Affinity selection of DNA-binding protein complexes using mRNA display.

Abstract

Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes.

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Affinity selection of DNA-binding protein
complexes using mRNA display
Seiji Tateyama, Kenichi Horisawa, Hideaki Takashima, Etsuko Miyamoto-Sato,
Nobuhide Doi and Hiroshi Yanagawa*
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University,
3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
Received October 31, 2005; Revised December 21, 2005; Accepted January 27, 2006
ABSTRACT
ComprehensiveanalysisofDNA–proteininteractions
is important for mapping transcriptional regulatory
networks on a genome-wide level. Here we present
a new application of mRNA display for in vitro sel-
ection of DNA-binding protein heterodimeric com-
plexes. Under improved selection conditions using
a TPA-responsive element (TRE) as a bait DNA,
known interactors c-fos and c-jun were simultan-
eously enriched about 100-fold from a model library
(a 1:1:20000 mixture of c-fos, c-jun and gst genes)
after one round of selection. Furthermore, almost all
kinds of the AP-1 family genes including c-jun, c-fos,
junD, junB, atf2 and b-atf were successfully selected
from an mRNA display library constructed from a
mousebrainpolyA+RNAaftersixroundsofselection.
These results indicate that the mRNA display selec-
tion system can identify a variety of DNA-binding
protein complexes in a single experiment. Since
almost alltranscription
oligomeric complexes to bind with their target DNA,
thismethodshouldbemostusefultosearchforDNA-
binding transcription factor complexes.
factors form hetero-
INTRODUCTION
The specific interactions between cis-regulatory DNA ele-
ments and transcription factors are critical components of
transcriptional regulatory networks (1,2). The whole genome
and complete cDNA sequences contain a large number of
transcription factors and their binding DNA sequences, and
thus comprehensive analysis of DNA-transcription factor
interactions is expected to provide a deep understanding of
the mechanisms of cell proliferation, developmental processes
in tissue morphogenesis and disease. Currently, combined use
of chromatin immunoprecipitation (ChIP) assay with DNA-
microarrays (ChIP-chip) (3–5) is the most widely used high-
throughput method for discovering cis-regulatory DNA
elements for a transcription factor. In contrast, development
of high-throughput methods for discovering transcription
factors for a cis-regulatory DNA element remains at an
early stage. Although the yeast one-hybrid method (6,7)
and phage display (8–10) are attractive candidates,thesemeth-
ods are not easily scalable because of the use of living cells. In
addition, as over-expression of transcription factors often
affects cellular metabolisms, such transcription factors are
difficult to screen. In order to circumvent these difficulties,
we focused on a totally in vitro mRNA display technology
(11–17), in vitro virus (IVV) (11–14), for the discovery of
DNA–protein interactions. In mRNA display, a library of
genotype (mRNA)–phenotype (protein) linking molecules
(IVV) is constructed in which mRNA is covalently bound
to protein through puromycin during cell-free translation.
After affinity selection via the protein moiety of the IVV,
the mRNA moieties of the selected molecules are amplified
by means of RT–PCR. Therefore, even very low-copy number
proteins can be identified by iterative affinity selection from a
library with high diversity and complexity, routinely in the
range of 1013members (11–17).
We previously demonstrated that the IVV selection system
is effective for selection of protein–protein interactions in the
case of c-Jun and c-Fos bait proteins (13). In this study, we
show for the first time that the IVV selection system is also
useful for selection of DNA–protein complex interactions
(Figure 1). As a model bait DNA, we chose a TPA-
responsive element (TRE; TGAC/GTCA), which is a common
feature of promoter and enhancer sequences of many mam-
malian genes, such as collagenase I, SV40, interleukin 2,
CD44 and TNFa (18). We show here that IVVs for c-Fos
and c-Jun can form a heterodimer complex and interact
with TRE in a sequence-specific manner, and that almost
all kinds of the AP-1 family proteins can be enriched and
selected with TRE-immobilized beads from an IVV library
constructed from a mouse brain cDNA library.
*To whom correspondence should be addressed. Tel: +81 45 566 1775; Fax: +81 45 566 1440; Email: hyana@bio.keio.ac.jp
? The Author 2006. Published by Oxford University Press. All rights reserved.
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Nucleic Acids Research, 2006, Vol. 34, No. 3 e27
doi:10.1093/nar/gnj025

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MATERIALS AND METHODS
Preparation of bait DNA-immobilized affinity beads
Bait DNA was prepared by hybridization of two complement-
ary chemically synthesized oligonucleotide DNAs (19–22)
(Supplementary Table 1). Briefly, a mixture of equal amounts
of oligonucleotides dissolved in DNA-binding buffer [10 mM
Tris–HCl (pH 8.0), 1 mM EDTA, 1 M NaCl and 0.1% Triton
X-100] was mixed, heated at 95?C for 10 min, and cooled to
room temperature for 2–3 h. Then, 150 pmol of the DNA
mixture was added to 20 ml of either Streptavidin- or
NeutrAvidin-immobilized agarose beads (Pierce) and incub-
ated at 4?C for 1 h. The beads were washed with DNA-binding
buffer twice and equilibrated with selection buffer [50 mM
Tris–HCl (pH 7.6), 50 mM NaCl, 1 mM EDTA, 5 mM MgCl2,
5 mM DTT and 2% glycerol].
Preparation of positive and negative control
IVV constructs
DNAtemplatesforc-Fos (encoding c-Fos protein, aminoacids
118–212), c-Jun (encoding c-Jun protein, amino acids
237–334), and glutathione S-transferase (GST) (encoding
GST protein, amino acids 1–160) IVV formation were pre-
pared by means of two PCR steps using the primers listed in
Supplementary Table 1. The first PCR was performed using
either a mouse brain cDNA library (Takara Shuzo) or a pGEX-
4T-3 GST fusion vector (Amersham) as templates with the
primers 50T7c-Fos and 30c-FosFlag, 50T7c-Jun and 30c-Jun-
Flag, and 50T7GST and 30GSTFlag, respectively. The second
PCR was performed using the first PCR products as templates
with the primers 50FWT7 containing SP6 promoter, transla-
tional enhancer from tobacco mosaic virus (12–14), and T7 tag
and 30RV30 containing Flag tag and A tail. After purifi-
cation with a QIAquick PCR purification kit (Qiagen), the
DNA templates were transcribed with a RiboMAX large-
scale RNA production system SP6 (Promega). The resulting
mRNA was purified with an RNeasy RNA purification kit
(Qiagen) and ligated to a Fluoro-PEG Puro spacer [p(dCp)2-
T(Fluor)p-PEGp-(dCp)2-puromycin] (12–14) with a T4 RNA
ligase (Takara Shuzo). The ligated mRNA was again purified
with the RNeasy RNA purification kit.
Preparation of IVV template library
An IVV template library was prepared according to our pre-
vious protocols(12–14) with some modifications. Briefly,1 mg
ofmousebrainpolyA+mRNA(BDBiosciencesClontech)was
reverse-transcribed using a SuperScript double-strand cDNA
synthesis kit (Invitrogen) and 2 pmol of a 30random primer
(Supplementary Table 1) according to the manufacturer’s
instructions. The cDNA was ligated with an adaptor DNA
Figure 1. Schematic representation of the IVV selection procedure. (1) A cDNA library is transcribed and ligated with a Fluoro-PEG Puro spacer. (2) The IVV
templateRNAlibraryistranslatedinawheatgermcell-freetranslationsystem.(3)TheconstructedIVVlibraryispurifiedwithanti-FLAGM2antibody-immobilized
beadstoremoveuntranslatedmRNAandimpuritiescontainedinthewheatgermmixture.(4)ThepurifiedIVVare(5)subjectedtoaffinityselectionwithmutatedbait
DNA-immobilized beads to eliminate non-specific binders to DNA. (6) Unbound IVV are then subjected to affinity selection with bait DNA-immobilized beads.
(7) After washing, DNA-binding IVV are eluted by DNase I digestion. (8) The mRNA portions of the selected IVV are reverse-transcribed, PCR-amplified and
(9) subjected to the next round of selection or (10) identified by cloning and sequencing.
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(Supplementary Table 1) using a Ligation High kit (Toyobo).
The ligated cDNA was purified with the QIAquick PCR puri-
fication kit and PCR-amplified using 50FW and 30RV36 pri-
mers (Supplementary Table 1) and a TaKaRa Ex Taq hot start
version (Takara Shuzo). The PCR product was purified with
the QIAquick PCR purification kit, size-fractionated with a
CHROMA SPIN-1000 (BD Biosciences Clontech), and
transcribed in the same manner as described above. The
resulting mRNA was purified with the RNeasy RNA purifica-
tion kit, size-fractionated with the CHROMA SPIN-1000, and
ligatedtotheFluoro-PEG Purospacer with the T4RNA ligase.
The ligated mRNA was again purified with the RNeasy RNA
purification kit.
Affinity selection
IVV formation was performed as described previously (12–
14) with some modifications. A100 ml aliquot of a PROTEIOS
wheat germ cell-free protein synthesis mixture (Toyobo), con-
taining20pmol oftheligatedRNA and80UofaSuper RaseIn
RNase inhibitor (Ambion), was incubated at 24?C for 2 h. The
constructed IVVs were added to 60 ml of anti-FLAG M2
antibody-immobilized agarose beads (Sigma) equilibrated
with 40 ml of FLAG binding buffer [50 mM HEPES-NaOH
(pH 8.3), 150 mM NaCl and 0.25% Triton X-100] and mixed
on a rotator at 4?C for 1 h. The beads were washed with 400 ml
of FLAG binding buffer four times and treated with 50 ml of
FLAG elution buffer [50 mM Tris–HCl (pH 7.6), 50 mM
NaCl, 1 mM EDTA, 5 mM MgCl2, 2% glycerol, 5 mM
DTT, 1 mg of poly·d(I-C) and 1 mg/ml of FLAG M2 peptide
(Sigma)] at 4?C for 1 h. In the case of selection from a mouse
brain cDNA library, the purified IVVs were pre-selected with
mutated TRE bait DNA-immobilized agarose beads (Supple-
mentary Table 1) (21). After incubation at 4?C for 1 h, the
flow-through fraction containing unbound IVVs was subjected
to affinity selection.
The resulting IVVs were added to the bait DNA-
immobilized agarose beads and mixed on a rotator at 4?C
for 2 h. Streptavidin and NeutrAvidin agarose beads were
alternately used to avoid enrichment of Streptavidin- or
NeutrAvidin-specifically bound molecules. The beads were
washed with 50 ml of the selection buffer (described above)
seven times followed by 50 ml of DNase buffer [40 mM
Tris–HCl (pH 8.0), 10 mM NaCl, 10 mM CaCl2and 6 mM
MgCl2] once. Then, 50 ml of DNase buffer containing 3 U of
DNase I (Promega) was added and the mixture was incubated
at room temperature for 10 min. The reaction was terminated
by addition of 8 ml of 0.25 M EGTA. The resulting supernatant
was used as a template for RT–PCR. The RT–PCRs were
performed with a OneStep RT–PCR kit (Qiagen) using
50FW and 30RV30 primers (Supplementary Table 1). The
resulting RT–PCR product was used for the next round of
selection as described above or analyzed by quantitative
real-time PCR.
Quantitative real-time PCR analysis
Real-time PCR was performed with a LightCycler FastStrand
DNA master SYBR green I kit (Roche) using gene-specific
primers (Supplementary Table 2) (13,18,22–28). The standard
DNA was prepared by either PCR-amplification from
the cloned plasmids using M13-FW and M13-RV primers
(Supplementary Table 1) or RT–PCR from the mouse brain
polyA+mRNA using gene-specific primers (Supplementary
Table 2).
Cloning and sequencing
After selection, RT–PCR products were cloned using a Qiagen
PCR cloning kit (Qiagen) and sequenced with an ABI PRISM
3100 Genetic Analyzer (Applied Biosystems). The sequences
were clustered using the CLUSTALW program and subjected
to nucleotide–nucleotide BLAST (BLASTN) search to identi-
fy the protein represented by each cluster (13).
Pull-down assay
TheDNA templateswerepreparedbyPCR-amplificationfrom
the cloned plasmids using 50FW and 30RVXho27 primers
(Supplementary Table 1). For c-Jun, c-Fos, JunD, JunB and
B-ATF, the shortest clone was chosen in each case. The PCR
products were transcribed, translated and affinity-purified as
described in ‘Affinity selection’ except that DNase treatment
was omitted. Thebound proteinswere eluted with SDS–PAGE
sample buffer [0.1 M Tris–HCl (pH 6.8), 4% SDS, 0.2%
bromophenol blue and 20% glycerol] at 90?C for 5 min and
used for western blot analysis, which was performed with an
ECF western blotting kit (Amersham Biosciences) and mouse
anti-T7·tag monoclonal antibody (Novagen).
Electrophoretic mobility shift assay (EMSA)
EMSA was performed according to previous reports (20–22)
with minor modifications. Cy5-labeled double-stranded probe
DNA,non-specific double-stranded competitor DNA (21), and
non-labeled double-stranded probe DNA (Supplementary
Table 1) were prepared by hybridization as described
above. Proteins produced from the DNA templates of selected
clones were partially purified from the wheat germ cell-free
protein synthesis mixture by using anti-FLAG M2 antibody-
immobilized agarose beads (Sigma). The purity and concen-
tration of the proteins were monitored on 10–20% SDS–PAGE
with the ECF western blotting kit and the mouse anti-T7·tag
monoclonal antibody. The assay mixture contained, in a total
volume of 9 ml, 50 mM Tris–HCl (pH 7.6), 50 mM NaCl,
1 mM EDTA, 5 mM MgCl2, 5 mM DTT, 5% glycerol, an
appropriate amount of proteins (?20 ng), and 0.5 mg of poly-
·d(I-C). The mixture was incubated at room temperature for
5 min. Then, 100 fmol of Cy5-labeled probe DNA was added
and incubation was continued for 20 min. In the case of
competition assay, either non-specific competitor DNA or
non-labeled probe DNA was added to the mixture. The sam-
ples were electrophoresed on 4–20% gradient polyacrylamide
gel (Daiichi Pure Chemicals, Japan) with 0.25· TBE buffer.
Gels were run at 4?C for 2 h at 150 V and analyzed with a
Molecular Imager FX (Bio-Rad Laboratories).
RESULTS AND DISCUSSION
Establishment of the improved IVV selection system
To determine whether the IVV selection system is effective
for discovery of DNA-binding proteins (Figure 1), we first
performed a model experiment using a bait DNA containing
TRE and c-Fos and c-Jun proteins as prey. To obtain model
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libraries, DNA templates for c-fos,c-jun and gst were mixed in
ratios of 1:1:2 and 1:1:200. The model libraries were tran-
scribed, ligated to the Fluoro-PEG Puro spacer, and translated
in a wheat germ cell-free translation system. The resulting
IVV were then incubated with the bait DNA-immobilized
beads, washed and eluted by DNase I treatment. The eluates
were amplified byRT–PCR and the products were analyzed by
quantitative real-time PCR. As a result, both c-fos and c-jun
were simultaneously enriched about 100-fold after one round
of selection (Figure 2A, lanes 2 and 7), while no enrichment
was observed in the absence of the bait DNA (Figure 2A, lanes
3 and 8), in the presence of 400 mM free puromycin as an
inhibitor of the formation of IVV (Figure 2A, lanes 4 and 9),
and in the presence of a mutated TRE (CCGAATT) bait DNA
(Figure 2A, lanes 5 and 10). When c-fos and c-jun were inde-
pendently added to the model library, no enrichment and slight
enrichment (about 10-fold) were observed, respectively
(Figure 2B, lanes 5 and 8). These results indicate that the
peptide portions of c-Fos and c-Jun IVVs can form a heterodi-
mer complex and bind to the bait DNA in a sequence-specific
manner. This observation is consistent with previous reports
that c-Fos cannot bind to DNA and that a c-Fos/c-Jun het-
erodimer, rather than c-Jun/c-Jun homodimer, preferentially
binds to DNA (18,19,21,22).
Furthermore, we performed an iterative selection using a
1:1:20000 model library in the presence and absence of the
bait DNA. After four rounds of selection in the presence of
the bait DNA, c-fos and c-jun were enriched to a detect-
able level and gst was decreased (Figure 2C, lanes 2, 4, 6
and 8). The final ratio of c-fos, c-jun and gst was about
60:60:1, indicating that c-fos and c-jun had been enriched
about 1.2 · 104-fold versus gst (Figure 2D). On the other
hand, no significant enrichment of c-fos and c-jun was
observed in each round of selection in the absence of the
bait DNA (Figure 2C, lanes 3, 5, 7 and 9). These results
indicate that even low-copy number genes of DNA-binding
proteins can be detected by repeating the selection round in the
IVV selection system.
Selection of DNA-binding proteins from a cDNA library
Next we applied the established IVV selection system to the
selection of TRE-binding proteins from a mouse brain cDNA
library. Pre-selection of the cDNA-derived IVV library was
performed using anti-FLAG M2 antibody-immobilized beads
to remove untranslated mRNA and impurities contained in
thewheatgermcell-free
[Figure 1, step (3)] and using the mutated TRE bait DNA-
immobilized beads to eliminate non-specific binders to DNA
[Figure 1, step (5)]. Then, two different kinds of selection,
i.e. selection in the presence of bait DNA [bait (+) selection],
and selection in the absence of bait DNA [bait (?) selection],
were performed to distinguish non-specific binders to the
agarose beads (13,23). Selection was continued until almost
all of the selected clones were found to be positives or positive
candidates. After six rounds of the selection, the resulting
libraries, 6th bait (+) and 6th bait (?), were cloned and 477
and 98 randomly chosen clones were sequenced, respectively.
Clones found in both the bait (+) and the bait (?) libraries, or
that contained stop codons, were removed. The remaining
451 clones were subjected to nucleotide–nucleotide BLAST
translationsystemmixture
(BLASTN) search to identify the encoded proteins. Of the 451
clones, 39 corresponded to either 30-untranslated regions (30-
UTR) in mRNA sequences or incorrect reading frames. The
remaining 412 clones were then clustered into six sequence
groups and four individual sequences (Table 1).
The six sequence groups were known TRE-binding
proteins, c-Jun, c-Fos, JunD, JunB, ATF2 and B-ATF. Each
Figure 2. Affinity enrichment of DNA-binding protein complexes. (A) PCR
profilesofthe1:1:2and1:1:200modellibrariesareshown:initiallibrary(lanes
1and6),librariesafteroneroundofselectionoftheIVVlibraryinthepresence
(lanes 2 and 7) and absence (lanes 3 and 8) of the bait DNA, libraries after one
round of selection in the presenceof 400 mM free puromycinand the bait DNA
(lanes4and9)andlibrariesafteroneroundofselectionoftheIVVlibraryinthe
presenceofthemutatedTREbaitDNA(lanes5and10).(B)PCRprofilesofthe
c-fos, c-jun and gst (1:1:200), c-fos and gst (1:100), and c-jun and gst (1:100)
model libraries are shown: initial library (lanes 1, 4 and 7), libraries after one
round of selection in the presence (lanes 2, 5 and 8) and absence (lanes 3, 6
and 9) of the bait DNA. (C) PCR profile of each round of the 1:1:20000 model
library in the iterative selection. Initial library (lane 1) and libraries after first
(lanes 2 and 3), second (lanes 4 and 5), third (lanes 6 and 7) and fourth (lanes 8
and 9) rounds of selection in the presence (+; lanes 2, 4, 6 and 8) and absence
(?;lanes,3,5,7and9)ofthebaitDNA.(D)Contentsofthec-fos,c-junandgst
genesinlibrariesateachroundoftheiterativeselection.DNA(1010molecules,
4–5 ng) was subjected to quantitative real-time PCR with specific primers
(Supplementary Table 2).
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cluster except for ATF2 consists of plural protein sequences
(Table 1). All of the six selected protein sequences contained
the conserved basic region for TRE-interaction and the heptad
repeat of leucine residues required for dimerization. To deter-
mine whether the selected protein fragments indeed specific-
ally bind with the bait DNA, we performed pull-down assay
and EMSA. As expected, c-Jun, JunD and JunB showed inter-
action with the bait DNA. ATF2 showed faint but significant
interactions in pull-down assay and EMSA (Figure 3). The
relative binding activity was enhanced in the presence of
c-Jun. c-Fos and B-ATF themselves did not bind with the
bait DNA, but did bind in the presence of c-Jun (Figure 3).
In our experiments using EMSA, we found that binding
affinities of c-Fos/c-Jun/TRE, c-Fos/JunB/TRE, c-Fos/JunD/
TRE and c-Jun/B-ATF/TRE complexes ranged from 12.5 to
50 nM, whereas that of c-Jun/ATF2/TRE complex was about
200 nM. This result is not in conflict with previous reports
on the selection of protein–protein interactions by mRNA
display: the binding affinities of most selected proteins are
<1 mM (16,17).
The four individual sequences were Eef1a1, SGT1,
6330407J23Rik and a hypothetical protein (named Hypothet-
ical protein 1). Hypothetical protein 1 and 6330407J23Rik
have not yet been registered as an open reading frame
(ORF) in the public databases, though they are present in
the mouse genome sequence and mRNA sequence, respect-
ively. The other two are known proteins, in-frame, and within
the native ORF, but have not been reported to bind with TRE,
c-Fos or c-Jun. In DNA-binding experiments with these pro-
teins, faint but possibly unspecific interactions were observed
in pull-down assays, but no interaction was observed in EMSA
(Figure 3). These interactions were not enhanced in the pres-
ence of other selected proteins. Search of the PSORTII pro-
gram and other public databases revealed that Eef1a1 and
SGT1 proteins are not located in the nucleus, implying that
these proteins may not act as transcription factors. Although
these four clones may be false-positives, further studies would
be required to clarify whether Hypothetical protein 1 and
6330407J23Rik exist in cells and have biological significance,
and whether these four proteins have a biologically relevant
interaction with TRE in vivo.
Confirmation of the enrichment of other
known candidates
On the basis that IVVs specifically interacting with TRE are
enriched in the selection (13,23), we further performed quant-
itative real-time PCR analysis (23) to determine whether not
only c-jun, c-fos, junD, junB, atf2 and b-atf, but also other
known positive candidates (18,24–28), fosB, fra1, fra2, atf3
and atf7, were enriched in the 6th bait (+) library as compared
with the initial library. As shown in Figure 4, significant
enrichments were confirmed for fosB, fra1, fra2 and atf7 as
well as all the selected clones listed in Table 1. None of the
candidates was enriched in any round of the bait (?) libraries.
In the case of atf3, significant enrichment could not be detec-
ted. The enrichment efficiencies of fosB, fra1, fra2 and atf7
were lower than those of c-jun, c-fos, junD, junB, atf2 and b-
atf, but higher than those of hypothetical protein 1, eef1a1,
sgt1 and 6330407J23Rik (Figure 4). The rates of successfully
cloned known positives and successfully enriched known
Table 1. Characterization of the selected clones
Gene symbol
c-jun
c-fos
junD
junB
atf2
b-atf
hypothetical protein 1
eef1a1
sgt1
6330407J23Rik
Accession no.
NM_010591
NM_010234
NM_010592
NM_008416
NM_009715
NM_016767
CAA01058705
NM_010106
NM_026474
NM_026138
Number of clones
173
113
53
27
23
13
6
2
1
1
Types of selected
protein fragments
10
4
3
4
1
2
1
1
1
1
Locus on mRNA
sequence (base)
1556–1903
488–775
820–1131
971–1348
1265–1513
270–566
1728–1790a
1408–1477
783–845
1864–1968
Locus on the shortest
mRNA sequence (base)
1682–1858
551–763
892–1086
1115–1315
1265–1513
270–488
1728–1790a
1408–1477
783–845
1864–1968
Leucine heptad repeats
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
TRE-binding motifs
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Pull-down assayb
+++
?/+++c
+++
+++
+/++c
?/+++c
+
+
+
?
EMSA
Yes
No/yesc
Yes
Yes
Yes
No/yesc
No
No
No
No
aLocus on Mus musculus whole genome shotgun assembly contig 47331.
bThe relative binding activities are designated by plus and minus signs and represent the percentages of total protein that bound: +++, >10%; ++, 1–10%; +, 0.1–1%; ?, not detected.
cIn the presence of c-Jun.
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Figure 3. Interactionbetween the selectedclonesand TRE.Approximately20 ng of the purifiedproteinswere mixed with100 fmolof the Cy5-labeledprobe DNA
andincubatedatroomtemperaturefor20min(lanes1,4,5,8,11,14,15,16,17,18,19,22,25,28,31,34,35,36and37).Either5pmol(lanes2,6,9,12,20,23,26,29
and 32)of non-specific competitorDNA or5 pmol(lanes3, 7, 10,13, 21, 24,27, 30 and33) ofnon-labeled probeDNA wasadded.Openarrowheads,homodimeric
complexes;solidarrowheads,heterodimericcomplexes; asterisks,aheterodimericcomplexofc-Fosandc-Jun.Theupperbandoffreeprobes(seeninalllanes)isa
dsDNA consisting of two complementary oligonucleotide DNAs both labeled with Cy5 at their 50ends, and the lower one (seen in lanes 3, 7, 10, 13, 21, 24, 27, 30
and 33) is an unexpectedly formed hybrid of non-labeled and labeled oligonucleotide DNAs.
Figure 4. Quantitative real-time PCR analysis of the selected clones in the initial, 6th bait (+), and 6th bait (?) libraries. Contents of DNAs of selected clones and
b-actinasanegativecontrolin5ng(about1010molecules)oftheeachlibrarywereanalyzedwithspecificprimers(SupplementaryTable2)usingaLightCycler.The
enrichment rates of c-jun, c-fos, junD, junB, atf2, b-atf, hypothetical protein 1, eef1a1, sgt1, 6330407J23Rik, fosB, fra1, fra2 and atf7 were 3.2 · 105-, 3.0 · 105-,
5.4 · 104-, 9.6 · 104-, 4.8 · 103-, 3.7 · 104-, 1.3 · 102-, 13-, 1.6 · 102-, 1.7 · 102-, 9.2 · 102-, 8.0 · 102-, 8.7 · 102-, and 8.3 · 102-fold, respectively.
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positives are 6/11 and 10/11, respectively. Although almost all
these AP-1 family proteins (ten kinds) were significantly
enriched, the question arises, why were c-Jun, c-Fos, JunD,
JunB, ATF2 and B-ATF successfully cloned, while the others,
so-called false-negatives, were not? As shown in Figure 4, the
contents of c-jun, c-fos, junD, junB, atf2, b-atf, fosB, fra1, fra2
and atf7 in the 6th bait (+) library were about 37, 19, 9.4, 3.1,
3.0, 1.5, 0.0034, 0.0011, 0.00088 and 0.0066%, respectively.
Because 412 clones were cloned and analyzed in this study,
only genes whose content is more than 0.25% would be cloned
theoretically. Indeed, the contents of all of the selected clones
quantified by real-time PCR were at least 0.25%. Excess selec-
tion rounds do not result in enrichment of false-negatives,
because the pool is dominated by the positives (such as c-
fos and c-jun). If the affinities of false-negatives are much the
same as those of the positives or lower than those of the
positives, the false-negatives cannot exclude the positives
from the saturated library. Thus, enrichment of such false-
negative clones would be detected by the combination of
the IVV system with a DNA microarray (29), as a sensitive
and high-throughput alternative to the process of cloning and
sequencing. Another possible reason for the occurrence of
false-negatives may be their low contents (fosB, fra1, fra2
and atf7, less than 0.00001%; atf3, less than 0.000001%),
or low contents of in-frame genes (fosB, fra1, fra2 and
atf7, less than 0.0000017%, atf3, less than 0.00000017%, the-
oretically), in the initial library; in this case, the use of a
normalized library constructed from tissues (30,31) or a
full-length cDNA clone library may reduce the number of
false-negatives.
In view of the enrichment factor of 100 for c-jun and c-fos
found in the selection of model libraries (Figure 2), the ques-
tion arises, why do those two proteins show only rather low
enrichment factors of 3.2 · 105and 3.0 · 105, respectively,
after a total of six rounds (Figure 4)? When the enrichment
factors for c-fos and c-jun in the selection of the cDNA library
were monitored in each round of selection, the factor of
50–170 in the 2nd round was comparable with that in the
model selection, while the factors of 2–15 in the 1st and 3–
6th rounds were relatively low. One possible reason for the
lower enrichment efficiencyin the 1stround of selectionis that
the initial library contains not only in-frame gene fragments,
but also out-of-frame gene fragments from the same region as
the in-frame gene fragments, because the IVV library was
constructed by random-priming PCR of mRNA. Probably
only in-frame gene fragments are enriched and out-of-frame
gene fragments are removed in the 1st round of selection, and
the selected in-frame gene fragments are then enriched in the
latter rounds of selection. As the selection rounds proceed,
DNA-binders other than c-fos and c-jun are also enriched in
the pool and then compete with each other. In contrast, such
competitors were absent in the model selection (only the gst
gene was present).
Advantages of IVV selection system
The use of our IVV selection system for selection of DNA–
protein interactions as described here has several advantages
over previous techniques, such as one-hybrid and phage dis-
play systems, mainly due to the greater flexibility of the
in vitro selection conditions and the high diversity and
complexity of the IVV library which contains about 1013inde-
pendent molecules. First, the IVV selection system is available
for selection of DNA-binding protein heterodimeric com-
plexes. Since almost all transcription factors form heterooli-
gomeric complexes to bind with their target DNA, our results
indicate that the IVV selection system would be more useful
than previous techniques to search for DNA-binding transcrip-
tion factor complexes. Phage display systems were recently
utilized for selection of DNA-binding proteins (8–10), but no
DNA-binding protein that forms a heterooligomeric complex
was obtained. The yeast one-hybrid method (6,7) is not neces-
sarily suitable for the detection of transcription factors that can
form heterodimers and then bind with their target DNAs, but
which cannot bind to the DNAs as monomeric proteins or
homodimeric protein complexes. For example, c-Fos and
B-ATF themselves do not show TRE-binding ability, and
would be identified as negatives in the yeast one-hybrid
system.
Second, the IVV selection system, a totally in vitro
technique, is available for enrichment of many kinds of bind-
ers, such as c-Jun, c-Fos, JunD, JunB, ATF2, B-ATF, FosB,
Fra1, Fra2 and ATF7 in a single experiment, probably because
the diversity and complexity of the IVV library is not limited
by the use of living cells. In particular, over-expression of
transcription factors that are usually expressed at a low
level is often toxic to the host cells in phage display and
yeast one-hybrid systems.
Furthermore, the IVVselection system affordsa remarkably
low false-positive rate. Even if all four novel proteins are
false-positives, the false-positive rate is only 2% (10 clones
per 412 clones; Table 1). Purification using the anti-FLAG M2
antibody-immobilized beads and especially pre-selection of
the IVV library with the mutated TRE bait DNA-
immobilized beads were very effective to reduce both
false-positives and false-negatives: when these processes
were omitted, the number of false-positive clones reached
?50% and only c-jun, c-fos and junD were cloned (data not
shown).
In summary, we have demonstrated the utility of the IVV
system for selection, analysis and mapping of DNA-
transcription factor interactions. The greater flexibility of
the selection conditions of the IVV system, and the greater
diversity and complexity of the IVV library allow easier selec-
tion of a variety of protein complexes with low rates of false-
positives, as compared with current techniques such as phage
display and the yeast one-hybrid method. Therefore, our sys-
tem should contribute to the large-scale analysis of DNA-
transcription factor interactions for mapping of transcriptional
regulatory networks on a genome-wide level.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
TheauthorsthankMsA.Utsumiforexperimentalsupport.This
work was supported in part by a Grant-in-Aid for Scientific
Research, and a Special Coordination Fund from the Ministry
of Education, Culture, Sports, Science and Technology, Japan.
PAGE 7 OF 8
Nucleic Acids Research, 2006, Vol. 34, No. 3 e27

Page 8

Funding to pay the Open Access publication charges for this
article was provided by Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science, and Technology, Japan.
Conflict of interest statement. None declared.
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