linked cells were centrifuged and washed once with phosphate-buffered
saline. The cells were suspended in sodium dodecyl sulfate (SDS) lysis buffer (1%
SDS, 10 mM EDTA, and 50 mM Tris-HCl at pH 8.1) containing protease
inhibitor cocktail (Sigma) and sonicated to obtain average fragment sizes of 200
to 500 bp. Solubilized chromatin was clarified by centrifugation for 10 min at
13,000 rpm at 4°C, and the supernatant was diluted 10-fold in ChIP dilution
buffer (1% Triton X-100, 1 mM EDTA, 150 mM NaCl, and 15 mM Tris-HCl, pH
8.1). The diluted chromatin was precleared for 60 min with protein A (see Fig.
4) or G (see Fig. 8) agarose beads that had been blocked with salmon sperm
DNA and bovine serum albumin. The precleared chromatin was incubated with
anti-dimethyl H3-K4, anti-dimethyl H3-K9, anti-trimethyl H3-K9, anti-acetyl H3,
and anti-SB antibodies at 4°C for 12 to 16 h. Immunocomplexes were bound to
protein A or G agarose beads at 4°C for 30 min. The beads were washed once
each with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150
mM NaCl, and 20 mM Tris-HCl at pH 8.1), high-salt wash buffer (0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris-HCl at pH 8.1), and
LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, and
10 mM Tris-HCl at pH 8.1) and twice with Tris-EDTA buffer (1 mM EDTA
and 10 mM Tris-HCl at pH 8.0). The DNA-protein immunocomplexes were
eluted from the protein A or G beads with elution buffer (1% SDS and 0.1 M
NaHCO3), and the eluates were treated with proteinase K for 1 h at 56°C and
extracted once with phenol-chloroform, and the DNA was precipitated with
ethanol. The precipitated DNA was resuspended in 50 �l of Tris-EDTA. The
immunoprecipitated and input DNAs were analyzed by PCR using primer pairs
specific for the Oct4 promoter sequence (5�-GGAGGTGCAATGGCTGTCTT
GTCC-3� and 5�-CTGCCTTGGGTCACCTTACACCTCAC-3�), the Mage-a2
promoter sequence (5�-TTGGTGGACAGGGAAGCTAGGGGA-3� and 5�-CG
CTCCAGAACAAAATGGCGCAGA-3�), the Hsp68 minimal promoter se-

quence (5�-GCGCCGCTCTGCTCTGCTTCTCTTGTCTTC-3� and 5�-CCCCC
TCAGGAATCCGTACTCTCCAGTGAA-3�), and the LacZ sequence (5�-GC
GCCCATCTACACCAACGTAACCTATCCC-3� and 5�-CGCGGCTGAAAT
CATCATTAAAGCGAGTGG-3�).

Cell fractionation assay. Rapidly growing ES cells stably transfected with
either empty vector or SB10 transposase expression vector were harvested using
the Nuclear and Cytoplasmic Extraction Reagent (NE-PER; Pierce, Rockford,
IL) according to the instructions provided by the manufacturer. The respective
cytoplasmic, nuclear, and pellet fractions were analyzed by Western blot analysis.
Immunodetections of tubulin, Sp1, and HP1 were used as controls to identify
cytoplasmic, nuclear/cytoplasmic, and pellet fractions, respectively.

Excision assay of the transposon located in the Pax1 locus. For the transposon
excision assay, 2 � 105 ES cells in either the presence or absence of 1 �g/ml of
DOX were transfected with 2 �g of pCMV-SB11 (15) using TransFast (Promega,
Madison, WI) and cultured for 48 h on mouse embryonic fibroblasts treated with
mitomycin C (feeder cells). The transfected ES cells were harvested, and the
feeder cells were removed by incubation on gelatin-coated dishes for 20 min at
37°C. Unattached ES cells were recultured on gelatin-coated dishes for another
3 days. Genomic DNA extracted from the feederless culture was used as a
template in the following semiquantitative PCR analysis. Different amounts of
template DNA (100 ng, 10 ng, and 1 ng) were amplified by nested PCR using the
following primers: Puro-L4 (5�-GCGCGTGAGGAAGAGTTCTTGCAGCTCG
GT-3�) and PGKp-U1 (5�-TGGCCTCTGGCCTCGCACACATTCCACATC-
3�) for the first PCR and Puro-L3 (5�-GTCGGCGAACGCGGCGGCGAGGG
TGCGTAC-3�) and PGKp-U2 (5�-CGCGCCACCTTCTACTCCTCCCCTAGT
CAG-3�) for the second PCR. PCR amplifications were performed using
HotStarTaq (QIAGEN, Hilden, Germany) under the following conditions: 95°C
for 15 min, followed by 35 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 40 s,
with a final extension at 72°C for 5 min.

For control experiments (see Fig. 6B, right), the Fas ligand locus was amplified
by single-round PCR using the following primer pairs: Fex1u (5�-ATGCAAGT
GAGTGGGTGTCTCACAG-3�) and Fex1l (5�-AAACTGACCCTGGAGGAG
CCCAAGA-3�). PCR amplifications were performed using HotStarTaq under
the following conditions: 95°C for 15 min, followed by 35 cycles of 94°C for 15 s,
60°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min.

Immunofluorescence microscopy. ES and NIH 3T3 cells were transfected with
pCMV-SB11. After 48 h, these cells were trypsinized and seeded on a 24-well
plate coated with gelatin. After 5 h, the cells were fixed with 4% paraformalde-
hyde and permeabilized with 0.1% Triton X-100 before immunoreaction with
rabbit anti-SB polyclonal antiserum (dilution, 1:1,000), followed by Alexa594-
conjugated donkey anti-rabbit immunoglobulin G (dilution, 1:1,000). Counter-
staining was performed using 4�,6�-diamidino-2-phenylindole (DAPI). These
samples were observed under fluorescence microscopy (Olympus IX70; Olym-
pus, Tokyo, Japan).

Determination of transposon insertion sites by ligation-mediated (LM) PCR.
Genomic DNA was isolated from tTR and control clones after transfection with
SB11 transposase expression vector. After 1 �g of genomic DNA was digested
with a 4-base restriction enzyme (RsaI or HaeIII), splinkerettes (6) were ligated
to the cleaved ends. The ligation products were purified with phenol-chloroform,
followed by ethanol precipitation. Samples were digested with BglII to prevent
amplification from the donor site before being used as templates for subsequent
PCRs. We performed nested PCR with first primers specific for splinkerettes
(Spl-P1, 5�-CGAATCGTAACCGTTCGTACGAGAA-3�) and the transposon
vector (T/DR2, 5�-CTGGAATTGTGATACAGTGAATTATAAGTG-3�) using
the HotStarTaq system (QIAGEN) under the following conditions: 50-�l PCR
volume; 1 cycle at 95°C for 15 min, 30 cycles at 95°C for 30 s, 55°C for 30 s, and
72°C for 1 min, and 1 cycle at 72°C for 5 min. For the second nested PCR, we
used 1 �l of the first nested-PCR product as a template under similar conditions,
using second-PCR primers specific for splinkerettes (Spl-P2, 5�-TCGTACGAG
AATCGCTGTCCTCTCC-3�) and the transposon vector (T/BAL, 5�-CTTGTG
TCATGCACAAAGTAGATGTCC-3�). Nested-PCR product bands were puri-
fied with a Gel Extraction Kit (QIAGEN), followed by sequencing using the
primer Spl-P2 or T/BAL.

RESULTS

Preferential localization of SB transposase to heterochro-
matin. Transposition of DNA-type transposons generally con-
sists of its excision and subsequent reinsertion into the host
genome. Before excision occurs, two important steps are re-
quired: step 1, transposase binding, and step 2, synaptic-com-

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plex formation (Fig. 1A). We have previously shown that CpG
methylation of the SB transposon enhanced transposition ef-
ficiency, and we postulated that either one of the above-men-
tioned steps or both are affected by CpG methylation, followed
by heterochromatin formation. To investigate whether the nu-
clear localization of SB transposase accounts for the transpo-
sition enhancement, we transiently transfected the SB trans-
posase gene into ES cells. As revealed by immunofluorescence
analysis, SB transposase preferentially colocalized with intense
DAPI staining, which is indicative of heterochromatin (25, 33)
(Fig. 1B). Similar results were also observed in NIH 3T3 cells
(data not shown).

To further characterize the SB transposase, we determined
the intracellular distribution of SB transposase and investi-
gated whether SB transposase was tightly bound to nuclear
components, like HP1 family members (31). The transposase
was first introduced into ES cells, and cytoplasmic/nuclear ex-
tracts and pellet fractions were subsequently analyzed by West-
ern blot analysis. As shown in Fig. 1C, transcription factors,

such as Sp1, could be efficiently extracted from the nuclear
fraction, whereas chromatin-associated proteins, such as
HP1�/�/�, could not be extracted. A large amount of the
introduced SB transposase was found in the pellet fraction,
indicating that SB transposase is tightly associated with chro-
matin (Fig. 1C). To investigate whether SB transposase coop-
eratively functioned with the HP1 family in heterochromatin,
we examined possible physiological interaction between SB
transposase and the HP1 family using coimmunoprecipitation
analysis. However, in our hands, we could not detect any in-
teraction (data not shown).

Our immunostaining and biochemical analyses suggest that
SB transposase has an affinity for the heterochromatic regions.

Generation of ES cells bearing one copy of transposon vec-
tor inserted into a defined locus. In our previous study, we
introduced an in vitro-methylated transposon into cultured
cells and examined its transpositional efficiency (42). Since
modification of the transposon was performed in “test tubes,”
the enhanced transposition could be the result of unusual

FIG. 3. Schematic representation of tTR expression vector and characterization of stably expressed tTR cells. (A) tTR expression vector under
the control of the CAG promoter and its control vector. Three individual clones generated by each vector were selected for further analyses. Hygro,
hygromycin. (B) Sensitivity to G418. The tTR and control clones were cultured in G418-containing medium (150 �g/ml) for 6 days. Resistant
colonies were stained with Giemsa solution. Clones expressing tTR completely perished, while all control clones survived. (C) X-Gal staining.
X-Gal staining showed that tTR clones were negative while control clones were positive. These data suggest that the LacZ gene was completely
silenced by tTR-induced heterochromatin.

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mechanisms. To investigate this phenomenon further, modifi-
cation of the transposon was introduced by using an in vivo
mechanism. Since the KRAB domain can recruit causative
heterochromatic proteins, such as HP1 and Kap1 (1), tTR,
which contains the KRAB domain, may induce heterochro-
matic conformation with its binding proteins around the in-
serted tetO sequences. We generated a transposon vector with
tetO sequences positioned near the IR/DRs (Fig. 2A), and this
single transposon copy was inserted into the Pax1 locus, 50 kb
upstream of the first exon, by insertion-type homologous re-
combination. Southern analysis was performed to confirm suc-
cessful insertion of a single transposon copy bearing tetO se-
quences in the Pax1 locus (Fig. 2B).

Gene silencing by tTR expression around the transposon
insertion locus. To generate ES cell lines expressing tTR,
pCAG-tTR-IRES-Hygro (Fig. 3A) was transfected into ES
cells bearing a single transposon copy in the Pax1 locus. As a
control, we also generated stable cell lines that contained only
the hygromycin resistance gene (Fig. 3A). From each transfec-
tion, three individual clones were isolated, and they were
named tTR no. 1 to 3 or control no. 1 to 3 for experimentally
or control-transfected clones, respectively.

To evaluate the degree of heterochromatic change induced
by tTR expression, all clones were cultured in ES medium
supplemented with G418 for 6 days. Although the distance
between tetO sequences and the PGK promoter of the Neo
cassette is more than 3 kb, tTR clones were G418 sensitive,
while control clones were G418 resistant (Fig. 3B). In addition,
the X-Gal staining assay revealed no detectable LacZ gene
expression in tTR clones, while control clones were all LacZ
positive (Fig. 3C). Since the transcriptional activity of the
Hsp68 minimal promoter is very low, the PGK promoter lo-
cated immediately upstream of Hsp68 may enhance its activity.
To examine whether the effect of gene silencing was continu-
ously maintained without tTR binding to tetO, we cultured the

ES cell lines expressing tTR with DOX for 3 weeks and looked
for LacZ expression. The absence of any LacZ-positive colo-
nies suggests that the gene silencing of the LacZ gene contin-
ued for at least 3 weeks without tTR binding to tetO (data not
shown). These data, shown in Fig. 3B and C, suggest that the
gene silencing induced by tTR expression was maintained
around the transposon/tetO region.

Histone modification and DNA methylation status of the
transposon locus. tTR, consisting of TetR and the KRAB
domain, can bind to tetO sequences in the genome and coor-
dinate the machinery required for the establishment of a highly
localized heterochromatin-like silenced state in euchromatic
loci, which are epigenetically heritable (1). Thus, to examine
heterochromatic markers at the transposon locus, we per-
formed ChIP analysis of the targeted locus using antibodies
specific to dimethylated H3-K9, trimethylated H3-K9, acety-
lated H3, and dimethylated H3-K4 (Fig. 4). We performed
these analyses in the presence of DOX for 3 days to keep tTR
away from tetO and generate more natural heterochromatin
conformation in the transposon locus. The recovered DNAs
from tTR and control clones were analyzed by semiquantita-
tive PCR using gene-specific primer pairs for the Hsp68 min-
imal promoter, the LacZ coding region, and the internal con-
trol locus Oct4 promoter region (16) for the euchromatic
region and the Mage-a2 promoter region (35) for the hetero-
chromatic region. Although both control loci were detected at
similar levels by these antibodies in all clones tested, the LacZ
and Hsp68 regions were highly enriched by dimethylated
H3-K9 and trimethylated H3-K9 antibodies in tTR-positive
clones, but not in control clones. Furthermore, in the LacZ
locus, control clones were moderately enriched by acetylated
H3 antibodies and more enriched by dimethylated H3-K4 an-
tibodies than tTR-positive clones. These results suggest that
dynamic changes in histone modifications were effectively in-
duced by tTR around the transposon/tetO region.

FIG. 4. ChIP analyses of the transposon locus. Formaldehyde cross-linked chromatin from tTR (no. 1) and control (no. 1) ES clones cultured
with DOX for 3 days to exclude tTR from tetO was immunoprecipitated without antibody (No Ab) or with anti-dimethyl H3-K9, anti-trimethyl
H3-K9, anti-dimethyl H3-K4, or anti-acetyl H3 antibody. Fivefold serial dilutions of input and precipitated DNAs from tTR and control clones
were used for semiquantitative PCR. Oct4 and Mage-a2 were used as controls for euchromatic and heterochromatic regions, respectively.

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To further investigate epigenetic changes associated with
heterochromatin formation, we analyzed the methylation sta-
tus of CpG dinucleotides within the transposon containing tetO
sequence by Southern blotting using methylation-sensitive re-
striction enzymes. As shown in Fig. 5, DNA from tTR clones
double digested with SacI and methylation-sensitive HpaII
yielded a 2.8-kb band that corresponded to the band with
SacI-only digestion, while DNA from the control clones
yielded a 0.75-kb band (Fig. 5, middle). DNAs from both tTR
and control clones double digested with SacI and methylation-
insensitive MspI always yielded a 0.75-kb band (Fig. 5, right).
These results suggest that tTR effectively induced DNA meth-
ylation around the transposon/tetO region.

Taken together, tTR effectively induced not only histone
modifications, but also DNA methylation in the transposon
locus.

Effect of heterochromatin on excision of the transposon. To
test whether heterochromatic formation influences transposi-
tion efficiency, we transiently transfected a transposase gene
(SB11) into tTR and control clones and analyzed excision by
nested PCR. As shown in Fig. 6A, left, a 498-bp PCR band is
expected when the transposon is excised from the Pax1 locus.
Before the excision analysis, we tested the sensitivity of our
PCR conditions and found that they were sensitive enough to
amplify a single excision event (Fig. 6A, right). Using the same
PCR conditions, we performed the excision analysis in the
presence of DOX for 3 days. Excision was detected with as
little as 1 ng template in tTR clones. In contrast, excision in

control clones was detected only at the 100-ng level (Fig. 6B,
left). Therefore, the heterochromatin conformation induced by
tTR resulted in approximately 100-fold-higher efficiency of
excision events than that with control clones. DNA templates
from tTR and control clones were of similar quality for PCR
amplification (Fig. 6B, right). Expression of SB proteins was
not affected by the expression of tTR (Fig. 6C). Thus, tTR can
enhance the excision of transposons after introducing hetero-
chromatin conformation change.

Analyses of reinsertion events of the SB transposon. To
evaluate transposition events, including reinsertions, puromy-
cin selection could also be available. If the transposon mobi-
lizes to other loci, a functional PGK-puromycin gene is gener-
ated (Fig. 6A, left). After puromycin selection, transposon
reinsertion sites would be identifiable. However, the transpo-
son locus is silenced, probably due to the recruitment of het-
erochromatic proteins through the tetO site (Fig. 3B and C).
Therefore, even if transposition occurs, the clones would still
be puromycin sensitive. To revert the heterochromatic confor-
mation, ES cells were treated with histone deacetylase inhibi-
tor, trichostatin A (10), and cytosine methylation inhibitor,
5-aza-dC (9). Our preliminary experiments revealed that ES
cells were very sensitive to these drugs, even at very low doses,
indicating that trichostatin A and 5-aza-dC could not be used
to revert the heterochromatic conformation in our present
experiments (data not shown).

To examine reinsertion events without puromycin selection,
we set up a new strategy (Fig. 7A). First, we inoculated 100 or

FIG. 5. Analysis of methylation status within the transposon. (Top) Detailed restriction map of methylation-sensitive restriction enzymes. The
LacZ probe used is shown as a box under the LacZ coding region. (Bottom) Genomic DNA from tTR-positive clones was not digested with
methylation-sensitive HpaII, indicating that the region was highly methylated. On the other hand, genomic DNA from control clones was
completely digested. Genomic DNAs from tTR and control clones were digested with methylation-insensitive MspI (shown on the right). The
tTR-positive and control clones were cultured with DOX for 3 days.

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FIG. 6. Effect of heterochromatin on the excision of the transposon by transiently transfected SB transposase. (A) Schematic representation
of the excision assay and sensitivity of PCR conditions. (Left) SB transposase (pCMV-SB11) was transfected into tTR and control clones in the
presence of DOX. After 48 h, feeder cells were removed, and the remaining cells were cultured for 3 days on a gelatin-coated dish; genomic DNA
was extracted, and nested PCR was performed. The excised transposon locus yielded a 498-bp PCR product. Arrowheads, primers for nested PCR;
asterisk, excision footprint. (Right) A single copy of the excised DNA could be detected by this nested-PCR condition. An excision-positive ES

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1,000 tTR-expressing ES cells and 1,000 control ES cells into
each well of a 96-well culture plate and looked for excision
events in these cells after brief expansion. As shown in Fig. 7B,
31 out of 96 wells from the 100-cell/well group were excision
positive. A single excision event could be detected by our PCR
conditions (Fig. 6A), suggesting that most excision-positive
wells from the 100-cell/well group, if not all, had one excision
event, and thus, a particular reinsertion site could be analyzed.
Consistent with Fig. 6B, all wells from control ES cells were
excision negative. Secondly, ES cells from excision-positive
wells from the 100-cell/well group were further expanded, and
reinsertion sites were determined by LM-PCR after the puri-
fication of genomic DNA. We could determine reinsertion
sites from 9 out of 24 excision-positive clones (Fig. 7C). Dis-
tribution of insertion sites in tTR clones occurred mostly on
the same chromosome as the donor site (Fig. 7C), which is
consistent with our previous studies (19, 24). Using control ES
cells, we collected excision-positive cells by puromycin selec-
tion and found that about half of the puromycin-resistant
clones were reinsertion negative (Kokubu et al., unpublished),
implying that approximately half of the excised transposons fail
to land in the genome. This is consistent with the above-
mentioned frequency of reinsertion events (9 out of 24) in tTR
clones, suggesting that heterochromatic formation at the SB
transposon does not influence the reinsertion process. These
data demonstrate that enhanced excision efficiency by tTR-
induced heterochromatin formation at the SB transposon leads
to a high rate of transposition.

Recruitment of SB transposase to tTR-induced heterochro-
matinized transposons. To directly evaluate whether SB trans-
posase has a preference for tTR-induced heterochromatinized
transposons, we performed a ChIP assay using anti-SB trans-
posase polyclonal antibody. As shown in Fig. 8, SB transposase
was clearly recruited more in the heterochromatinized trans-
poson than in the nonheterochromatinized one. Thus, en-
hanced transposition by heterochromatin conformation at the
SB transposon could be explained by enhanced recruitment of
SB transposase to heterochromatin conformation.

DISCUSSION

Over the past few years, several studies of SB transposable
elements (TEs) have been done with regard to their applica-
tion for insertional mutagenesis (3, 19). However, only a few
studies have been conducted to understand the exact molecu-
lar mechanisms underlying the interaction between SB trans-
posase and its recognition sites for transposition (26, 43). A
better understanding of the molecular mechanisms involved in
transposition will enable researchers to further develop and
improve the SB system for mutagenesis by increasing the rate
of transposition and mobilization in the genome.

Comparison between our previous and present studies. Het-
erochromatin and DNA methylation are generally thought to
suppress the mobilization of transposable elements, both au-
tonomous DNA transposons and retrotransposons, by inhibit-
ing the expression of catalytic enzymes for transposition (17,
29, 38, 41). In our previous work, we found that heterochro-
matin formation might facilitate the transposition reaction
when transposase was supplied in trans (42). However, there is
an important question that needs to be addressed: whether the
in vitro methylation of DNA and subsequent chromosomal
integration resemble the native cellular heterochromatiniza-
tion. That is, the unexpected modification might have caused a
bias change in the frequency of SB transposase recruitment to
IR/DRs. In order to exclude this possibility, we took advantage
of a tetracycline-controlled transrepressor protein, tTR, con-
sisting of a tetracycline repressor (TetR) fused to the KRAB
domain of human KOX1, to establish highly localized hetero-
chromatin states in the euchromatic region which are heritably
and epigenetically maintained (1). Using this system in our
present study, the transposition frequency with heterochroma-
tin conformation was increased 100-fold compared to nonhet-
erochromatin conformation (Fig. 6B). Compared with our pre-
vious results, the excision efficiency was similarly enhanced and
was approximately 100-fold higher than controls. However, the
minimum template DNA used for nested PCR in our previous
study was 10 ng (42). In contrast, approximately 1 ng template
was used in the present study (Fig. 6B). The PCR conditions
in both studies were capable of detecting a single excision
event (Fig. 6A) (42), suggesting that the excision efficiency
was approximately 10-fold higher in the present study. This
increased efficiency may indicate that recruitment of the
repressor molecules caused a more natural heterochromatin
conformation. Detailed analysis is necessary to clarify the
difference in efficiency between the two studies. For exam-
ple, the integration sites of the transposon vector, the ver-
sions of the transposase genes (SB10 versus SB11), and the
cell lines used were different.

One or 10 nanograms of genomic DNA corresponds to ap-
proximately 150 or 1,500 cells, respectively. This transposition
frequency is lower than that in mouse germ cells (i.e., 0.03 to
0.2 transposition/transposon/germ cell) (8, 11, 18). Our previ-
ous (42) and present studies have caused only a “local” intro-
duction of heterochromatin conformation. In contrast, a tan-
dem array of SB transposon transgenes would be widely
heterochromatinized in mice. This different state of genome
conformation might explain the effectiveness of SB transposi-
tion in mouse germ cells.

Which step of the transposition event is enhanced by het-
erochromatic conformation? The initial transposition event
can be divided into at least two major processes, as shown in

clone was obtained, and genomic DNA was prepared. The indicated copy numbers of excision-positive genomic DNAs were mixed with 100 ng
of excision-negative genomic DNA. (B) High excision frequencies of the tTR-positive clones at the defined genomic locus. (Left) Genomic DNA
from each clone (1, 10, and 100 ng) was used as a template for nested PCR. Ten PCR amplifications were carried out for each clone. Note that
about 100-fold-higher frequency of excision events was detected in tTR-positive clones than in control clones. Two independent experiments were
performed, and a representative result is presented. M, molecular marker. (Right) Control experiment to evaluate the quality of isolated DNA. The Fas
ligand locus was amplified by single-round PCR. (C) Expression of SB transposase after transfection. tTR and control clones were transfected with the
SB11 transposase expression vector. The Western blot analysis after 48 h posttransfection is shown. Tubulin was used as a loading control.

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FIG. 7. Analyses of reinsertion events in the presence of puromycin selection. (A) Experimental procedure to analyze for reinsertion events.
After the transfection of SB transposase, tTR and control clones were inoculated into 96-well culture plates with the indicated cell numbers.
Excision-positive cell populations were expanded, genomic DNA was isolated, and LM-PCR analyses were performed to identify reinsertion sites.
(B) Screening for excision events. Excision-positive wells derived from 100-cell/well groups theoretically contained one excision event, and insertion
sites were determined by LM-PCR. M, molecular marker. (C) Flanking transposon genomic sequences. The original donor site is located on
chromosome 2, and reinsertion chromosomes are shown.

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Fig. 1A: step 1, binding of the transposase to its recognition
sites within the IRs, and step 2, the formation of a synaptic
complex to pair the two ends of IR/DRs and to hold them
together with transposase subunits. The exact step(s) enhanced
by the heterochromatin should be determined in future stud-
ies. In the present study, we demonstrated by immunostaining
assay that SB transposase was colocalized with intense DAPI
staining in the heterochromatic regions (Fig. 1B). Further-
more, SB transposase was recruited to the tTR-induced het-
erochromatic regions (Fig. 8). These findings suggest that SB
transposase prefers heterochromatic to euchromatic regions.
In other words, heterochromatic conformation can enhance
step 1. With puromycin selection, we determined more than
100 reinsertion sites using a control clone (Kokubu et al.,
unpublished). Although there are many local hopping events
which are commonly observed in the SB transposition (11, 13,
19, 24), we could not recognize any site preference within the
local hopping regions, implying that the SB transposon would
not prefer heterochromatin to euchromatin for reinsertion.
However, detailed examination is required for a definitive con-
clusion.

It has been reported that the HMGB1 protein interacts with
SB transposase and induces a local distortion of the DNA upon
binding in vitro, indicating the enhancement of the above-
mentioned step 2 (43). However, how the HMGB1 protein is
involved in the SB transposition in vivo must be determined.

When we tried to introduce SB transposase in the absence of
DOX, tTR-expressing and control clones had the same level of
transposon excision efficiency (data not shown). Why did the
excision efficiency change in tTR-expressing clones in the pres-
ence or absence of DOX? We hypothesized that tTR proteins
and their associated proteins may accumulate around the tetO
sequence, hindering the recruitment of SB proteins to the
IR/DRs (step 1) because of the close proximity between tetO
and IR/DRs. However, in the presence of DOX, tTR proteins
and their associated proteins were dissociated from the tetO
sequence, thus allowing access of the SB protein to the IR/
DRs. Alternatively, the binding of tTR and associated proteins
may disturb the bending of IR/DRs essential for synaptic-
complex formation and thus inhibit step 2.

Taking these data together, we hypothesize that the hetero-
chromatin conformation recruits SB transposase and enhances

its binding to the SB transposon that is a prerequisite for
subsequent reactions, such as excision and reinsertion.

The biological significance of these findings. Clarification of
the relationship between TEs and the host genome is becoming
an important issue, because mammalian-genome-sequencing
projects have revealed that approximately one-half of the total
number of genomes consists of TEs (30). One can easily imag-
ine that expansion of TEs would be deleterious to the host
genome due to chromosomal rearrangement mediated by TEs.
Expanded TEs reside mainly in the heterochromatin confor-
mation (22). A possible explanation of this phenomenon is that
heterochromatin causes a reduced rate of recombination. Dur-
ing evolution, species have selected against such high rates of
recombination.

Positive roles of TEs in genome evolution have also been
discussed (30). More recently, Kazazian proposed that mobile
elements are drivers of genome evolution (23). Viewed in this
light, the maintenance of TEs in heterochromatin might be an
important feature of genome evolution. The current findings
are that SB transposase has a preference for heterochromatin
conformation and is able to mobilize TEs efficiently in trans
when chromatin is in this conformation state. Therefore, TEs
might have evolved to counteract the host genome defense
strategy by accumulating in heterochromatin.

ACKNOWLEDGMENTS

We thank Z. Ivics for providing the anti-SB antibody. We are grate-
ful to M. Kouno, T. Hayakawa, K. Yae, S. Kouno, M. Tachibana, Y.
Shinkai, and H. Tojo for their helpful advice and excellent technical
support. We thank current members of our laboratory for their sup-
port and advice, particularly Y. Odan for her excellent secretarial
support.

This work was supported by grants from the New Energy and In-
dustrial Technology Development Organization of Japan; the Cosme-
tology Research Foundation; RIKEN, the Institute of Physical and
Chemical Research; and a grant-in-aid for Science Research from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan.

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