2. ing to stimulate or repress target gene transcription (3–5).
NRs are well known to possess a characteristic modular
structure, and TCRs are recruited to specific domains of
NRs in a ligand-independent or -dependent manner (3–
5). Although the DNA-binding domain (DBD) of NRs is
necessary for binding to their cognate hormone-respon-
sive elements located near proximal promoters as well as
in enhancer elements far distant from transcription start
sites, many previous studies indicate that the DBD could
also function as an interaction surface with other proteins
(6–10). Using yeast two-hybrid (YTH) screen with the
DBD and a part of the hinge region of thyroid hormone
receptor (TR)  as bait, we previously isolated PSMC3
(also known as Tat-binding protein1), one of the 6 AT-
Pase subunits consisting of 19S regulatory subunit of 26S
proteasome and showed that Tat-binding protein1 func-
tions as a coactivator of TR as well as androgen receptor
(11–13).
Peroxisome proliferator-activated receptor (PPAR)␥, a
member of the NR family, is well-recognized to function
as a master regulator of adipocyte differentiation and
thereby believed to play fundamental roles in lipid and
glucose metabolism in the body (14–16). A number of
TCRs are shown to directly interact with different do-
mains of PPAR␥ including DBD and involved in adipo-
genesis in vivo and in vitro (17–19). In an additional
attempt to identify proteins that interact with the DBD
and a part of the hinge region of human PPAR␥ using
YTH screen, we isolated a partial cDNA of KIAA1769, a
human expression sequence tag with unknown function
at the time of experiments, and termed PPAR␥-DBD-in-
teracting protein 1 (PDIP1) (20). Surapureddi et al (21)
independently identified the rat orthlogue of KIAA1769
in biochemically purified liver proteins that associates
with liganded full-length PPAR␣ and designated as
PPAR␣-interacting cofactor complex 285 (PRIC285). In
several human cell lines, we identified the expression of 2
PDIP1 isoforms that differ in their N-terminal amino acid
(aa) alignment due to alternative splicing (the short iso-
form named as PDIP1␣ that corresponds to KIAA1769
[PRIC285 isoform2] and the long isoform as PDIP1
[PRIC285 isoform1]) (20). Cotransfection of PRIC285/
PDIP1␣ or PDIP1 could enhance PPAR␥ and PPAR␣-
mediated transcriptional activation and also augment
transcription mediated by several other NRs in cell cul-
ture systems (20, 21). Recently, Hugo Gene Nomencla-
ture Committee has approved HELZ2 (helicase with zinc
finger 2, transcriptional coactivator) as the symbol name
for PRIC285/PDIP1, and here we designated PDIP1 as
HELZ2 and PDIP1␣/PRIC285 as HELZ2␣. However,
the molecular mechanism whereby HELZ2 enhances
PPAR␥-mediated gene transcription remains to be
elucidated.
To gain further insights into the molecular function of
HELZ2, we attempted to isolate HELZ2-associating pro-
teins in HeLa cells by employing immunoprecipitation
(IP) with epitope-tagged HELZ2 followed by mass spec-
trometry analyses in the present study. We here isolated
the TR-associated protein 3 (THRAP3) (also known as
TR-associated protein, 150-kDa, TRAP150) (22) as a
protein stably associating with HELZ2. We character-
ized the interaction domains and functional cooperation
in PPAR␥-mediated gene transcription between HELZ2
and THRAP3 and identified novel physiologic roles of
Thrap3 in adipocyte differentiation.
Materials and Methods
Cell cultures
HeLa cells were cultured in DMEM supplemented with 10%
fetal bovine serum (Biowest, Rue de la Caille, France) and am-
picillin/streptomysin (GIBCO, Life Technologies Corporation
Japan, Tokyo, Japan) at 37°C under 5% CO2 atmosphere.
3T3-L1 cells were obtained from Health Science Research Re-
sources Bank (Osaka, Japan) and cultured in DMEM containing
10% fetal bovine serum and antibiotics. Adipogenic differenti-
ation was induced using insulin, dexamethasone (Sigma-Aldrich
Japan, Osaka, Japan), and isobutylmethylxanthine (Enzo Life
Science, Ann Arbor, Michigan) as previously described (20).
Plasmids
The expression vectors for human HELZ2 (pSVSPORT-
HELZ2) and mouse PPAR␥2 (pSVSPORT-PPAR␥2) were de-
scribed previously (20). The HELZ2 cDNA was excised by
EcoRI and XbaI from pSVSPORT-HELZ2 (20) and ligated to
the EcoRI/XbaI site of p3ϫFLAG-CMV10 (pCMV10-
HELZ2) (Sigma-Aldrich Japan). The full-length cDNA encod-
ing human THRAP3 was amplified by RT-PCR using HeLa cell
RNA and ligated to pCR2.1 vector (Invitrogen, Life Technolo-
gies Corp., Gaithersburg, Maryland) (pCR2.1-THRAP3).
THRAP3 cDNA was then excised by EcoRI from pCR2.1-
THRAP3 and inserted to EcoRI site of pcDNA3.1 vector (Invit-
rogen, Life Technologies Corp) (pcDNA3.1-THRAP3). Partial
THRAP3 cDNAs (aa 168–995 [⌬N], aa 1–591 [⌬C], and aa
168–591 [⌬NC]) were excised from pCR2.1-THRAP3 by
BamHI/SalI, EcoRI/MluI, and BamHI/MluI, respectively, and
ligated in flame into pVP16 vector (Takara Bio Inc., Otsu, Ja-
pan). Partial cDNAs encoding THRAP3 ⌬N, ⌬C, and ⌬NC
were also amplified by PCR and subcloned in flame into
pcDNA4/HisMax vector (Invitrogen, Life Technologies Corp.).
The full-length HELZ2 cDNA was amplified by PCR and li-
gated in flame into pAcGFP1-N1 and -C1 vectors (CLON-
TECH Laboratories, Inc, Palo Alto, California). PCR-amplified
THRAP3 cDNA was ligated into pDsRed-Monomer-C1 vector
(CLONTECH). The nucleotide sequences of all constructs were
verified by sequencing.
770 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780
3. Preparation of nuclear extract (NE),
immunoprecipitation (IP), and liquid
chromatography-tandem mass spectrometry
(LC-MS/MS)
pCMV10-HELZ2 or p3ϫFLAG-CMV7-bacterial alkaline
phosphatase (FLAG-BAP) (Sigma-Aldrich Japan) was trans-
fected to HeLa cells in a 10-cm dish using Lipofectamine 2000
(Invitrogen, Life Technologies Corp), and NE was prepared af-
ter 24 hours according to the method as described previously
(23). One milligram of NE was subjected to immunoprecipita-
tion with anti-FLAG M2 antibody (Ab)-conjugated agarose
(Sigma-Aldrich Japan) overnight at 4°C, and washed, and
bound proteins were eluted using excess amounts of FLAG pep-
tide (Sigma-Aldrich Japan). The released proteins were then re-
solved in SDS-PAGE, and the gel was stained with silver staining
kit (Life Technologies Corp.). The specific bands not observed
in the eluent from FLAG-BAP-transfected cells were cut out and
subjected to aa analysis by nano LC-MS/MS on a ThermoFisher
LTQ Orbitrap XL (Nextgensciences, Ann Arbor, Michigan). In
other experiments, preparation of NE and IP were carried out
using Universal magnetic Co-IP kit (Active Motif Japan, Tokyo,
Japan). The protein concentration was determined by Brad-
ford’s method using albumin as standard.
IP and Western blot analysis
IP and immunoblotting were performed as we described pre-
viously (11–13, 20) using anti-FLAGM2 Ab (Sigma-Aldrich Ja-
pan), anti-THRAP3 Ab (H-300, Santa Cruz Biotechnology, Inc,
Santa Cruz, California), anti-PPAR␥ Ab (catalog no. 81B8, Cell
Signaling Technology Japan, Tokyo, Japan; and catalog no.sc-
7273, Santa Cruz Biotechnology), antirabbit polyclonal Helz2
Ab (no. 2332–2) generated by immunizing a polypeptide de-
rived from mouse Helz2 (Immuno-Biological Laboratories Co.,
Ltd., Fujioka, Japan), and anti-Xpress Ab (Invitrogen, Life
Technologies Corp). Anticyclophilin A Ab (Upstate Biotechnol-
ogy Inc., Lake Placid, New York), anti- actin Ab (Sigma-Al-
drich Japan), Living Colors GFP (green fluorescent protein) and
DsRed monoclonal Abs (CLONTECH) were used for Western
blotting.
Mammalian two-hybrid assay
Expression vectors for GAL4DBD-fused HELZ2 polypep-
tides (pMHELZ2) were previously constructed (20). Transient
transfection with 3ϫupstream activating sequence-thymidine
kinase-luciferase vector (3ϫUAS-TKLuc) (Takara Bio, Inc.) in
the absence or presence of pMHELZ2 and/or pVP16THRAP3
was carried out using the calcium phosphate precipitation
method in HeLa cells. The total amounts of transfected plasmids
were adjusted by adding empty pM or pVP16 vector. Luciferase
activity was measured after 24 hours of transfection and nor-
malized by protein concentration as described previously (11–
13, 20).
Glutathione S-transferase (GST) pull-down assay
pGEX4T1 vectors expressing GST-fused partial HELZ2
polypeptides were constructed previously, and GST-fused poly-
peptides were prepared in Escherichia coli as described else-
where (20). Proper synthesis of fusion proteins was verified by
SDS-PAGE analysis. 35
S-labeled full-length THRAP3 protein
was synthesized using in vitro transcription/translation system
(Promega Corp, Madison, Wisconsin) and pcDNA3.1-
THRAP3. Equivalent amounts of GST alone or GST-fused pro-
teins were incubated with 35
S-labeled THRAP3 for 1 hour at
4°C and, after extensive washing, subjected to SDS-PAGE as
described previously (20). The binding was quantitated using an
image analyzer (BAS-2000, Fujifilm Corp, Tokyo, Japan).
Fluorescence microscopy analyses
HeLa cells were grown on chamber slides and AcGFP1-
tagged HELZ2 and/or DsRed-tagged THRAP3 expression
vector was transfected in the presence of pSVSPORT-PPAR␥2
using Lipofectamine 2000 (Invitrogen, Life Technologies
Corp.). In other experiments, pCMV10HELZ2 was trans-
fected to HeLa cells with DsRedTHRAP3 and pSVSPORT-
PPAR␥2. After fixation, cells were incubated with anti-FLAG
M2Ab for 2 hours at 37°C. After washing, cells were incubated
with antimouse IgG-FITC (Sigma-Aldrich Japan) for 45 minutes
at 37°C. Direct and indirect fluorescence microscopy analysis
was performed 24 hours after transfection. The microscopic
images at 520–550 nm and 460–490 nm excitation wave-
lengths were merged using Adobe Photoshop Elements 8
(Adobe Systems, Inc., San Jose, California).
Luciferase assay and short interfering RNA (siRNA)
HeLa cells were cultured in 6-well plates and transfected
with a firefly luciferase reporter vector driven by 3 copies of
direct repeat with 1-bp spacer (DR1-TKLuc) (20) in the absence
or presence of pSVSPORT-PPAR␥2, pSVSPORT-HELZ2, or
pcDNA3.1-THRAP3 using the calcium phosphate precipitation
method. The total amounts of transfected plasmids were ad-
justed by adding empty pSVSPORT vector. Luciferase activity
was measured after 24 hours of incubation with 10 M trogli-
tazone (Daiichi Sankyo Co, Tokyo, Japan) or dimethylsulfoxide
as described elsewhere (11–13, 20). siGENOME SMRTpool
siRNA for human and murine THRAP3 and control siRNA
were obtained from Thermo Fisher Scientific (Lafayette, Colo-
rado). siRNA targeting murine Helz2 was obtained from Sigma-
Aldrich Japan. The knockdown efficiency of siRNA introduced
into HeLa cells was evaluated by Western blotting of whole cell
lysate (WCL) and/or qRT-PCR using total RNA prepared from
siRNA-transfected cells. Twenty-four hours after transfection
of siRNA, DR1-TKLuc was transfected in the presence or ab-
sence of expression vectors for PPAR␥ and/or HELZ2 by cal-
cium phosphate precipitation method, and a luciferase assay
was performed as described above.
Electroporation of siRNA and quantitative RT-PCR
(qRT-PCR)
Electroporation of siRNA into 3T3-L1 preadipocytes was
carried out using Gene Pulser II (Bio-Rad Laboratories, Inc.,
Hercules, California) at 100 V and 950 F as described (24). At
the indicated time, WCL was prepared as described and total
RNA was isolated using Isogen (Nippon Gene Co, Ltd, Tokyo,
Japan). qRT-PCR was preformed as described previously (25).
The TaqMan probes and PCR primers used were summarized in
Supplemental Table 1 published on the Endocrine Society’s
Journals Online web site at http://mend.endojournals.org. Oil
Red O staining was performed using a kit according to the
doi: 10.1210/me.2012-1332 mend.endojournals.org 771
4. manufacturer’s protocol (Cayman Chemical, Ann Arbor,
Michigan).
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed using a kit (EMD Millipore
Corp., MA) according to the protocol as we described previ-
ously (12, 13). Antibodies used in ChIP assays were as described
above. Primers for qRT-PCR to amplify the genomic region
containing PPAR␥-response elements (PPREs) in mouse Fabp2/
aP2 and Adipoq gene enhancers were as previously described
(26, 27).
Statistical analysis
Statistical analysis between 2 samples was performed using
unpaired t test, and multiple comparison was carried out using
ANOVA, followed by the Turkey’s multiple comparison tests.
Significance was set at P Ͻ .05.
Results
Identification of THRAP3 as a
factor associating with HELZ2
using coimmunoprecipitation
coupled with LC-MS/MS
To isolate HELZ2-associating
proteins, we transiently transfected
FLAG-tagged HELZ2 into HeLa
cells, and IP of NE was performed
using anti-FLAG Ab. A silver-
stained band at 150 kDa, not ob-
served in control IP from FLAG-
BAP-transfected cells, was excised
and subjected to LC-MS/MS analysis.
Six different polypeptides matching to
the aa alignments in THRAP3 (22)
were identified by database search
(Figure 1A). To verify the association
between HELZ2 and THRAP3, IP
was carried out using anti-FLAG Ab
with HeLa cell NE transfected with
FLAG-HELZ2, and subjected to im-
munoblotting using anti-THRAP3
Ab. As shown in Figure 1B, endoge-
nous THRAP3 was precipitated with
anti-FLAG Ab, but not with preim-
mune IgG. In a reciprocal manner,
FLAG-tagged HELZ2 was precipi-
tated with anti-THRAP3 Ab (Figure
1C). These findings clearly illustrate
thatHELZ2andTHRAP3stablyas-
sociate in the HeLa cell nuclei.
To next evaluate whether endog-
enous Thrap3 protein could associ-
ate with endogenous Helz2 or Pparg,
IP coupled with immunoblotting was
carried out using NE prepared from
differentiated 3T3-L1 cells. As shown in Figure 1D, endog-
enous Thrap3 protein could be immunoprecipitated with
anti-Helz2 Ab (left panel). Endogenous Ppar␥ could also be
immunoprecipitated with anti-Helz2 Ab (middle panel).
Moreover, endogenous Ppar␥ could be coprecipitated with
anti-Thrap3 Ab (right box). The association among endog-
enous Pparg, Helz2, or Thrap3 in 3T3-L1 cells did not ap-
parently increase by 24-hour treatment with troglitazone, a
synthetic ligand for PPAR␥, in our experimental condition
(Figure 1D, right panel and data not shown). We also stud-
ied the cellular colocalization of GFP-tagged HELZ2 with
DsRed-tagged THRAP3 using direct and indirect fluores-
cence microscopy analyses and found that HELZ2 and
THRAP3 could colocalize in the HeLa cell nucleus (data not
Figure 1. A, Identification of THRAP3 as a HELZ2-interacting protein in HeLa cells using LC-
MS/MS. Transfection, preparation of NE, and IP were performed as described in Materials and
Methods. Silver-stained SDS-PAGE gel is shown. The positions of molecular size markers are
indicated in kilodaltons (kDa) and “Input” represents 3% of input NE. A band around 150 kDa in
FLAG-HELZ2-transfected cells, not observed in FLAG-BAP-transfected cells, was cut out and
subjected to aa analysis using LC-MS/MS. B, Endogenous THRAP3 was coprecipitated with FLAG-
HELZ2. HeLa cells were transfected with 3ϫ FLAG-HELZ2, and NEs were prepared after 24
hours. IP of 500 g NE was carried out using anti-FLAG Ab or preimmune IgG, and IP samples
were subjected to SDS-PAGE. Immunoblotting (IB) was performed using anti-THRAP3 Ab
(␣THRAP3). “Input” represents 4% of input NE. C, HELZ2 was coprecipitated with endogenous
THRAP3. IP of 500 g of 3ϫFLAG-HELZ2-transfected NE was carried out using ␣THRAP3 or
IgG. IB was performed using anti-FLAG Ab (␣FLAG). “Input” represents 4% of input NE. D,
Association of endogenous Thrap3 with Helz2 and Pparg in differentiated 3T3–L1 cells. Five
hundred micrograms of NE prepared from troglitazone (Tro)-treated differentiated 3T3–L1 cells
were subjected to IP using indicated Abs followed by IB employing indicated Abs. “Input”
represents 4% of input NE.
772 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780
5. shown). These findings taken together indicated that endog-
enous Pparg, Thrap3, and Helz2 could stably associate each
other in the nucleus of differentiated 3T3-L1 cells.
HELZ2 interacts with the serine (S)/arginine (R)-
rich domain and the Bcl2-associated transcription
factor (BCLAF) 1-homologous region in THRAP3
Human THRAP3 protein consists of 995 amino acids
and possesses an S/R-rich domain (SR domain) at its N
terminus and also carries a region homologous to
BCLAF1 at its C terminus (28) (Figure 2A). To next de-
termine the region necessary for
binding to HELZ2 in THRAP3,
the full-length and truncated forms
of THRAP3 were expressed as
VP16-fusion proteins, and their in-
teraction with GAL4DBD-fused
HELZ2 (pMHELZ2) were exam-
ined using a mammalian two-hybrid
assay in HeLa cells. As shown in Fig-
ure 2B, the activity of 3ϫUAS-TK-
Luc was significantly activated by
cotransfection of pMHELZ2 with
pVP16THRAP3. Significant, but
lesser, activation was observed by
cotransfection of pMHELZ2 with
pVP16⌬N that lacks the SR domain.
In contrast, cotransfection of
pMHELZ2 with pVP16⌬C that
lacks the region homologous to
BCLAF1 or pVP16⌬NC that lacks
both SR domain and BCLAF1 ho-
mologous region did not activate the
reporter gene. To further confirm
the THRAP3 domain necessary for
interaction with HELZ2, vector
expressing HisMax-tagged ⌬N, ⌬C,
or ⌬NC THRAP3 was cotransfected
with the FLAG-tagged HELZ2 ex-
pression vector, and IP was carried
out using anti-FLAG Ab followed by
immunoblotting with anti-Xpress
Ab that recognizes HisMax-tag.
Expression levels of FLAG-tagged
HELZ2 and truncated THRAP3
proteins were verified by immuno-
blotting using anti-FLAG Ab and
anti-Xpress Ab, respectively (left
panel in Figure 2C). As shown in
the right panel of Figure 2C,
FLAG-tagged HELZ2 protein as-
sociated only with the full-length
THRAP3, but not with N- or C-
terminal truncated THRAP3 protein. These findings
collectively indicated that HELZ2 requires both the
N-terminal SR domain and the C-terminal BCLAF1
homologous region to effectively interact with
THRAP3.
THRAP3 directly binds the helicase motifs in
HELZ2
To next evaluate the region necessary for direct bind-
ing to THRAP3 in HELZ2, partial polypeptides of
Figure 2.A, Schematic representation of VP16 fusion constructs carrying the full-length
and truncated versions of THRAP3. SR domain, and the region homologous to BCLAF1 in
THRAP3 are indicated. ⌬N and ⌬C lack aa 1–167 and aa 592–995, respectively. ⌬NC lacks
both aa 1–167 and aa 592–995. B, HELZ2 interacts mainly with the C-terminal BCLAF1
homologous region in THRAP3 in mammalian two-hybrid (MTH) assay. MTH assay was
performed using pMHELZ2, and pVP16 fused full-length or truncated THRAP3 vector in
HeLa cells. Data are expressed as fold activation of the luciferase activity in the presence of
pMHELZ2 and empty VP16 vector, and represent mean Ϯ SEM from 3 independent
experiments. Asterisks denote significant difference between indicated groups (P Ͻ .05). C,
HELZ2 requires both the SR domain and the region homologous to BCLAF1 to associate
with THRAP3 in IP assays. FLAG-HELZ2 and HisMax-tagged full-length or truncated THRAP3
expression vectors were transfected to HeLa cells. WCL was prepared and subjected to IP
with Xpress AB (␣Xpress) followed by IB using FLAG AK (␣FLAG) (right panel). IB using
␣FLAG and ␣Xpress of 10% of input WCL was shown in the left panel.
doi: 10.1210/me.2012-1332 mend.endojournals.org 773
6. HELZ2 were serially synthesized as GST-fusion proteins
(Figure 3A), and GST pull-down assays were carried out
using in vitro translated 35
S-labeled full-length THRAP3
protein. Proper synthesis of GST-fused proteins was ver-
ified by SDS-PAGE analysis (Figure 3B). After extensive
washing, the partial polypeptides corresponding to aa
563-1059 (helicase motif 1) and aa 2135–2649 (helicase
motif 2), but not other 4 polypeptides, significantly re-
tained with 35
S-labeled THRAP3 (Figure 3C). These data
indicated that THRAP3 directly binds two helicase motifs
in HELZ2.
THRAP3 and HELZ2 cooperatively enhance
PPAR␥-mediated gene activation
We next examined whether cotransfection of THRAP3
affects the PPAR␥-mediated gene transcription that was
augmented by HELZ2 using a transient transfection as-
say in HeLa cells (Supplemental Figure 1A). The activity
of DR1-TKLuc was stimulated by cotransfected PPAR␥
in the presence of troglitazone. In agreement with our
previous observation (20), the activation by liganded
PPAR␥ was significantly augmented by cotransfection of
HELZ2. Although cotransfection of THRAP3 did not
enhance the PPAR␥-mediated gene
activation in the absence of trans-
fected HELZ2, cotransfection of
THRAP3 with HELZ2 further en-
hanced the promoter activity. In the
absence of cotransfected PPAR␥,
cotransfection of HELZ2 and
THRAP3 did not stimulate the re-
porter gene activity, indicating that
PPAR␥ is required for the coopera-
tive enhancement of the promoter
activity by HELZ2 and THRAP3.
As expected from the findings in
mammalian two-hybrid assays (Fig-
ure 2B), THRAP3 lacking the C-ter-
minal BCLAF1 homologous domain
(THRAP3⌬C) did not reveal coop-
erative enhancement of the reporter
activity with HELZ2 (Supplemen-
tal Figure 1B).
To next examine whether knock-
down of endogenous THRAP3 affects
enhancement of PPAR␥-mediated gene
transcription by HELZ2, siRNA-tar-
geting THRAP3 (siTHRAP3) was in-
troduced 24 hours before transfection
of the reporter and expression vectors
for PPAR␥ and HELZ2. Lipofection
of siTHRAP3 reduced levels of en-
dogenous THRAP3 protein by ap-
proximately 30% after 24 hours, and the suppressive effect
lasted for at least 4 days (Supplemental Figure 1C and data
not shown). Knockdown of THRAP3 significantly attenu-
ated the enhancement of PPAR␥-mediated gene transcrip-
tion by HELZ2 (Supplemental Figure 1D). These findings
suggest that endogenous THRAP3 is required for enhance-
ment of PPAR␥-mediated gene transcription by HELZ2.
Knockdown of Thrap3 attenuates terminal
differentiation of 3T3-L1 cells
Because PPAR␥ is established to function as a master
regulator of adipocyte differentiation (14–16), we next
questioned whether siRNA-mediated knockdown of
Thrap3 affects differentiation of 3T3-L1 preadipocytes.
In Western blot analyses, Thrap3 protein was expressed
at a constant level throughout the differentiation of
3T3-L1 adipocytes (Figure 4A) similarly with Helz2 as we
previously described (20). Electroporation of siThrap3,
but not siControl (siCtr), into 3T3-L1 cells 3 days before
hormonal induction apparently reduced levels of Thrap3
mRNA and protein on day 0, and the effect lasted at least
until day 4 (Figure 4, B and C). As depicted in Figure 4D,
Figure 3. A, Schematic representation of GST-fused HELZ2 polypeptides. The positions of the
ATP binding site, two helicase motifs, and RNase B domain in HELZ2 are indicated. Five serial
HELZ2 polypeptides (aa 1–579, aa 563–1059, aa 1060–1619, aa 1620–2134, and aa 2135–
2649) were bacterially synthesized, and proper synthesis of GST-fusion proteins was verified by
SDS-PAGE. Arrowheads indicate individual GST-fused protein, and an asterisk represents
nonspecific protein. B, THRAP3 binds helicase motifs in HELZ2 in GST pull-down assays. 35
S-
labeled THRAP3 protein was synthesized and incubated with equivalent amounts of GST-fused
HELZ2 polypeptides. After extensive washing, bound proteins were resolved in SDS-PAGE.
“Input” represent 10% of input 35
S-labeled THRAP3 protein, and a representative
autoradiography is shown above. Data represent mean Ϯ SEM from 3 independent experiments
and are expressed as percentage (%) of input THRAP3 protein. Asterisks indicate significant
difference between indicated samples (P Ͻ .05).
774 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780
7. accumulation of lipid droplets stained with Oil Red O
was apparently reduced in siThrap3-introduced
3T3-L1 cells on day 5. The expression of Pparg as well
as Cebpa and Fabp4/aP2, two well-known PPAR␥ tar-
get genes (29, 30), significantly increased 4 days after
differentiation induction in siCtr-introduced 3T3-L1
cells, but the expression of these 3 genes was signifi-
cantly attenuated in siThrap3-introduced cells. In con-
trast, the expression of Crebbp gene, which has been
reported to be unchanged during differentiation of
3T3-L1 cells (20, 31), was not affected by introduction
of siThrap3 (Figure 4E).
We next evaluated whether knockdown of Thrap3 af-
fects the expression of several transcription factors essen-
tial in the early stage of adipogenic differentiation (32–
34). The knockdown efficiency of Thrap3 was monitored
Figure 4. A, Levels of Thrap3 protein during 3T3–L1 cell differentiation[b]. WCL were prepared before and after induction of differentiation of
3T3–L1 cells. IB was performed using anti-THRAP3 Ab and anti--actin Ab. B, The experimental design to evaluate effects of knockdown of Thrap3
in 3T3–L1 cell differentiation. After electroporation of siRNA, differentiation of 3T3–L1 cells was induced, and total RNA and WCL were prepared
on the indicated days. C, Effect of siThrap3 on levels of Thrap3 mRNA and protein on day 0 and day 4. siCtr and siThrap3 were introduced into
3T3–L1 cells as indicated above. WCL was prepared on the day before differentiation induction (Day 0) and 4 days later (Day 4). IB of the same
membrane was performed using anti-THRAP3 Ab and anti--actin Ab. D, Differentiation of 3T3–L1 cells is attenuated by knockdown of Thrap3.
Five days after differentiation induction of 3T3–L1 cells in which siCtr or siThrap3 was introduced, accumulation of lipid droplets was
microscopically analyzed by Oil Red O staining. Bars represent 100 m. The experiment was repeated twice with similar results. E, Expression
changes of Pparg, Cebpa, Crebbp, and Fabp4/aP2 genes in siCtr or siThrap3-introduced 3T3–L1 cells on days 0 and 4. Introduction of siRNA into
3T3–L1 cells was carried out at the same time as in Figure 4C. Total RNA was isolated on day 4 and subjected to qRT-PCR for Pparg, Cebpa,
Crebbp, and Fabp4/aP2 genes. Actb gene was amplified in parallel and used as an internal control. Data represent the relative mRNA expression
and mean Ϯ SEM from triplicate samples. Experiments were repeated twice with similar results. Asterisks indicate significant difference between
indicated groups (P Ͻ .05). ns, not statistically significant (P Ͼ .05). F, Expression changes of genes involved in early adipocyte differentiation in
siCtr or siThrap3-introduced 3T3–L1 cells. siCtr and siThrap3 were introduced as described above, and total RNA was isolated at the indicated time
course after differentiation induction. The expression of Thrap3, Cebpd, Cebpb, Kfl5, and Krox20 was quantitated using qRT-PCR in parallel. Data
represent the mRNA expression relative to that of Actb and mean Ϯ SEM from triplicate samples. The experiment was repeated twice with similar
results. G, Effect of troglitazone on differentiation of siThrap3-introduced 3T3–L1 cells. siCtr and siThrap3 were introduced as described above,
and 10 M troglitazone (TRO) or dimethylsulfoxide (DMSO) was added on day 0, 2, and 4. Oil Red O staining of cells was carried out on day 5.
Bars represent 300 m. The experiment was repeated once with similar results. Ins, insulin; IBMX, isobutylmethylxanthine.
doi: 10.1210/me.2012-1332 mend.endojournals.org 775
8. to quantitate Thrap3 mRNA level in a parallel qRT-PCR
(Figure 4F). In agreement with previous findings reported
by others (32–34), the expression of Krox20, Klf5,
Cebpb, and Cebpd immediately increased after hormonal
induction in siCtr-treated 3T3-L1 cells, whereas no sig-
nificant differences in the expression of these genes were
observed in siThrap3-introduced cells in the indicated
time periods (Figure 4F).
To next examine whether pharmacologic activation of
Pparg could bypass the effect of Thrap3 knockdown on
adipocyte differentiation, siThrap3-introduced 3T3-L1
cells were treated with troglitazone, and accumulation of
fat droplets was monitored using Oil Red O staining. As
depicted in Figure 4G, accumulation of lipid droplets
could not be completely restored in siThrap3-introduced
3T3-L1 cells even in the presence of troglitazone when
compared with siCtr-introduced cells. Taken together,
these findings suggest that Thrap3 plays pivotal roles
mainly in terminal differentiation of 3T3-L1 adipocytes,
but not at early stage of differentiation.
Recruitments of Ppar␥, Helz2, and Thrap3 to the
PPREs in the Fabp4/aP2 and Adipoq gene
enhancers
To lastly examine whether endogenous Helz2 and
Thrap3 could be recruited to the PPREs in Fabp4/aP2 and
Adipoq gene enhancers (26, 27) in differentiated 3T3-L1
cells, troglitazone was added on day 4, and ChIP assays
Figure 4. (Continued).
776 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780
9. were performed 24 hours later. As shown in Figures 5, A
and B, recruitments of Pparg, Helz2, and Thrap3 to the
PPREs in two different enhancer regions were signifi-
cantly increased in the presence of troglitazone. In con-
trast, when endogenous Helz2 or Thrap3 protein was
knocked down by preintroduction of siHelz2 or
siThrap3, respectively, ligand-dependent recruitments of
Pparg, Helz2, or Thrap3 to the enhancer region in Fabp4/
aP2 gene were diminished (Figure 5, C and D). These
findings demonstrated that endogenous Helz2 and
Thrap3 could be corecruited in a ligand-dependent man-
ner with Pparg to the PPREs in Fabp4/aP2 and Adipoq
gene enhancers in differentiated 3T3-L1 cells. In addition,
Helz2 and Thrap3 appeared to cooperatively stabilize the
binding of Pparg to DNA element in the presence of
ligand.
Discussion
HELZ2, possessing characteristic structures including
two ATP-binding motifs, an RNaseB domain, and dual
DNA/RNA helicase motifs, belongs
to the DNA2/NAM7 helicase family
involved in gene transcription, RNA
processing, and DNA repair (35,
36). Initially, PRIC285 and PDIP1
were independently isolated as a
protein interacting with PPAR␣ and
PPAR␥, respectively (20, 21). In cell
culture system, HELZ2 has been
shown to augment ligand-depen-
dent gene activation mediated by
PPAR␥ (20, 21), whereas siRNA-
mediated knockdown of endoge-
nous HELZ2 attenuated PPAR␥-
mediated gene activation in HeLa
cells (20). Interestingly, HELZ2 has
been suggested to be involved in the
pathogenesis of type III familial par-
tial lipodystrophy caused by
heterozygous PPARG mutation in
human (37). However, the detailed
mechanism whereby HELZ2 en-
hances PPAR␥-mediated gene tran-
scription remains to be elucidated.
As a step toward understanding the
molecular function of HELZ2, we
identified THRAP3 as a protein sta-
bly associating with HELZ2 in the
present study. THRAP3 was origi-
nally identified as a component of
the Mediator/TRAP complex that
interacts with the liganded TR␣ (22). However, in recent
studies, THRAP3 has been reported to play roles in pre-
cursor-mRNA alternative splicing as well as in DNA
damage response apart from its transcription-regulatory
function (38–40). These cumulative data suggest that
THRAP3 is a multifunctional protein involved in gene
transcription, RNA processing, and DNA repair similarly
with HELZ2. Nonetheless, direct roles of THRAP3 in
PPAR␥-mediated transcriptional regulation or in the dif-
ferentiation program of adipocytes have never been
explored.
In the present study, transfection of THRAP3 in the
absence of HELZ2 did not enhance PPAR␥-mediated
gene activation, whereas its cotransfection with HELZ2
cooperatively augmented gene transcription activated by
PPAR␥. siRNA-mediated knockdown of endogenous
THRAP3 abolished the enhancement of PPAR␥-medi-
ated gene activation by HELZ2. A truncated form of
THRAP3 lacking the BCLAF1 homologous region that
was necessary for interaction with HELZ2 failed to en-
hance transcriptional activation mediated by PPAR␥ in
Figure 5. A and B, Recruitments of Pparg, Helz2, and Thrap3 to the PPREs in Fabp4/aP2 and
Adipoq gene enhancers in differentiated 3T3–L1 cells. Troglitazone (TRO) (10 M) or
dimethylsulfoxide (DMSO) was added 3 days after differentiation induction of 3T3–L1 cells and
ChIP assay was started 24 hours later. Data represent promoter occupancy of three proteins in
the absence or presence of TRO expressed as a percentage of input DNA samples (mean Ϯ SEM,
n ϭ 3). The ChIP assay was repeated once with similar results. C and D, Recruitments of Pparg,
Helz2, and Thrap3 to the PPRE in Fabp4/aP2 gene enhancer in siHelz2 or siThrap3-introduced
3T3–L1 cells. siHelz2, or siThrap3 was introduced by electroporation on day 3, and TRO or DMSO
was added on day 4. ChIP assay was started 24-hours later. Knockdown efficiency of siHelz2
evaluated by qRT-PCR was approximately 40% on day 0. Data represent promoter occupancy of
3 proteins in the absence or presence of TRO expressed as described above (mean Ϯ SEM, n ϭ
3). The ChIP assay was repeated once with similar results. ns, not statistically significant (P Ͼ .05).
doi: 10.1210/me.2012-1332 mend.endojournals.org 777
10. the presence of cotransfected HELZ2. These findings
indicate that HELZ2 requires direct interaction with
THRAP3 to augment PPAR␥-mediated gene activation
and that THRAP3 may function as an adaptor molecule
to tether other TCRs to PPAR␥-bound HELZ2.
Accumulated evidence, established largely by using im-
mortal cell lines such as 3T3-L1 cells, demonstrates that
differentiation of adipocytes from fibroblast-like precur-
sor cells induced by hormonal stimuli is orchestrated by
coordinated and sequential expression of a variety of
transcription factors at early and late stages of adipogen-
esis (16, 41, 42). However, knowledge about roles of
TCRs required for gene-regulatory functions of these
transcription factors remains to be limited in a complex
process of adipocyte differentiation. Among these adipo-
genic transcription factors, genome-wide studies utilizing
a ChIP-chip method recently uncovered that PPAR␥ and
C/EBP␣ function to optimize adipocyte maturation by
coordinately activating transcription of a number of com-
mon target genes in differentiated 3T3-L1 cells (43, 44).
In the present study, we showed that accumulation of
lipid-laden droplets and the expression of Pparg as well as
Cebpa and Fabp4/aP2, well-characterized PPAR␥ target
genes (29, 30), were significantly attenuated by knock-
down of Thrap3 in 3T3-L1 cells. In contrast, the expres-
sion of transcription factors involved in the early adipo-
genesis including Krox20, Klf5, Cebpb, or Cebpd (31–33)
was not significantly altered by introduction of siThrap3.
The present ChIP assay revealed that Helz2 and Thrap3
could be corecruited, in a ligand-dependent manner, to
Prarg on the DR1-elements in Fabp4/aP2 and Adipoq
gene enhancers in differentiated 3T3-L1 cells. In addition,
troglitazone treatment could not fully restore the differ-
entiation of siThrap3-introduced 3T3-L1 cells. These
findings collectively suggest that Thrap3 might be neces-
sary for terminal differentiation of adipocytes by enhanc-
ing Pparg-mediated gene transcription, rather than early
adipogenesis.
Surapureddi et al (21) previously reported that
PRIC285 (Helz2␣) was biochemically copurified with
several components of the Mediator/TRAP complex such
as TRAP230, Sur-2, and TRAP100 by using liganded
PPAR␣ as a bait, raising the possibility that HELZ2 may
cooperatively stimulate PPAR␥-mediated gene activation
with the Mediator/TRAP complex. Several core subunits
of the Mediator/Trap complex have been reported to play
indispensable roles both at early and late stages of adi-
pocyte differentiation (47–50). Med1/Trap220 and
Med14/Trap170 are shown to be required for adipocyte
differentiation in part by activating PPAR␥-mediated
gene activation through direct interaction with the C-ter-
minal ligand-binding domain and the N-terminal A/B do-
main of PPAR␥, respectively (47, 48). The expression of
Cebpb at early stage of adipocyte differentiation was sup-
pressed by knockdown of Med14/Trap170 (49), and
knockdown of Med23/Trap150 reduced the expression
of Krox20 in 3T3-L1 cells (50), thereby speculated to
attenuate adipocyte differentiation. In the present study,
we showed that the expression of Cebpb or Krox20 was
not affected by knockdown of Thrap3. These findings
taken together suggest that several core subunits of the
Mediator/Trap complex play nonoverlapping and over-
lapping roles with Thrap3 in early and late stages of adi-
pocyte differentiation, respectively.
PPAR␥ has been established to stimulate the expres-
sion of a downstream transcription factor, CCAAT en-
hancer binding protein (C/EBP)␣, through direct tran-
scriptional stimulation of Cebpa (32). The enhanced
expression of C/EBP␣ by PPAR␥ reciprocally activates
the expression of an upstream Pparg by directly binding
to the CCAAT box present in the Pparg2 promoter, re-
sulting in robust enhancement of terminal differentiation
of adipocytes (32). Moreover, it has recently been re-
ported by utilizing a ChIP-chip assay that PPAR␥ by itself
up-regulates the expression of Pparg2 by binding to the
DR-1 element located in the Pparg2 promoter in 3T3-L1
cells (45). Taken together, it was speculated that reduced
transactivation function of PPAR␥ caused by Thrap3
knockdown in the present study could result, in part, in
malfunction of these feed-forward loops, thereby sup-
pressing the expression of Pparg, Cebpa, and Fabp4/aP2
and maturation of 3T3-L1 cells. Alternatively, because
glucocorticoid receptor and TR could stimulate the ex-
pression of Pparg2 and Cebp␣ during early adipogenesis
(43, 46, 51), it is possible that the reduced expression of
Pparg, Cebpa, and Fabp4/aP2 might result from impaired
transactivation function of glucocorticoid receptor
and/or TR by knockdown of Thrap3. Because Helz2 and
Thrap3 are readily expressed in 3T3-L1 preadipocytes
before induction of differentiation, further study is nec-
essary to investigate whether Helz2/Thrap3 complex can
enhance other NR-mediated gene activation at early dif-
ferentiation stages.
Acknowledgments
We thank Aya Osaki, Yuko Tagaya, and Atsuko Miura (De-
partment of Medicine and Molecular Science, Gunma Univer-
sity Graduate School of Medicine, Maebashi, Japan) and Kat-
suya Dezaki (Division of Integrative Physiology, Department of
Physiology, Jichi Medical University School of Medicine,
Shimotsuke City, Japan) for their helpful technical assistance.
This work was supported by a Grant-in-Aid from the Min-
778 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780
11. istry of Science, Education, Sports and Culture of Japan (to T.S.)
and the Global Center of Excellence Program of Japan (to
M.M.).
Address all correspondence and requests for reprints to:
Tetsurou Satoh, M.D., Ph.D., Department of Medicine and
Molecular Science, Gunma University Graduate School of
Medicine, 3–39-15 Showa-machi, Maebashi 371-8511, Japan,
E-mail: tsato@gunma-u.ac.jp.
Disclosure Summary: The authors have nothing to disclose.
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780 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780
![ing to stimulate or repress target gene transcription (3–5).
NRs are well known to possess a characteristic modular
structure, and TCRs are recruited to specific domains of
NRs in a ligand-independent or -dependent manner (3–
5). Although the DNA-binding domain (DBD) of NRs is
necessary for binding to their cognate hormone-respon-
sive elements located near proximal promoters as well as
in enhancer elements far distant from transcription start
sites, many previous studies indicate that the DBD could
also function as an interaction surface with other proteins
(6–10). Using yeast two-hybrid (YTH) screen with the
DBD and a part of the hinge region of thyroid hormone
receptor (TR)  as bait, we previously isolated PSMC3
(also known as Tat-binding protein1), one of the 6 AT-
Pase subunits consisting of 19S regulatory subunit of 26S
proteasome and showed that Tat-binding protein1 func-
tions as a coactivator of TR as well as androgen receptor
(11–13).
Peroxisome proliferator-activated receptor (PPAR)␥, a
member of the NR family, is well-recognized to function
as a master regulator of adipocyte differentiation and
thereby believed to play fundamental roles in lipid and
glucose metabolism in the body (14–16). A number of
TCRs are shown to directly interact with different do-
mains of PPAR␥ including DBD and involved in adipo-
genesis in vivo and in vitro (17–19). In an additional
attempt to identify proteins that interact with the DBD
and a part of the hinge region of human PPAR␥ using
YTH screen, we isolated a partial cDNA of KIAA1769, a
human expression sequence tag with unknown function
at the time of experiments, and termed PPAR␥-DBD-in-
teracting protein 1 (PDIP1) (20). Surapureddi et al (21)
independently identified the rat orthlogue of KIAA1769
in biochemically purified liver proteins that associates
with liganded full-length PPAR␣ and designated as
PPAR␣-interacting cofactor complex 285 (PRIC285). In
several human cell lines, we identified the expression of 2
PDIP1 isoforms that differ in their N-terminal amino acid
(aa) alignment due to alternative splicing (the short iso-
form named as PDIP1␣ that corresponds to KIAA1769
[PRIC285 isoform2] and the long isoform as PDIP1
[PRIC285 isoform1]) (20). Cotransfection of PRIC285/
PDIP1␣ or PDIP1 could enhance PPAR␥ and PPAR␣-
mediated transcriptional activation and also augment
transcription mediated by several other NRs in cell cul-
ture systems (20, 21). Recently, Hugo Gene Nomencla-
ture Committee has approved HELZ2 (helicase with zinc
finger 2, transcriptional coactivator) as the symbol name
for PRIC285/PDIP1, and here we designated PDIP1 as
HELZ2 and PDIP1␣/PRIC285 as HELZ2␣. However,
the molecular mechanism whereby HELZ2 enhances
PPAR␥-mediated gene transcription remains to be
elucidated.
To gain further insights into the molecular function of
HELZ2, we attempted to isolate HELZ2-associating pro-
teins in HeLa cells by employing immunoprecipitation
(IP) with epitope-tagged HELZ2 followed by mass spec-
trometry analyses in the present study. We here isolated
the TR-associated protein 3 (THRAP3) (also known as
TR-associated protein, 150-kDa, TRAP150) (22) as a
protein stably associating with HELZ2. We character-
ized the interaction domains and functional cooperation
in PPAR␥-mediated gene transcription between HELZ2
and THRAP3 and identified novel physiologic roles of
Thrap3 in adipocyte differentiation.
Materials and Methods
Cell cultures
HeLa cells were cultured in DMEM supplemented with 10%
fetal bovine serum (Biowest, Rue de la Caille, France) and am-
picillin/streptomysin (GIBCO, Life Technologies Corporation
Japan, Tokyo, Japan) at 37°C under 5% CO2 atmosphere.
3T3-L1 cells were obtained from Health Science Research Re-
sources Bank (Osaka, Japan) and cultured in DMEM containing
10% fetal bovine serum and antibiotics. Adipogenic differenti-
ation was induced using insulin, dexamethasone (Sigma-Aldrich
Japan, Osaka, Japan), and isobutylmethylxanthine (Enzo Life
Science, Ann Arbor, Michigan) as previously described (20).
Plasmids
The expression vectors for human HELZ2 (pSVSPORT-
HELZ2) and mouse PPAR␥2 (pSVSPORT-PPAR␥2) were de-
scribed previously (20). The HELZ2 cDNA was excised by
EcoRI and XbaI from pSVSPORT-HELZ2 (20) and ligated to
the EcoRI/XbaI site of p3ϫFLAG-CMV10 (pCMV10-
HELZ2) (Sigma-Aldrich Japan). The full-length cDNA encod-
ing human THRAP3 was amplified by RT-PCR using HeLa cell
RNA and ligated to pCR2.1 vector (Invitrogen, Life Technolo-
gies Corp., Gaithersburg, Maryland) (pCR2.1-THRAP3).
THRAP3 cDNA was then excised by EcoRI from pCR2.1-
THRAP3 and inserted to EcoRI site of pcDNA3.1 vector (Invit-
rogen, Life Technologies Corp) (pcDNA3.1-THRAP3). Partial
THRAP3 cDNAs (aa 168–995 [⌬N], aa 1–591 [⌬C], and aa
168–591 [⌬NC]) were excised from pCR2.1-THRAP3 by
BamHI/SalI, EcoRI/MluI, and BamHI/MluI, respectively, and
ligated in flame into pVP16 vector (Takara Bio Inc., Otsu, Ja-
pan). Partial cDNAs encoding THRAP3 ⌬N, ⌬C, and ⌬NC
were also amplified by PCR and subcloned in flame into
pcDNA4/HisMax vector (Invitrogen, Life Technologies Corp.).
The full-length HELZ2 cDNA was amplified by PCR and li-
gated in flame into pAcGFP1-N1 and -C1 vectors (CLON-
TECH Laboratories, Inc, Palo Alto, California). PCR-amplified
THRAP3 cDNA was ligated into pDsRed-Monomer-C1 vector
(CLONTECH). The nucleotide sequences of all constructs were
verified by sequencing.
770 Katano-Toki et al Roles of THRAP3 in Adipocyte Differentiation Mol Endocrinol, May 2013, 27(5):769–780](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)