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DJBP: A Novel DJ-1-Binding Protein, Negatively Regulates theAndrogen Receptor by Recruiting Histone DeacetylaseComplex, and DJ-1 Antagonizes This Inhibition byAbrogation of This Complex

Takeshi Niki,1,3 Kazuko Takahashi-Niki,1,3 Takahiro Taira,1,3 Sanae M.M. Iguchi-Ariga,2,3

and Hiroyoshi Ariga1,3

1Graduate School of Pharmaceutical Sciences and 2College of Medical Technology, Hokkaido University, Kita-ku,Sapporo, Japan; and 3CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan

AbstractDJ-1 was identified by us as a novel oncogene that

transforms mouse NIH3T3 cells in cooperation with ras .

We later identified PIAS (protein inhibitor of activated

STAT)xA as a DJ-1-binding protein, and found that DJ-1

restored androgen receptor (AR) transcription activity

that was repressed by PIASxA. To further characterize

the function of DJ-1, we cloned cDNA encoding a novel

DJ-1-binding protein, DJBP, by a yeast two-hybrid

system. DJBP mRNA was found to be specifically

expressed in the testis. In addition to the binding of

DJBP to the COOH-terminal region of DJ-1, DJBP was

also found to bind in vitro and in vivo to the DNA-

binding domain of the AR in a testosterone-dependent

manner and to be colocalized with DJ-1 or AR in the

nucleus. Furthermore, a co-immunoprecipitation assay

showed that the formation of a ternary complex between

DJ-1, DJBP, and AR occurred in cells in which DJ-1

bound to the AR via DJBP. It was found that DJBP

repressed a testosterone-dependent AR transactivation

activity in monkey Cos1 cells by recruiting histone

deacetylase (HDAC) complex, including HDAC1 and

mSin3, and that DJ-1 partially restored its repressed

activity by abrogating DJBP-HDAC complex. These

results suggest that AR is positively regulated by DJ-1,

which antagonizes the function of negative regulators,

including DJBP.

IntroductionDJ-1 was identified as a protein that transforms mouse

NIH3T3 cells in cooperation with activated ras (1). The human

DJ-1 gene, which is comprised of seven exons, is mapped to

1p36.2–36.3, where many chromosome aberrations in cancer

have been reported (2). DJ-1 was found to be more strongly

expressed in the testis than other tissues and also in sperm,

suggesting that DJ-1 has at least two functions, one function

in somatic cells, especially in germ cells, and one function in

sperm.

With regard to the function of DJ-1 in somatic cells, we have

identified PIAS (protein inhibitor of activated STAT)xa/ARIP3

(androgen receptor-interacting protein 3) as a DJ-1-binding

protein (3). PIASxa/ARIP3 belongs to the PIAS family of

proteins, including PIAS1, PIAS3, PIASxa, PIASxh, and

PIASy/g, and was characterized at first as a testis-specific AR

coregulator (4, 5). We have shown that PIASxa inhibits the

transcription activity of the AR by binding to the DNA-binding

domain of AR and that DJ-1 antagonizes this inhibition by

sequestering PIASXa from the AR in CV-1, Cos1, and TM4

Sertoli cells, indicating that DJ-1 is a positive regulator of the

AR (3). Although PIAS family proteins have been shown to

play roles in SUMO-1 conjugation (6–11) and in the regulations

of STAT (12, 13), nuclear receptors (4, 5, 14–17), and K

channels (18), the contribution of DJ-1 to such functions is not

clear.

RS, another name for human DJ-1, has been reported by

another group as a protein of an RNA-binding protein

regulatory subunit (19). It has recently been reported that

DJ-1/RS was strongly expressed and secreted from cells to

serum in about half of breast cancer patients, some of whom

possessed an antigen against DJ-1/RS, indicating that DJ-1/RS

is a biomarker for breast cancer (20). It has also been reported

that pI of DJ-1 changed from 6.2 to 5.8 by treatment of cells

with H2O2, paraquat, or lipopolysaccharide that resulted in the

production of reductive oxygen species, suggesting that DJ-1

might be a sensor to oxidative stresses (21, 22).

With regard to the function of DJ-1 in sperm, a rat

homologue of human DJ-1 has been identified by other

laboratories as a key protein related to infertility of male rats

that had been exposed to sperm toxicants such as ornidazole

and epichlorohydrin (23–26). DJ-1/RS/CAP1 has been shown

to be expressed in the sperm head and to be translocated to

the cytoplasm of sperm after treatment of rat sperm with the

above toxicants (27, 28). Furthermore, we and others have

reported that an anti-SP22 or anti-DJ-1 antiserum inhibited

fertility in vitro , suggesting that DJ-1 plays a role in

Received 7/16/02; revised 11/1/02; accepted 12/19/02.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: Ministry of Education, Science, Sport, Culture and Technologyof Japan.The nucleotide sequence reported in this paper has been submitted to the

DDBJ/GenBank/EBI Data Bank with accession number AB073862.Requests for reprints: Hiroyoshi Ariga, Graduate School of PharmaceuticalSciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812,Japan. Phone: 81-11-706-3745; Fax: 81-11-706-4988.E-mail: [email protected] D 2003 American Association for Cancer Research.

Vol. 1, 247–261, February 2003 Molecular Cancer Research 247

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fertilization (29, 30). The results suggest that DJ-1 affects both

the production of sperm by activation of the AR to express

male-specific genes and the fertilization function of mature

sperm.

The AR is a member of the nuclear receptor superfamily and

plays a role as a ligand-dependent transcription factor. After a

ligand binds to the AR, the AR is translocated into the nucleus

and binds to the androgen-responsive element, ARE, on the

androgen-activating gene that affects development, growth, and

regulation of male reproductive functions (31–34). The AR,

like other nuclear receptor family proteins, possesses domains

for transactivation, DNA-binding domain (DBD), and ligand-

binding domain (LBD) at the NH2-terminal, central, and

COOH-terminal regions, respectively. Transcriptional activity

of the AR is known to be regulated by various coregulators that

bind to the respective domain of AR. More than 30 proteins of

such coregulators have been reported (Ref. 35, see homepage at

http://ww2.mcgill.ca/androgendb).

To further characterize the function of DJ-1, we cloned

cDNA encoding a novel DJ-1-binding protein, DJBP, by a yeast

two-hybrid system using a human testis library in this study.

DJ-1, DJBP, and AR were found to form a ternary complex, in

which DJ-1 bound to AR via DJBP. DJBP repressed

testosterone-dependent AR transactivation activity and DJ-1

restored its repressed activity, suggesting that DJBP and DJ-1

have mutually antagonizing functions with respect to the AR.

ResultsCloning of cDNA Encoding DJBP From a Human TestiscDNA Library

To identify proteins that bind to DJ-1, a yeast two-hybrid

screening was carried out using a human testis cDNA library

with full-sized DJ-1 as a bait, and cDNA encoding a novel DJ-

1-binding protein, named DJBP, was cloned. DJBP cDNA

comprises 2119 bp and encodes 570 amino acids (Fig. 1). DJBP

contains three LXXLL-like motifs, LXXLL, YLXXL, and

ILXXL, located at amino acid numbers 74, 92, and 235,

respectively. The LXXLL motif is known to be the sequence

that is necessary for coactivators to interact with nuclear

receptors. To examine the expression of DJBP mRNA in human

tissues, Northern blotting was carried out using a premade

tissue blot with labeled DJBP cDNA as a probe (Fig. 2). Two

distinct bands, of 1.9 and 7 kb in length, the former of which

corresponds to the length of the DJBP cDNA obtained, were

detected only in the testis (Fig. 2, lane 12). DJ-1 was also

confirmed to be strongly and moderately expressed in the testis

and other tissues, respectively, as described previously (1).

Interaction of DJBP With DJ-1To assess the association of DJBP with DJ-1 in vivo , human

293T cells were transfected with expression vectors for Flag-

tagged DJ-1 (F-DJ-1) with or without HA-tagged DJBP (HA-

DJBP). Forty-eight hours after transfection, cell extracts were

prepared and subjected to an immunoprecipitation reaction with

an anti-Flag antibody. The precipitates were then blotted with

an anti-HA or -Flag antibody (Fig. 3A). The results showed that

HA-DJBP was coprecipitated with F-DJ-1 in cells cotransfected

with F-DJ-1 and HA-DJBP but not in cells transfected with

HA-DJBP alone (Fig. 3A, lanes 1 and 2), respectively),

indicating that DJBP binds to DJ-1 in 293T cells. To examine

the binding of DJP to DJ-1 in a physiological condition, the

proteins in the extracts prepared from bovine testes were first

immunoprecipitated with an anti-DJ-1 antibody or non-specific

IgG and the precipitates were immunoblotted against an anti-

DJBP antibody (Fig. 3B). The anti-DJ-1 antibody did

precipitate DJ-1 (Fig. 3B, lane 2 in lower panel). DJBP, on

the other hand, was detected in the immunoprecipitate with the

anti-DJ-1 antibody but not with IgG (Fig. 3B, lanes 1 and 2),

respectively). These results clearly indicate that DJBP binds to

DJ-1 in cells.

To examine the interaction of DJBP with DJ-1 in vitro ,

F-DJ-1 and DJBP were expressed in E. coli as glutathione

S-transferase (GST)-fusion proteins and purified after releasing

GST by digestion of GST-fusion proteins with PreScission

protease. Purified DJBP was mixed with or without F-DJ-1 and

subjected to immunoprecipitation reaction with the anti-Flag

antibody followed by the detection of proteins in the

precipitates with an anti-DJBP antibody (Fig. 3C). The results

showed that DJBP was coprecipitated with F-DJ-1 in the

mixture containing F-DJ-1 and DJBP but not in the mixture

containing DJBP alone (Fig. 3C, lanes 1 and 2), respectively),

indicating that DJBP directly binds to DJ-1.

To determine the DJ-1-binding region in DJBP, a yeast two-

hybrid assay was carried out using various deletion mutants of

DJBP fused to the GAL4-activating domain and DJ-1 fused to

the LexA DNA-binding domain as a bait (Fig. 3D). DJ-1 was

found to bind to COOH-terminal regions of DJBP spanning

amino acid numbers 193–570, 250–570, and 372-570 but not

to NH2-terminal regions, indicating that amino acids 372–570

of DJBP are bound by DJ-1. This was also confirmed by the

results of an in vitro pull-down assay using GST or GST-fusion

proteins of DJBP containing amino acids 1–570 (full length)

and 371–570, in which DJBP and its deletion mutant but not

GST were bound by DJ-1 (Fig. 3E).

Colocalization of DJBP and DJ-1To determine the localization of DJ-1 and DJBP, an

expression vector for HA-DJBP was transfected into monkey

Cos1 cells, and the cells were stained with anti-HA and anti-

DJ-1 antibodies to show the introduced HA-DJBP and

endogenous DJ-1, respectively, and visualized with FITC and

rhodamine-conjugated secondary antibodies under a confocal

laser microscope (Fig. 4). The results showed that endogenous

DJ-1 and HA-DJBP were localized mainly in the nucleus and

both proteins were colocalized in the nucleus (Fig. 4, A–C,

respectively).

Association of DJBP With the Androgen ReceptorTo determine whether DJBP associates with AR in vivo ,

293T cells cultured in the presence of 100 nM testosterone were

transfected with expression vectors for HA-DJBP with or

without F-AR, and 48 h after transfection, cell extracts were

immunoprecipitated with an anti-Flag antibody and the

precipitated protein was detected with an anti-HA or the anti-

Flag antibody (Fig. 5A). The results showed that HA-DJBP was

coprecipitated with F-AR in cells cotransfected with both F-AR

DJBP, a Negative Regulator of the Androgen Receptor248



FIGURE 1. Nucleotide and amino acidsequences of DJBP. The nucleotide sequence ofDJBP cDNA was determined by a dideoxy methodusing Long Reader 4200 (LI-Cor, Lincoln, Nebraska)and Gene Rapid (Amersham BioScience, Bucks,UK) autosequencers, and the deduced amino acidsequence is shown. LXXLL motifs within the aminoacid sequence are underlined .

Molecular Cancer Research 249



and HA-DJBP but not in cells transfected with HA-DJBP alone

(Fig. 5A, lanes 1 and 2), respectively), indicating that DJBP

binds to AR in 293T cells. To assess the direct interaction

between DJBP and AR, a GST pull-down assay was carried out

using purified GST-DJBP and GST. Purified proteins were

reacted in the presence of 100 nM testosterone with 35S-AR

synthesized in vitro in a reticulocyte lysate, and the bound

proteins were visualized by fluorography (Fig. 5B). The results

showed that labeled AR bound to DJBP but not to GST,

indicating that DJBP directly interacts with the AR (Fig. 5B,

lanes 1 and 2), respectively). Because it is known that some

coregulators for the AR bind to the AR in a ligand-dependent

manner, testosterone (a ligand of the AR)-dependent binding

activity of DJBP to AR was examined by a liquid h-galactosidase assay in yeast. Because DJBP binds to the

COOH-terminal half of the AR, as shown in Fig. 6B, this

truncated form of the AR, ARDN, was used in this assay

(Fig. 5C). The results showed that h-galactosidase activity

obtained between DJBP and ARDN increased with increase in

the dose of testosterone. To further confirm the testosterone-

DJBP

DJ-1

Actin

lane 1 2 3 4 5 6 7 8 9 10 11 12

9

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FIGURE 2. Northern blot analysis ofDJBP in various human tissues. HumanNorthern RNA blot (12 major tissues,Origene, Rockville, MD) was hybridizedwith labeled cDNAs of DJBP (upperpanel ), DJ-1 (middle panel ), and h-actin(lower panel ) as probes. Positions of sizemarkers are shown on the right in theupper panel .

FIGURE 3. Interaction of DJBP with DJ-1. A. 293T cells were transfected with pcDNA3-F-DJ-1 and pcDNA3-HA-DJBP. At 48 h after transfection, cellextracts were prepared and subjected to an immunoprecipitation reaction using an anti-Flag antibody (M2, Roche Applied Science, Rotkreuz, Switzerland).The precipitates were blotted with an anti-HA or the anti-Flag antibody. B. The proteins in the extract from bovine testes were first immunoprecipitated with ananti-DJ-1 antibody or non-specific IgG. The proteins in the precipitates were separated in a 12.5% polyacrylamide gel and blotted with an anti-DJBP antibodyor the anti-DJ-1 antibody. Lane 1, the extract used for the binding reaction was applied to the same gel (Input). C. F-DJ-1 and DJBP were expressed inEscherichia coli as GST-fusion proteins and purified after releasing GST by digestion of GST-fusion proteins with PreScission protease (AmershamBioScience) as described in ‘‘Materials and Methods.’’ One microgram of each purified protein was mixed and subjected to an immunoprecipitation reactionusing an anti-Flag antibody. The precipitates were blotted with an anti-DJBP antibody. Lane 3, the DJBP used for the reactions (Input) was run in parallel.D. Yeast L40 cells were transformed with expression vectors for LexA, LexA-DJ-1, and various deletion mutants of DJBP fused to the GAL4-activationdomain. The h-galactosidase activity of each colony was measured. E. DJBP(1– 570), DJBP(372 – 570), and DJ-1 were expressed in E. coli as GST-fusionproteins and purified. GST-free DJ-1 was then prepared after releasing GST from GST-DJ-1 by digestion with PreScission protease. DJ-1 and GST-DJBPswere mixed together and subjected to a pull-down assay as described in ‘‘Materials and Methods.’’ The proteins bound to GST-DJBPs were blotted with ananti-DJ-1 antibody. Lane 4, the DJ-1 used for the reactions (Input) was run in parallel.




dependent binding activity of DJBP to the AR in vivo , 293T

cells cultured in the various amounts of testosterone were

transfected with expression vectors for Flag-DJBP with or

without the AR, and 48 h after transfection, cell extracts were

immunoprecipitated with the anti-Flag antibody and the

precipitated protein was detected with the anti-Flag antibody

or an AR antibody (Fig. 5D). The results showed that AR was

coprecipitated in a testosterone-dependent manner with F-DJBP

in cells cotransfected with both AR and F-DJBP but not in cells

transfected with the AR alone (Fig. 5D, lanes 1, 2, 4, 5, 7, 8, 10,

and 11), indicating that DJBP binds to the ligand-binding form

of the AR in 293T cells.

To determine the AR-binding region of DJBP, 293T cells

were cotransfected with the AR and various Flag-tagged

deletion mutants of DJBP, and the AR that co-immunopreci-

pitated with DJBP in cells were detected with an anti-AR

antibody after the immunoprecipitation of cell extracts with an

anti-Flag antibody (Fig. 6A). In addition to the full-sized DJBP,

DJBP deletion mutants of amino acids 372–570 and 476–570

also immunoprecipitated the AR, whereas DJBP deletion

mutants of amino acids 1–371 and 372–475 did not (Fig. 6A,

lanes 1, 3, 5, 2, and 4), respectively), indicating that the AR

binds to amino acids 476–570 of DJBP which do not contain

LXXLL motifs. To determine the DJBP-binding region of the

AR, on the other hand, Cos1 cells were cotransfected with the

HA-tagged DJBP and various Flag-tagged deletion mutants of

the AR, and DJBP that co-immunoprecipitated with ARs in

cells was detected with an anti-HA antibody after the

immunoprecipitation of cell extracts with the anti-Flag antibody

(Fig. 6B). Deletion constructs of the AR are schematically

shown in Fig. 6C. The results showed that while two mutants of

the AR, DC and LBD, were not bound by DJBP, the deletion

mutants of the AR, DN and DBD, were, like the wild-type AR,

bound by DJBP (Fig. 6B, lanes 2, 5, 3, 4, and 1), respectively).

These results indicate that DJBP binds to the DNA-binding

domain of the AR and that the LXXLL motif is not necessary

for DJBP to interact with the AR. To examine the colocalization

of DJBP with the AR in cells, Cos1 cells were cotransfected

with F-DJ-1 and HA-DJBP in the presence of 100 nM

testosterone, and transfected cells were stained with anti-AR

and anti-HA antibodies at 48 h after transfection. HA-DJBP

was found to be colocalized with the AR in the nucleus (data

not shown).

Repression of AR Transactivation Activity by DJBP andRestoration of Its Repression by DJ-1

Because we showed in this study that DJBP binds to both

DJ-1 and AR and in a previous study that DJ-1 does not directly

bind to the AR (3), we then examined the relationships among

DJBP, DJ-1, and AR. 293T cells cultured in the presence of

100 nM testosterone were transfected with various combinations

of F-AR, HA-DJBP, and DJ-1-HA, the cell extracts were

immunoprecipitated with an anti-Flag antibody, and the

precipitants were blotted with anti-HA and Flag antibodies

(Fig. 7A, lanes 1–8). HA-DJBP was co-immunoprecipitated

with F-AR as expected (Fig. 7A, lanes 2 and 3). DJ-1-HA, on

the other hand, was co-immunoprecipitated with F-AR in cells

transfected with both HA-DJBP and F-AR but not in cells

transfected with F-AR alone (Fig. 7A, lanes 3 and 4),

respectively). Expression levels of the respective proteins of

F-AR, HA-DJBP, and DJ-1-HAwere comparable in transfected

cells (Fig. 7A, see Input). Furthermore, the amounts of DJ-1

and HA-DJBP co-immunoprecipitated with F-AR were found

to be stimulated in the presence of 100 nM testosterone (Fig. 7A,

lanes 9 and 10). Thus, these results clearly indicate that DJ-1,

DJBP, and AR form a ternary complex, in which DJ-1

associates with the AR via DJBP.

To then assess the relationship between the biological

activities of DJ-1, DJBP, and AR, monkey Cos1 cells were

transfected with pARE2-TATA-Luc as a reporter and with or

without pcDNA3-F-AR together with various amounts of

pcDNA3-HA-DJBP or pEGFP-N1 in the presence or absence

of 100 nM testosterone. The plasmid, pCMV-h-gal, was also

cotransfected into cells to normalize the transfection efficiency.

Forty-eight hours after transfection, cell lysates were prepared

and their luciferase activities were measured (Fig. 7B). A

testosterone-dependent transcription activity of the AR was first

confirmed in the transfected cells without pcDNA3-HA-DJBP

(data not shown). It was found that neither DJBP nor EGFP

responded to pARE2-TATA-Luc reporter in the absence of the

AR. The results then showed that while introduction of EGFP

that did not bind to the AR, pEGFP-N1, did not affect the AR

activity, DJBP repressed the testosterone-dependent transcrip-

tion activity of the AR in a dose-dependent manner and about

70% of the AR activity was repressed after 1 Ag of pcDNA3-

HA-DJBP was transfected into cells (Fig. 7B), suggesting that

DJBP acts as a negative regulator of the AR. DJ-1-HA or EGFP

was then cotransfected into Cos1 cells together with 1 Ag of

pcDNA3-HA-DJBP and pARE2-TATA-Luc, and the luciferase

activities were determined (Fig. 7C). The results showed that

while introduction of vector, pEGFP-N1, did not affect the AR

activity, DJ-1 partially restored AR transcription activity

repressed by DJBP in a dose-dependent manner. Because

DJ-1 directly binds to DJBP but not the AR, these results

suggest that DJ-1 changes the AR from a transcriptional

negative form to an active form as we reported in the case of

DJ-1 and PIASxa (3).

Association of DJBP With Histone Deacetylase ComplexBecause transcriptional repression pathways of genes are

often connected with histone deacetylase (HDAC) or corepres-

sor complex, it is possible that the transcriptional repression of

the AR by DJBP uses the same or similar pathway connected

with HDAC. To explore this possibility, an expression vector

for FLAG-tagged DJBP was transfected into human 293T cells

cultured in the presence of 100 nM testosterone. Forty-eight

hours after transfection, the cell extract was prepared and the

proteins in the extract were first immunoprecipitated with the

anti-FLAG antibody or non-specific IgG. The precipitates were

immunoblotted against an anti-AR antibody (Fig. 8A). It was

first confirmed that the anti-FLAG antibody did not precipitate

proteins in the extracts from cells into which FLAG-DJBP had

not been transfected, and that the anti-FLAG antibody

precipitated FLAG-DJBP (Fig. 8A, lanes 3 and 1), respec-

tively). The endogenous AR, on the other hand, was detected in

the immunoprecipitate with the anti-FLAG antibody but not

with IgG (Fig. 8A, lanes 1 and 2), respectively), indicating

again that DJBP was associated with the AR in 293T cells. The




same blot as that shown in Fig. 8A was reprobed with anti-

HDAC1 and-mSin3A antibodies (Fig. 8A). Endogenous

HDAC1 and mSin3A were detected in the immunoprecipitates

with the anti-FLAG antibody but not with IgG, indicating that

DJBP and the AR are complexed with a corepressor complex,

including HDAC1 and mSin3A. It was found that neither

HDAC1 nor mSin3A directly bound to DJBP by the pull-down

assay, in which GST-DJBP was reacted with 35S-HDAC1 or35S-mSin3A synthesized in vitro in a reticulocyte lysate (data

not shown), suggesting that the DJBP-bound AR or the DJBP-

AR complex recruits the corepressor complex.

To confirm the existence of the HDAC complex in the

transrepression pathway of the AR to DJBP, TSA, an inhibitor

of HDAC1, was added to the cells transfected with the AR and

various amounts of DJBP in the presence of 100 nM

testosterone, and the luciferase activity was measured

(Fig. 8B). The results showed that TSA abrogated the DJBP-

repressed transcription activity of the AR and further increased

the transcription activity of the AR to the level comparable to

that obtained after introduction of the AR alone, suggesting that

repression of the AR transcription activity by DJBP is carried

out by the HDAC complex.

Abrogation of the DJBP-HDAC Complex by DJ-1To determine the mechanism underlying the abrogation of

the DJBP-repressed AR transcription activity by DJ-1, FLAG-

tagged DJBP and various amounts of HA-tagged DJ-1 or

EGFP were transfected into human 293T cells cultured in the

presence of 100 nM testosterone. Forty-eight hours after

transfection, the cell extract was prepared and the proteins in

the extract were first immunoprecipitated with an anti-FLAG

antibody. The precipitates were immunoblotted against anti-

HA, anti-HDAC1, anti-GFP, and anti-FLAG antibodies as

described in Fig. 8A (Fig. 9). It was first confirmed that the

constant amounts of endogenous HDAC1 were expressed and

that DJ-1-HA and EGFP were expressed in a dose-dependent

manner in transfected cells (Input, Fig. 9, lanes 9–16). It was

also confirmed that FLAG-DJBP was precipitated with the

anti-FLAG antibody (Fig. 9, lanes 1–3, 5, and 6). While EGFP

was not detected in the precipitates, DJ-1-HAwas also detected

with the anti-HA antibody in the immunoprecipitates from cells

that had been transfected with FLAG-DJBP (Fig. 9, lanes 2

and 3), indicating that DJBP was associated with DJ-1 in

ectopic expressed 293T cells. It was found that endogenous

HDAC1 was, however, first detected in the immunoprecipitates

from cells that had been transfected with FLAG-DJBP alone

and then the amounts of immunoprecipitated HDAC1

decreased in a dose-dependent manner (Fig. 9, lanes 1–3),

indicating that DJ-1 abrogated the DJBP-HDAC complex,

thereby leading to the restoration of the AR transcription

activity repressed by DJBP.

DiscussionIn this study, we identified a novel DJ-1-binding protein,

named DJBP, by a yeast two-hybrid system using a human

testis cDNA library. DJBP contains three LXXLL-like motifs

that are known to be necessary for coactivators to interact with

nuclear receptors. Expressions of DJBP mRNAs of 2 and 7 kb

in length were found to be limited to the testis of human tissues,

FIGURE 4. Colocalization of DJBP with DJ-1 in Cos1 cells. Cos1 cells were transfected with pcDNA3-HA-DJBP, stained with anti-HA (A) and anti-DJ-1(B) antibodies at 48 h after transfection, and visualized under a confocal laser microscope. C. Figs. A and B are merged.




and the cDNA cloned in this study appears to correspond to the

2-kb mRNA. Although the cDNA corresponding to the 7-kb

mRNA is not available at present, it might be of an alternative

splicing form of DJBP mRNAs.

From the results of various binding experiments in vitro and

in vivo , it was found that DJBP directly binds to both DJ-1 and

AR in a testosterone-dependent manner and that DJ-1

associates with AR via DJBP in a ternary complex in cells.

Because AR binds to the COOH-terminal region spanning

amino acids 476–570 of DJBP, in which three LXXLL-like

motifs are not present, it is thought that these motifs in DJBP

are not necessary for binding to the AR, like several AR-

binding proteins, including BRCA1 (36), Cyclin E (37), RAF

(38), TFIIH (39), CAK (39), RB (40), Ubc9 (41), c-Jun (42),

and Cyclin D1 (43). DJBP, on the other hand, binds to the

DNA-binding domain of the AR (AR-DBD). Because the

FIGURE 5. Interaction of DJBP with AR. A. 293T cells cultured in the presence of 100 nM testosterone were transfected with pcDNA3-F-AR and pcDNA3-HA-DJBP. Forty-eight hours after transfection, the proteins in cell extracts were precipitated with an anti-Flag antibody and blotted with anti-HA or -Flagantibodies. B. GST-DJBP and GST were expressed in E. coli and purified as described in ‘‘Materials and Methods.’’ GST-DJBP or GST was mixed with35S-labeled AR synthesized in vitro in reticulocyte lysate and subjected to a pull-down assay. Labeled proteins bound to GST-DJBP or GST were visualizedby fluorography. Lane 4, the AR used for the reactions (Input) was run in parallel. C. Yeast L40 cells were transformed with various combinations ofLexA-DJBP, GAD-ARDN, LexA, and GAD. The h-galactosidase activity of each colony was measured in the presence of various concentrationsof testosterone. D. 293T cells cultured in the various amounts of testosterone were transfected with pcDNA3-AR and pcDNA3-F-DJBP. Forty-eight hoursafter transfection, the proteins in cell extracts were precipitated with an anti-Flag antibody and blotted with anti-AR or -Flag antibodies.

FIGURE 6. Determination ofregions of binding between DJBPand AR. A. 293T cells cultured inthe presence of 100 nM testosteronewere transfected with various dele-tion mutants of Flag-tagged DJBPand AR. Forty-eight hours aftertransfection, the proteins in the cellextract were precipitated with ananti-Flag antibody and blotted withan anti-AR antibody. Lane 7, thecell extract used for the reactions(Input ) was run in parallel. B. Cos1cells cultured in the presence of 100nM testosterone were transfectedwith various deletion mutants ofFlag-tagged AR and HA-taggedDJBP. Forty-eight hours after trans-fection, the proteins in the cellextract were precipitated with ananti-Flag antibody and blotted withan anti-HA antibody. Lane 6, thecell extract used for the reactions(Input ) was run in parallel. C. TheAR and its deletion mutants usedfor the reactions are schematicallyshown, and the DJBP-binding activ-ities are shown on the right .




FIGURE 7. Effect of transcription activity of the AR by DJBP and DJ-1. A. 293T cells cultured in the presence of 100 nM testosterone were transfectedwith various combinations of pcDNA3-F-AR, pcDNA3-HA-DJBP, and pEF-DJ-1-HA (lanes 1 –8). Lanes 9 and 10, plasmids used in lane 3 were transfectedinto 293T cells in the absence or presence of 100 nM testosterone. Forty-eight hours after transfection, the proteins in the cell extract were precipitated with ananti-Flag antibody and blotted with anti-Flag and anti-HA antibodies. In the lower two panels (Input), the cell extract used for the reactions was run in paralleland blotted with the anti-HA antibody. B. Cos1 cells cultured in the presence of 100 nM testosterone were transfected with 0.12 Ag of pCMV-h-gal, 0.03 Ag ofpcDNA3-F-AR, and 1.2 Ag of pARE2-TATA-Luc together with various amounts of pcDNA3-HA-DJBP or pEGFP-N1 (HA-DJBP + AR or EGFP + AR,respectively). Cos1 cells were also transfected with the same DNAs as those above except for omitting pcDNA3-F-AR (HA-DJBP or EGFP, respectively). Forty-eight hours after transfection, cell lysates were prepared and their luciferase activities were measured. C. Cos1 cells cultured in the presence of 100 nM

testosterone were transfected with 0.12 Ag of pCMV-h-gal, 0.03 Ag of pcDNA3-F-AR, 1.2 Ag of pARE2-TATA-Luc, and 1 Ag of pcDNA3-HA-DJBP together withvarious amounts of pEF-DJ-1-HA or pEGFP-N1. Forty-eight hours after transfection, cell lysates were prepared and their luciferase activities were measured.




amino acid sequences of DBD among nuclear receptor family

proteins were well conserved, DJBP might also bind to other

nuclear receptors. Of the proteins that bind to AR-DBD,

SNURF (44), Ubc9 (41), and ARIP3/PIASXa (4) have been

reported to associate with multiple nuclear receptors, including

the AR.

DJBP was found to repress the transcription activity of AR

to 30% of that without DJBP. The mechanism underlying this

repression of AR activity by DJBP was assessed by the

findings that DJBP recruited the corepressor complex,

including HDAC1 and mSin3A. These results suggest that

an active form of the AR changes into an inactive form by

translocation to an inactive structure within the chromatin.

Repression activity of DJBP toward the AR was sensitive to

TSA, a specific inhibitor of HDAC, indicating that HDAC is

involved in the transrepression pathway of the AR. The

introduction of DJ-1 relieved the repressed the AR tran-

scription activity by abrogation of DJBP-HDAC complex. It

was reported that TSA augments dihydrotestosterone induc-

tion of AR levels (45). Although the results in this report

suggested that the androgen-induced chromatin remodeling

and transcription occur in the AR-responsive genes, the

results in our study here is a first report to identify the AR-

HDAC complex bridged by DJBP.

FIGURE 8. Binding of DJBP to the ARin the HDAC complex in vivo and effect oftrichostatin A (TSA ) on the repressionactivity of DJBP toward the AR. A. 293Tcells cultured in the presence of 100 nM

testosterone were transfected with anexpression vector for FLAG-DJBP bythe calcium phosphate precipitation tech-nique, and the cell extract was prepared48 h after transfection. Proteins in theextract were first precipitated with an anti-FLAG mouse monoclonal antibody ornon-specific IgG, and the precipitateswere immunoblotted with an anti-ARrabbit polyclonal antibody (N-20, SantaCruz Biotechnology, Santa Cruz, CA).Proteins in the extract from non-trans-fected cells were similarly treated withantibodies. The same blot as thatdescribed above was reprobed with ananti-HDAC1 antibody (H-51, Santa CruzBiotechnology) and an anti-mSin3A anti-body (AK-11, Santa Cruz Biotechnology).Lanes 4 and 5, the proteins used for thereactions (Input ) were run in parallel. B.Cos1 cells cultured in the presence of 100nM testosterone were transfected with0.12 Ag of pCMV-h-gal, 0.03 Ag ofpcDNA3-F-AR, and 1.2 Ag of pARE2-TATA-Luc together with various amountsof pcDNA3-HA-DJBP. Two-hundrednanomolars of TSA was added to theculture 40 h after transfection. After 48 h,cell lysates were prepared, and theluciferase activities in the lysates weremeasured.




Functions of the AR are regulated by various complex

formations. PIASxa, a member of the PIAS family of

proteins, was characterized at first as a testis-specific AR

coregulator, ARIP3 (4, 5). We have shown that PIASxa

inhibits the transcription activity of the AR by binding to the

DNA-binding domain of the AR and that DJ-1 antagonizes

this inhibition by sequestering PIASXa from the AR in CV-1,

Cos1, and TM4 Sertoli cells, indicating that DJ-1 is a positive

regulator of the AR (3). DJBP was found to bind to the AR,

leading to formation of a ternary complex between DJ-1,

DJBP, and the AR. Different to the mode of action of

PIASxa on the AR, DJBP was found to inhibit AR

transcription activity by recruiting histone-deacetylase

complexes, including HDAC1 and mSin3, and DJ-1 was

found to restore the repressed activity of the AR. These

findings suggest, as schematically shown in Fig. 10, that DJ-1

restores AR activity that has been repressed by different

mechanisms.

DJBP and DJ-1 were found to be expressed specifically and

strongly, respectively, in the testis. Furthermore, it was found

that DJ-1 was also expressed in sperm and played roles in

fertilization reactions. These results suggest that DJBP also

plays a role in spermatogenesis or fertilization along with DJ-1.

To clarify this possibility, we are investigating the expression

patterns, including location and timing, of DJBP in the testis

and sperm.

FIGURE 9. Abrogation of recruitmentof HDAC complex to DJBP by DJ-1.293T cells cultured in the presence of100 nM testosterone were transfectedwith 5 Ag of pcDNA3-F-DJBP, variousamounts of pEF-DJ-1-HA and pEGFP-N1 by the calcium phosphate precipita-tion technique, and the cell extract wasprepared 48 h after transfection. Proteinsin the extract were first precipitated withan anti-FLAG mouse monoclonal anti-body, and the precipitates were immuno-blotted with an anti-HA rabbit polyclonalantibody, an anti-HDAC1 antibody, or theanti-FLAG antibody as described in Fig. 8.The 0.25-, 1-, and 1-Ag pEF-DJ-1-HAor pEGFP-N1 was transfected into cellsin lanes 2, 3, and 4, respectively. Lanes9 – 16, the cell extracts used for thereactions (Input) were run in parallel.

AR

DJBP

DJ-1

HDAC

mSin3

corepressor complex

PIASxα

histone deacetylation

ARE target genes

FIGURE 10. Schematic drawings ofmechanisms by which DJ-1 antagonizesnegative modulators of the AR by DJBPand PIASxa.




Materials and MethodsCells

Human 293T and monkey Cos1 cells were cultured in

DMEM supplemented with 10% calf serum.

PlasmidsNucleotide sequences of the oligonucleotide used for PCR

primers were as follows:

DJBP-ATG(Bam), 5 V-GGATCCATGGGTCATTTT-

ACAA-3V;DJBP-ATG(Eco), 5V-GGGAATTCATGGGTCATTTT-

ACAA-3V;DJBP-193(Eco), 5V-GGGAATTCTATATAAATTGG-

AAAT-3V;DJBP-250(Eco), 5V-GGGAATTCATCACCCAGGAG-

TTTG-3V;DJBP-372(Eco), 5V-GGGAATTCGGCACTCCACCC-

TTGC-3V;DJBP-372(Bam), 5V-GGGGATCCGGCACTCCACCC-

TTGC-3V;AR-DN(Eco), 5 V-GGGAATTCGGACCTTATGGG-

GACA-3V;DJBP-372(Bam), 5V-GGGGATCCGGCACTCCACCC-

TTGC-3V;DJBP-476(Bam), 5V-GGGGATCCCAGAATGCACAC-

AAGA-3V;DJBP-475(Xho), 5V-GGGAGCTCGATCTTCATCCT-

GTGC-3V;AR-DBD(Eco), 5V-GGGAATTCTACGGAGCTCTC-

ACTT-3V;AR-DBD(Xho), 5V-GGCTCGAGTACAGTCATCTT-

CTGG-3V; andAR-LBD(Eco), 5V-GGGAATTCCAGAAGATGACT-

GTAT3V.

Combinations of plasmids constructed, primers, templates,

and restriction enzyme sites and plasmids inserted are shown in

Table 1. pGEX-F-DJ-1: The EcoRI-XhoI fragment of pGlex-

DJ-1 (3) was inserted into the EcoRI-XhoI sites of pGEX-6P-1-

F (46). pGAD-DJBP(1–371), pGAD-DJBP(193–371), and

pGAD-DJBP(250–371): the EcoRI-SmaI fragments of pGAD-

DJBP(1 – 570), pGAD-DJBP(93 – 570), and pGAD-

DJBP(250–570) were inserted into the EcoRI-SmaI sites of

pBluescript SK(-), and the EcoRI-XhoI fragments of these

plasmids were then inserted into the EcoRI-XhoI sites of

pGAD-GHX, respectively. pGLex-DJBP: The EcoRI-XhoI

fragment of pGAD-DJBP(1–570) was inserted into the

EcoRI-XhoI sites of pGLex (47). pcDNA3-F-DJBP(1–371):

The EcoRI-Xho I fragment of pGAD-DJBP(1–371) was

inserted into the EcoRI-XhoI sites of pcDNA3-F. pcDNA3-F-

AR-DC: The EcoRI-HindIII fragment of pcDNA3-F-AR was

subcloned into the EcoRI-SmaI sites of pBluescript SK(-), and

the EcoRI-XhoI fragments of this plasmid were then inserted

into the EcoRI-XhoI sites of pcDNA3-F. pcDNA3-F-AR-DN:

The EcoRI-XhoI fragment of pGAD-AR-DN was inserted into

the EcoRI-XhoI sites of pcDNA3-F.

AntibodyA fusion protein of GST and DJBP was expressed in E. coli

MN524 and purified as described previously (3). Rabbits were

immunized by the purified GST-DJBP. The anti-DJBP antibody

from the rabbit serum was prepared by the affinity chromatog-

raphy containing GST-DJBP, absorbed by GST, and used as the

polyclonal anti-DJBP antibody.

Cloning of DJBP cDNA From a Human Testis cDNALibrary by a Yeast Two-Hybrid System

Yeast L40 cells were co-transformed with expression vectors

for DJ-1 fused to the LexA DNA-binding domain, pGLex-DJ-1,

and human testis cDNAs from the MATCHMAKER cDNA

library (Clontech Laboratories, Inc., Palo Alto, CA), which

express human testis cDNAs fused to the GAL4-activating

domain. Approximately 1.5 � 106 colonies were screened for

expressions of HIS3 and LacZ.

Northern BlottingA human Northern RNA blot (12 major tissues, Origene)

was hybridized with 32P-labeled DJBP, DJ-1, and h-actincDNAs as probes under a highly stringent condition, and then

hybridized bands were detected by autoradiography.

Table 1. Plasmid Construction

Plasmid Template 5V-Primer 3V-Primer Restriction Enzyme Site Plasmid Inserted

pcDNA3-HA-DJBP pME18S-DJBP DJBP-ATG(Bam) SP6 BamHI, Xho I pcDNA3-HApGEX-DJBP pME18S-DJBP DJBP-ATG(Bam) SP6 BamHI, Xho I pGEX-6P-1pGAD-DJBP(1–570) pME18S-DJBP DJBP-ATG(Eco) SP6 EcoRI, Xho I pGAD-GHXpGAD-DJBP(193– 570) pME18S-DJBP DJBP-193(Eco) SP6 EcoRI, Xho I pGAD-GHXpGAD-DJBP(250– 570) pME18S-DJBP DJBP-250(Eco) SP6 EcoRI, Xho I pGAD-GHXpGAD-DJBP(372– 570) pME18S-DJBP DJBP-372(Eco) SP6 EcoRI, Xho I pGAD-GHXpGEX-DJBP(372–570) pcDNA3-HA-DJBP DJBP-372(Bam) SP6 EcoRI, Xho I pGEX-6P-1pGAD-AR-DN pCDNA3-F-AR AR-DN(Eco) SP6 EcoRI, Xho I pGAD-GHXpcDNA3-F-DJBP(1 – 570) pcDNA3-HA-DJBP DJBP-ATG(Bam) SP6 BamHI, Xho I pcDNA3-FpcDNA3-F-DJBP(372 –570) pcDNA3-HA-DJBP DJBP-372(Bam) SP6 BamHI, Xho I pcDNA3-FpcDNA3-F-DJBP(476 –570) pcDNA3-HA-DJBP DJBP-476(Bam) SP6 BamHI, Xho I pcDNA3-FpcDNA3-F-AR-DBD pCDNA3-F-AR AR-DBD(Eco) AR-DBD(Xho) EcoRI, Xho I pcDNA3-FpcDNA3-F-AR-LBD pCDNA3-F-AR AR-LBD(Eco) SP6 EcoRI, Xho I pcDNA3-F

Note: Combinations of plasmids constructed, primers, templates, and restriction enzyme sites and plasmids inserted are shown.




Interaction of DJBP With DJ-1 and AR in Vivo293T cells were transfected with pcDNA3-F-DJ-1 and

pcDNA3-HA-DJBP in the presence or absence of 100 nM

testosterone by the calcium phosphate method (48). Forty-eight

hours after transfection, the cell extract was prepared and

subjected to an immunoprecipitation reaction using an anti-

FLAG antibody conjugated with agarose beads (M2, Sigma

Chemical Co., St. Louis, MO). The precipitates were separated

on 12.5% SDS-PAGE and blotted with an anti-HA antibody

(12CA5, Roche). To examine formation of a ternary complex

between DJ-1, DJBP, and AR, 5 Ag of pcDNA3-F-AR, 2.5 Agof pcDNA3-HA-DJBP, and 1 Ag of pEF-DJ-1-HA were

transfected into 293T cells in the presence of 10 mM

testosterone, and then the same immunoprecipitation assay as

that described above was carried out.

Interaction of DJBP with DJ-1 and AR in VitroF-DJ-1 and DJBP were expressed in E. coli as GST-fusion

proteins and purified after releasing GST by digestion of GST-

fusion proteins with PreScission protease (Amersham

BioScience) as described previously (49). Purified F-DJ-1

and DJBP were mixed and subjected to an immunoprecipita-

tion reaction using an anti-Flag antibody followed by an anti-

DJBP antibody as described above. The anti-DJBP antibody

used in this reaction was proteins of the IgG faction prepared

from rabbits immunized with purified DJBP as an immunogen.

To examine the binding of DJBP with the AR, purified GST-

DJBP or GST was incubated with 35S-AR synthesized in vitro

using a reticulocyte lysate of a TnT-transcription-translation

coupled system (Promega, Madison, WI) and subjected to a

pull-down assay using an glutathione-Sepharose as described

previously (49).

ImmunofluorescenceCos1 cells were transfected with 1 Ag of pcDNA3-HA-

DJBP or pcDNA3-F-AR by the calcium phosphate method

(48). Forty-eight hours after transfection, the cells were fixed

with 4% paraformaldehyde, stained with anti-DJ-1 (3), anti-AR

(N-20, Santa Cruz Biotechnology), and HA antibodies, and

visualized under a confocal laser microscope.

b-Galactosidase Liquid AssayL40 cells were transformed with expression vectors for DJ-1

fused to the LexA DNA-binding domain, pGLex-DJ-1, and for

various deletion mutants of DJBP fused to the GAL4-activation

domain, pGAD-GHX-DJBPs. Transformed cells were cultured

in YPAD medium in the presence of testosterone for 4 h, and

h-galactosidase assay using cell extracts was carried out.

Luciferase AssayCos1 cells in 6-cm dishes were transfected with 1.3 Ag of

pARE2-TATA-Luc, a reporter plasmid, and 0.13 Ag of pCMV-

h-gal, an expression vector for h-galactosidase, by the calcium

phosphate method (48). Forty-eight hours after transfection, the

luciferase activity in cells was measured after normalization of

the transfection efficiency with h-galactosidase activities as

described previously (49).

AcknowledgmentsWe thank Yoko Misawa and Kiyomi Takaya for their technical assistance.

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2003;1:247-261. Mol Cancer Res Takeshi Niki, Kazuko Takahashi-Niki, Takahiro Taira, et al. accession number AB073862.submitted to the DDBJ/GenBank/EBI Data Bank with

The nucleotide sequence reported in this paper has been 2 2Science, Sport, Culture and Technology of Japan.

Ministry of Education, 1 1Abrogation of This ComplexComplex, and DJ-1 Antagonizes This Inhibition bythe Androgen Receptor by Recruiting Histone Deacetylase DJBP: A Novel DJ-1-Binding Protein, Negatively Regulates

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