PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

Experimental Cell Research 308 (2005) 65 – 77 www.elsevier.com/locate/yexcr

PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein Tetsuo Matsuuraa,b, Yohei Shimonoa, Kumi Kawaic, Hideki Murakamia, Takeshi Uranod, Yasumasa Niwab, Hidemi Gotob, Masahide Takahashia,c,* b

a Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Department of Gastroenterology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan c Department of Molecular Pathology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan d Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

Received 18 February 2005, revised version received 18 April 2005 Available online 23 May 2005

Abstract Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2h, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASxa and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis. D 2005 Elsevier Inc. All rights reserved. Keywords: RET finger protein; PIAS; SUMO; Nuclear localization; Transcription

Introduction The PIAS (protein inhibitor of activated STAT) protein family was originally characterized by their selective inhibition of STAT (signal transducer and activator of transcription) signaling [1,2]. They are composed of five members: PIAS1, PIAS3, PIASxa, PIASxh and PIASy. * Corresponding author. Department of Molecular Pathology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax: +81 52 744 2098. E-mail address: [email protected] (M. Takahashi). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.04.022

PIAS1 and PIAS3 were identified as selective inhibitors of STAT signaling [1,2]. In addition, PIASxa, PIASxh and PIASy were identified as homologous proteins with PIAS1 and PIAS3 [2], and then PIASxa was reported to be the androgen receptor interacting protein and to modulate the transcriptional activity of this receptor [3]. The discovery that Siz1/Ull1 of budding yeast, a member of the human PIAS family, acts as a SUMO ligase led to characterization of PIAS as a SUMO E3 ligase [4,5]. SUMO (small ubiquitin-like modifier) is a ubiquitin-like protein and consists of four family members, SUMO-1, -2, -3 and -4 [6 –11]. Although SUMO-1 is a 101 amino acid polypeptide sharing only 18% amino acid identity with ubiquitin,

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it contains a characteristic ubiquitin fold common to ubiquitin-like proteins [12,13]. As in the ubiquitination pathway, sumoylation is carried out through the sequential activities of E1, E2 and E3 enzymes. The E1 activating enzyme, Aos1 and Uba2, transfers activated SUMO-1 to the E2 conjugating enzyme, Ubc9, which forms thioester bonds with SUMO-1 [14 –17]. Several members of the SUMO E3 ligase family have been reported. These include the PIAS proteins and RanBP2 and the Polycomb group protein Pc [18,19]. Growing evidence supports the idea that, unlike in the ubiquitination process, the E3 ligase is dispensable and plays a role by enhancing the sumoylation of target proteins [20,21]. Although the functional significance of sumoylation is less well understood than ubiquitination, expanding evidence indicates that many proteins are sumoylated, and sumoylation may play a role in targeting proteins to specific subcellular locations, stabilizing target proteins and modulating the transcriptional activity of substrate proteins [6,8 – 10,22]. For example, sumoylation of the cytoplasmic nuclear import factor RanGAP1 leads to its translocation to the nuclear pore complex, and sumoylation of PML enhances the translocation of PML into PML nuclear bodies [23 –25]. Sumoylation of InBa inhibits its ubiquitination and becomes resistant to proteasome-mediated degradation [26]. Although the effect of sumoylation on the transcriptional activity of p53 is controversial [27,28], it has been reported that activities of various transcriptional factors, including Lef-1, Promyelocytic leukemia zinc finger (PLZF), Myb, Sp3, p300 and androgen receptor, are attenuated or repressed by sumoylation [29 – 34]. The Ret finger protein (RFP) was originally identified as a fusion protein with RET tyrosine kinase that possesses transforming activity [35]. RFP is composed of a characteristic RING finger, a B-box zinc finger and a coiled-coil region (RBCC motif), and a carboxyl-terminal RFP domain or B30.2 domain. The RBCC motif is suspected to be involved in protein –protein interactions, and RFP itself has been reported to associate with PML and int-6 and colocalizes with these proteins [36,37]. RFP is highly expressed in various human and rodent tumor cell lines, as well as in male germ cells. RFP protein is also found in the nuclei of peripheral and central neurons, hepatocytes, adrenal chromaffin cells and male germ cells [35,38]. Especially in testis, RFP is detected as a perinuclear cap structure in primary spermatocytes, suggesting that RFP has a specific role in spermatogenesis [38]. We have reported that RFP is a transcriptional co-repressor and associates with Enhancer of Polycomb 1 (EPC1) and Mi-2h [39,40]. We demonstrate here that RFP and PIAS proteins associate and co-localize in the nucleus, forming nuclear speckles that are reminiscent of PML nuclear bodies. RFP is sumoylated by PIAS proteins, and the transcriptional repressive activity of RFP is enhanced by them. Finally, immunohistochemical analysis shows that in testis RFP is highly restricted to a specific nuclear structure of primary spermatocytes, where PIASy and SUMO-1 also accumulate. These

findings suggest that PIAS proteins act as SUMO E3 ligases for RFP and that the association of PIAS and RFP may be important for transcriptional repression and spermatogenesis.

Materials and methods Plasmid constructs The RING finger B-box region, the coiled-coil domain region and the RFP domain region of RFP were cloned into the pAS2-1 vector as described previously [39]. PIAS1, PIAS3 and PIASxa cDNAs and SUMO-1 cDNA were cloned into the pcDNA3-Myc expression vector. PIASy was amplified by specific primers with a flanking BamHI site on the 5Vprimer and an XhoI site on the 3Vprimer, using a human testis cDNA library as a template. The resulting PCR product was subcloned into the pGEM-T vector (Promega) and sequenced. The product was subcloned into the pcDNA3-Myc expression vector. Each region of the PIAS cDNAs was amplified by PCR using specific 5Vprimers with an EcoRI site and 3Vprimers with an XhoI site. The resulting PCR products were subcloned into the pGEM-T vector (Promega) and sequenced. Each product was inserted into the EcoRI/XhoI sites of the pACT2 vector. The cDNAs of PIAS proteins and SUMO-1 were also cloned into the EcoRI/XhoI sites of the pFLAG-CMV2 expression vector and the BamHI/ XhoI sites of the pcDNA3.1 (+) vector, respectively. Yeast two-hybrid screening and interaction assays Yeast two-hybrid screening using the full-length RFP as bait was performed as described previously [39]. To characterize the interacting regions between RFP and PIAS proteins, each fragment of the RFP and PIAS cDNAs amplified by PCR was cloned into pAS2-1 and pACT2 vectors, respectively, and transformed into the yeast Y190 strain. Positive interactions were determined by two criteria: (1) growth on selective medium lacking leucine, tryptophan and histidine with 40 mM 3-AT and (2) h-galactosidase expression. Measurements were performed using at least three independent colonies. GAL4-fused reporter gene targeted transcriptional assay The generation of pGL3 luciferase reporter plasmids has been described previously [39]. Human embryo kidney (HEK) 293 cells were cultured in 24-well tissue culture plates and co-transfected with 40 ng of luciferase reporter plasmid, 40 ng of pRL-TK plasmid (Promega), 120 ng of pCMV-GAL4DNABD (binding domain)-RFP expression vector, 400 ng of pcDNA3.1 SUMO-1 expression vector and 400 ng of pcDNA3-Myc-PIAS expression vector by the LipofectAMINE 2000 method, as described by the manufacturer (Invitrogen). The total amount of DNA in each transfection experiment was kept to 1.0 Ag by adding control

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plasmids. The cells were harvested 48 h after transfection, and luciferase assays were performed as described previously [39]. Values were expressed as means and standard deviations using the results from three independent experiments. Co-transfection with the pRL-TK plasmid was used to normalize all luciferase values according to the manufacturer’s instructions. Antibodies The anti-RFP antibody was described previously [39]. The anti-FLAG monoclonal antibody (M2) was obtained from Sigma. The anti-SUMO-1, anti-Myc (9E10) and antiPML antibodies were purchased from Santa Cruz Biotechnology. The anti-PIASy antibody was purchased from IMGENEX.

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Slides were deparaffinized in xylene and rehydrated through graded alcohols. They were subjected to microwave pretreatment for 12 min in 10 mM citrate buffer (pH 6.0) and cooled at room temperature. Nonspecific binding sites were blocked with 10% goat serum for 30 min. The sections were incubated with primary antibodies (rabbit polyclonal antiRFP antibody or mouse monoclonal anti-SUMO-1 antibody) overnight at 4-C. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol for 15 min. The slides were incubated with secondary antibody conjugated to peroxidase-labeled polymer (EnVision+, Dako), and the reaction products were visualized using diaminobenzidine and H2O2. Counterstaining was performed with hematoxylin.

Results

Co-immunoprecipitation studies

Identification of PIAS proteins as RFP interacting proteins

HEK293 cells were plated in 60-mm dishes and were transfected with 8 Ag of the indicated plasmids (pcDNA3Myc-PIAS, pcDNA3-Myc-PIASDCT, pcFLAG-CMV-RFP and pcDNA3.1-Myc-SUMO-1) using the LipofectAMINE 2000 method (Invitrogen). After 48 h of incubation, immunoprecipitation and SDS (sodium dodecyl sulfate)12% polyacrylamide gel electrophoresis were performed as described previously [39]. The filters were incubated with anti-Myc antibody (Santa Cruz), anti-FLAG M2 antibody (Sigma) or anti-SUMO-1 antibody (Santa Cruz).

RFP has a characteristic RBCC motif that is composed of a RING finger, a B-box zinc finger and a coiled-coil domain. The RBCC motif is supposed to be involved in protein – protein interactions, and we have reported that RFP is involved in the transcriptional repression associated with Enhancer of Polycomb 1 and Mi-2h [39,40]. To identify additional RFP interacting proteins, we performed yeast two-hybrid screening using the full-length RFP as bait. pAS-RFP and a human testis cDNA library (CLONTECH) were transfected into the yeast Y190 strain, and the transformants were plated first on selective medium lacking tryptophan and leucine and then on selective medium lacking histidine, tryptophan and leucine with 40 mM 3AT (histidine jump-start method). Of approximately 5  106 independent clones, 50 clones were positive for expression of histidine and h-galactosidase. By searching the BLAST data base, we identified that 3 out of 50 positive clones encode the protein inhibitor of activated STAT (PIAS) xa and y genes. To clarify which domain was responsible for the interaction, we cloned the different regions of RFP and PIAS proteins (PIAS1, 3, xa, y) in-frame into pAS2-1 and pACT2 vectors, respectively (Figs. 1A and B). Each vector was transformed into the yeast Y190 strain, and interactions were assayed by growth on selective medium lacking histidine, tryptophan and leucine with 40 mM 3-AT and by hgalactosidase activity. As shown in Fig. 1C, a strong interaction was detected between the coiled-coil domain of RFP and the carboxyl-terminal region of PIAS1 that includes the acidic region. On the other hand, PIAS1 constructs without the acidic region had no binding activity with RFP. The constructs lacking the coiled-coil domain of RFP had no binding activity with PIAS1. We constructed panels of assessments between RFP and other PIAS proteins and obtained the same results, showing that the carboxyl-terminal regions of PIAS3, xa and y, which include an acidic region, bind with the coiled-coil

Immunofluorescence and confocal microscopy HEK293 cells were grown on poly-d-lysine-coated eight-chamber slide glasses (Falcon) for 24 h and transfected with 0.8 Ag of pcDNA3-Myc-PIAS expression plasmids using the LipofectAMINE 2000 method (Invitrogen). Cells were incubated for 48 h and fixed with methanol (5 min, 20-C) and acetone (30 s, 20-C). After nuclease digestion, the cells were stained as described previously [39]. SW480 human colorectal adenocarcinoma cells were grown on four-chamber slide glasses (Falcon) for 24 h and transfected with 1.6 Ag of pcDNA3-Myc-PIAS expression plasmids as described above. In order to perform double staining for RFP and PIAS proteins, the cells were stained with mouse monoclonal anti-Myc antibody and Alexa 488conjugated anti-mouse IgG antibody followed by incubation with rabbit polyclonal anti-RFP antibody and Alexa 594conjugated anti-rabbit IgG antibody. The staining patterns were analyzed with a confocal microscope (FLUOVIEW FV300, OLYMPUS). Immunohistochemistry Fresh testes sampled from normal mice were fixed in 10% neutral-buffered formalin and embedded in paraffin. Fivemicrometer sections were used for immunohistochemistry.

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Fig. 1. Identification of binding region for RFP – PIAS interaction. (A) Schematic representation of RFP plasmids used for yeast two-hybrid assay. The various regions of RFP were fused with the GAL4-DNA binding domain (GAL4BD) of the pAS2-1 vector, as illustrated. The protein motifs of RFP are indicated. RB, RING finger B-box; C-C, coiled-coil; RFPD, RFP domain. (B) Schematic representation of PIAS family proteins. The protein motifs of PIAS family proteins are fused with the GAL4-DNA activating domain (GAL4AD) of the pACT-2 vector as indicated. NT, amino terminal region; SP-R, SP-RING; CT, carboxylterminal region; AR, acidic region. (C) Results of yeast two-hybrid system binding assays. (upper panel) The association is considered positive (+) when the following two criteria were met: (1) growth on the selective medium lacking tryptophan, leucine and histidine in the presence of 40 mM 3-AT and (2) the hgalactosidase activity. Absence of cell growth or detectable color is considered negative ( ). Measurements were performed using at least three independent colonies. (lower panel) Colony assay for the interaction between the designated regions of PIAS1 and the coiled-coil region of RFP is shown.

region of RFP (Supplementary Fig. 1 and data not shown). These results indicated that the binding of RFP and PIAS proteins is mediated by the association between the acidic region of PIAS proteins and the coiled-coil region of RFP. PIAS proteins associate with RFP at their carboxyl-terminal region To verify the interaction between RFP and PIAS proteins, immunoprecipitation experiments were performed using 293 human embryo kidney cells (HEK293), in which the endogenous RFP was detected with high sensitivity by

Western blotting [39]. The plasmid containing Myc-PIAS was transfected into HEK293 cells, and the lysate was immunoprecipitated using an anti-RFP antibody. As shown in Fig. 2A, all four PIAS proteins examined co-precipitated with RFP. Distinct molecular weights of PIASxa (63 and 67 kDa) may reflect an additional modification of PIASxa. These interactions were not observed when normal rabbit immunoglobulins were used for immunoprecipitation (Fig. 2A, control). Yeast two-hybrid assays revealed that PIAS proteins associate with RFP at their carboxyl-terminal region that includes an acidic region (Fig. 1). To assess the role of this

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Fig. 2. Role of the carboxyl-terminal region of PIAS family proteins in interactions with RFP. (A) Co-immunoprecipitation of RFP and PIAS family proteins. PIAS family proteins were cloned into a Myc expression vector and transfected into 293 human embryo kidney (HEK293) cells. The whole cell lysates were immunoprecipitated with an anti-RFP rabbit polyclonal antibody or normal rabbit IgG as a control. The precipitated proteins were analyzed by Western blotting using an anti-Myc antibody. Input indicates the whole cell lysate used for immunoprecipitation. (B) Loss of the carboxyl-terminal region of PIAS family proteins impairs the association with RFP. PIAS DCT indicates the PIAS deletion construct without the carboxyl-terminal region including the acidic region (Fig. 1B). PIASDCTs were cloned into the Myc expression vector and transfected into HEK293 cells. Assays by immunoprecipitation and Western blotting were performed as described above.

region in the RFP – PIAS interaction, we constructed the carboxyl-terminal deletion constructs of PIAS proteins (PIASDCT). As shown in Fig. 2B, the PIASDCT constructs examined did not co-immunoprecipitated with RFP, demonstrating the importance of the carboxyl-terminal region of PIAS proteins in the RFP –PIAS interaction. From all of these results, we concluded that RFP associates with PIAS proteins and that the carboxyl-terminal region of PIAS that includes the acidic region is important for the binding between RFP and all PIAS proteins. RFP is sumoylated by PIAS proteins PIAS proteins possess a Siz-PIAS(SP)-RING motif, and they have been reported to act as SUMO E3 ligases [34,41].

We examined the possibility that RFP is a target protein for SUMO modification. Since the pool of endogenous free SUMO-1 is reported to be low [42], we transfected a MycSUMO-1 expression vector together with a FLAG-RFP expression vector into HEK293 cells and immunoprecipitated using an anti-FLAG monoclonal antibody. We immunoblotted the precipitates with the anti-FLAG antibody and detected the 59 kDa band that corresponds to the unmodified FLAG-RFP and the 90 kDa band that may correspond to the Myc-SUMO-1 modified RFP (Fig. 3A). Neither band could be detected by using the control normal mouse immunoglobulins for immunoprecipitation. To confirm that the upper 90 kDa band corresponds to the SUMO modified RFP, we blotted the same lysates with anti-Myc and anti-SUMO antibodies. As presented in Figs. 3B and C,

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Fig. 3. Sumoylation of RFP. The plasmids containing FLAG-tagged RFP and Myc-tagged SUMO-1 were transfected into HEK293 cells. The whole cell lysates were immunoprecipitated with an anti-FLAG mouse monoclonal antibody or normal mouse IgG as a control. The precipitated proteins were analyzed by Western blotting using anti-FLAG (A), anti-Myc (B) and anti-SUMO-1 (C) antibodies. Input indicates the whole cell lysates used for the assay.

both antibodies clearly recognize the upper 90 kDa band. We concluded therefore that RFP is modified by SUMO-1 in mammalian cells. We next sought the role of PIAS proteins in sumoylation of RFP. PIAS proteins function as SUMO E3 ligases and enhance sumoylation of target proteins, although SUMO E3 ligase is not necessary for the overall sumoylation reaction [20,21]. We transfected the Myc-SUMO-1 expression vector and the FLAG-PIAS expression vector into HEK293 cells. The cell extracts were immunoprecipitated with the antiRFP antibody or with control normal rabbit immunoglobulin and blotted with an anti-Myc antibody. Fig. 4A shows that all PIAS proteins examined enhanced the sumoylation of RFP, as compared to levels seen with only SUMO-1 expression. To confirm the enhancement of RFP sumoylation by PIAS proteins, we co-transfected into HEK293 cells the Myc-SUMO-1 and increasing amounts of the FLAG-PIASy expression vectors. The cell extracts were treated in the same way described above. As shown in Fig. 4B, the expression of PIASy clearly increased the sumoylated RFP in a dose-dependent manner. Moreover, we constructed the SP-RING mutants of PIAS1 and PIASy which do not have the SUMO ligase activity by replacing cysteine 350 in PIAS1 and cysteine 342 in PIASy with alanine (designated C350A and C342A, respectively) [4]. Transfection of these FLAG-tagged PIAS SP-RING mutants did not increase the sumoylation of RFP (Fig. 4C). We concluded from these results that all PIAS proteins examined function as SUMOE3 ligases of RFP and are involved in the enhancement of the SUMO-conjugating reaction to RFP. PIAS proteins translocate RFP in nuclear bodies One of the roles of sumoylation is proposed to be the translocation of target proteins. In particular, the sumoyla-

tion of PML enhances the accumulation of PML into nuclear bodies, and CtBP forms nuclear bodies by the coexpression of Pc, another SUMO E3 ligase [18,23]. To clarify the distributions of RFP and PIAS proteins, we expressed the Myc-PIAS proteins in HEK293 cells and SW480 human colorectal adenocarcinoma cells. We examined the localizations of Myc-PIAS proteins and endogenous RFP by immunofluorescence using an antiMyc monoclonal antibody and an anti-RFP polyclonal antibody. All PIAS proteins form relatively fine nuclear dots throughout the nucleoplasm in the nuclei of HEK293 cells (Fig. 5A). RFP presents the same distribution pattern as PIAS proteins, and superimposition of confocal images revealed that RFP and PIAS proteins largely co-localized in these nuclear dot-like structures (Fig. 5A). In SW480 cells, all PIAS proteins and RFP showed the same distribution pattern as that of HEK293 cells and colocalized in nuclear dots (Supplementary Fig. 2). As we reported before, endogenous RFP localizes in the nucleus, forming relatively large nuclear dots or a fine granular or homogenous pattern (Figs. 5A and B). Co-expression of PIAS proteins clearly changes the distribution of RFP by dispersing the large nuclear dots and enhances the accumulation of RFP into fine nuclear dot-like structures. In contrast, expression of the SP-RING mutants of PIAS1 and PIASy (PIAS1 C350A and PIASy C342A) did not induce the fine nuclear dot pattern (Fig. 5C), suggesting that sumoylation is necessary for proper localization of RFP. Double staining of Myc-PIAS transfected cells with antiRFP and anti-PML antibodies indicated that about half of RFP co-localizes with PML in nuclear dot-like structures (Fig. 5D, lower panel and Supplementary Fig. 2). Without co-expression of PIAS, RFP maintained large dot-like or granular structures (Fig. 5D, upper panel). We concluded that PIAS proteins enhance the sumoylation of RFP and re-

Fig. 4. PIAS proteins promote sumoylation of endogenous RFP. (A) Co-expression of PIAS proteins enhanced sumoylation of endogenous RFP. HEK293 cells were transfected with the plasmid encoding FLAG-tagged PIAS proteins and Myc-SUMO-1 as indicated. The whole cell lysates were immunoprecipitated with an anti-RFP antibody or normal rabbit IgG as a control. The precipitated proteins were analyzed by Western blotting using an anti-Myc antibody. In the bottom panel, each whole cell lysate was blotted using an anti-FLAG or an anti-RFP antibody to assess the amount of FLAG-PIAS or endogenous RFP in the lysates analyzed. (B) Dose-dependent promotion of sumoylation of RFP by PIASy. The plasmid encoding Myc-SUMO-1 and increasing amounts of FLAG-PIASy were transfected into HEK293 cells. Immunoprecipitation and Western blotting were performed as described above. (C) Effects of PIAS1 C350A and PIASy C342A mutants on sumoylation of RFP. Expression of these mutants did not enhance its sumoylation.

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72 T. Matsuura et al. / Experimental Cell Research 308 (2005) 65 – 77 Fig. 5. Co-localization of RFP with PIAS proteins in the nucleus. (A) The plasmids containing Myc-PIAS were transfected into HEK293 cells. Double immunostaining of RFP and PIAS proteins was performed with an anti-RFP polyclonal antibody and an anti-Myc monoclonal antibody followed by incubation with Alexa 594-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG antibodies. Horizontal images present the representative staining patterns of PIAS proteins (green) and RFP (red) in the same cells. The two images were merged digitally, and co-localization of two proteins is indicated by the yellow color in the right panel. Two proteins co-localized in fine nuclear dot-like structures. Arrows indicate large nuclear dots of RFP in the cells which did not overexpress PIAS. (B) HEK293 cells were transfected with a Myc-expression vector as a control. Double immunostaining using an anti-RFP polyclonal antibody and an anti-Myc monoclonal antibody was performed as described above. Endogenous RFP was localized in relatively large nuclear dots. (C) Effects of PIAS1 C350A and PIASy C342A mutants on the distribution of RFP. RFP showed large nuclear dots or a homogenous pattern in the cells expressing PIAS mutants. (D) Association of RFP with PML nuclear bodies. HEK293 cells were transfected with Myc (upper panel)- or Myc-PIASy (lower panel)-expressing plasmids. Double immunostaining of RFP and PML was performed with an anti-RFP polyclonal antibody and an anti-PML monoclonal antibody followed by incubation with Alexa 594-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG antibodies. The two images were merged digitally as described. Scale bar, 20 Am.

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distribute RFP in fine nuclear dot-like structures that are highly related to PML nuclear bodies. Transcriptional repressive ability of RFP is enhanced by PIAS proteins Sumoylation of transcriptional factors is being increasingly linked with transcriptional repression [43]. We have reported that RFP is involved in transcriptional repression, associating with Enhancer of Polycomb 1 (EPC1) and Mi-2h, a main component of the NuRD complex [39,40]. To evaluate the effect of sumoylation of RFP on transcriptional repression, we carried out a reporter gene assay. We used a luciferase reporter construct containing five tandem repeats of the GAL4 binding sites, a serum response element (SRE) and an SV40 promoter (Fig. 6A). We transfected HEK293 cells with the luciferase reporter plasmid, a vector expressing GAL4 DNA binding domain (GAL4BD)-fused RFP, a SUMO-1 expression vector and various PIAS expression plasmids. The luciferase activity of GAL4BD alone was set as 100%. As shown in Fig. 6A, GAL4BD-RFP repressed the luciferase activity by approximately 45%. Co-expression of SUMO-1 enhanced the repressive ability of RFP by 12%. Furthermore, co-expression of various PIAS proteins with SUMO-1 enhanced the repressive activity of RFP by 21 – 29% (Fig. 6A). To investigate whether the effects of SUMO-1 and PIAS on the transcriptional repressive activity of RFP are additive, we transfected the PIAS expression plasmids with or without the SUMO-1 plasmid into HEK293 cells. As shown in Fig. 6B, the effect of PIAS on the RFP activity was not affected by SUMO-1 co-transfection. This may be due to a low possibility that, in addition to GAL4-luciferase reporter and GAL4-BD RFP plasmids, both SUMO-1 and PIAS expression plasmids are introduced into the same cells. Alternatively, endogenous SUMO-1 may be sufficient for the sumoylation of RFP and enhancement of its repressive activity by PIAS. In contrast to the wild-type PIAS, expression of PIAS1 C350A or PIASy C342A mutant did not enhance the repressive activity of RFP (Fig. 6C). These findings suggest that the transcriptional repressive ability of RFP is enhanced by sumoylation and that PIAS proteins enhance both the sumoylation and the repressive activity of RFP. Localization of RFP, SUMO-1 and PIASy in the nuclear domain of primary spermatocytes In addition to tumor cell lines, RFP is highly expressed in testis [38]. Although PIAS mRNAs are reported to be expressed in various tissues, high expression levels were detected especially in testis [1,44]. The high expression of both RFP and PIAS proteins that act as SUMO E3 ligases in testis led us to check the expression pattern of RFP, SUMO-1 and PIAS. We performed immunohistochemical staining to examine the expression of RFP, SUMO-1 and

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PIASy in normal mouse testis obtained from 5-week-old mouse. As shown in Fig. 7A, RFP was expressed in the nuclei of primary spermatocytes and formed a nuclear structure juxtaposed with the inner nuclear membrane, as we described previously [38]. Interestingly, an anti-SUMO1 antibody also labeled this nuclear structure of primary spermatocytes (Fig. 7B). High-power views of these sequential sections indicate that anti-RFP and anti-SUMO1 antibodies seem to recognize the same nuclear structure of primary spermatocytes (Figs. 7C and D). In addition, this structure was positively stained by anti-PIASy antibody (Figs. 7E and F). Immunohistochemical examinations using the normal mouse or normal rabbit immunoglobulins presented no specific staining (Figs. 7G and H, data not shown). Because it was recently reported that SUMO-1 localizes to the XY body of primary spermatocytes that is a specialized chromatin territory [45,46], our findings suggest that RFP and PIASy are also associated with this nuclear domain and may play a role in spermatogenesis.

Discussion PIAS proteins bind with RFP and promote its SUMO1 conjugation We identified RFP as a transcriptional co-repressor that associates with Mi-2h, the main component of the NuRD complex, and with Enhancer of Polycomb 1 (EPC1) in the nucleus [39,40]. Yeast two-hybrid screening of a human testis cDNA library using the full-length RFP as bait identified PIASx and PIASy as interacting proteins. PIAS proteins were originally proposed to be inhibitors of STAT signaling, and recent growing evidence shows that PIAS proteins also function as SUMO E3 ligases [1,2,4,5]. Our data demonstrate that RFP and PIAS co-immunoprecipitate and co-localize in the nuclear foci, with the same distribution pattern as Pc, Polycomb group SUMO E3 protein [18]. We also showed that all PIAS family proteins examined enhanced the sumoylation of RFP. These results are consistent with other results that suggest PIAS family proteins are less important determinants for substrate specificity in sumoylation. For example, p53 and c-Jun are sumoylated by both PIAS1 and PIASxh, androgen receptor by PIAS1 and PIASxa and Mdm2 by PIAS1 and PIASxh [4,21,32,47]. We identified the acidic region of PIAS proteins as a region that interacts with RFP. PIAS proteins contain several conserved domains including a SAP box (SAF-A, Acinus, PIAS), a PINIT motif, a SP-RING and an acidic region. The SAP box interacts with the matrix attachment region [34]. The SAP box, PINIT motif and SP-RING are suspected to play roles in nuclear retention [48]. The SP-RING is also known to be essential for SUMO E3 ligase activity and proposed to be the binding site for the E2 enzyme Ubc9 [4].

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The acidic region is reported to be the binding site of PIAS3 to the nuclear coactivator TIF2 [49]. Our results strengthen the notion that the acidic region of PIAS proteins serves as the substrate binding region and the SP-RING domain contains the enzymatic activity by associating with the E2 enzyme Ubc9 and SUMO-1. Unfortunately, SUMO acceptor sites of RFP have not been determined yet. There are 21 lysine residues in RFP, but the surrounding sequences of each lysine residue do not match the consensus sequence (c-Lys-X-Glu in which c is a large hydrophobic amino acid and X can be any amino acid) for SUMO modification [22]. Thus, instead of determining the SUMO acceptor sites in RFP, we constructed the SP-RING mutants of PIAS1 and PIASy that do not have the SUMO ligase activity to confirm that RFP is sumoylated by PIAS. As expected, these mutants impaired sumoylation and translocation of RFP (Figs. 4C and 5C). SUMO conjugation enhances the transcriptional repressive activity of RFP SUMO is covalently attached to several kinds of target proteins, including nuclear body proteins, transcriptional factors and co-factors, signal transduction proteins and viral proteins. Sumoylation of target proteins is associated with several biological processes including protein targeting, change of subcellular localization and blocking ubiquitination at the same acceptor site [6,8,10,22]. A number of observations point to the possibility that sumoylation of transcriptional factors is associated with transcriptional repression by sequestration of transcriptional factors in nuclear bodies and/or by sumoylation of components of transcriptional complexes. For example, sumoylated Lef-1 and Sp3 have been shown to translocate to the PML body and nuclear speckles, respectively [33,34]. Our results demonstrate that PIAS proteins enhance the sumoylation and transcriptional repressive activity of RFP in a dosedependent manner. We observed that co-expression of PIAS proteins led to translocation of RFP into nuclear speckles in HEK293 cells and SW480 cells. These results suggest that nuclear sequestration is one of the mechanisms for enhancement of repressive activity of RFP. Alternatively, it is possible that the PIAS interaction modulates the direct interaction of RFP with other transcription factors. Recent results show that a number of proteins involved in transcription are also sumoylated, such as HDAC1, HDAC4, p300 and histone H4 [30,50 – 53]. Sumoylation

Fig. 7. Localization of RFP, SUMO-1 and PIASy in mouse testis. Normal mouse testis was stained immunohistochemically using anti-RFP, antiSUMO-1 and anti-PIASy antibodies. (A) and (B) The representative staining patterns of RFP and SUMO-1 in sequential sections of mouse testis. (C) and (D) High-power views of boxed areas in panels (A) and (B), respectively. The arrows indicate the accumulation of RFP and SUMO-1 in the nuclear domain (XY body) in the same primary spermatocytes. (E) The representative staining of PIASy in mouse testis. (F) Sequential sections were stained with anti-SUMO-1 (upper panel) and anti-PIASy (lower panel) antibodies. The arrows indicate the accumulation of SUMO-1 and PIASy in the nuclear domain (XY body) in the same primary spermatocytes. (G) Mouse testis was stained using normal mouse IgG as a control. (H) Highpower view of boxed area in panel (E). Scale bar, 20 Am.

of p300 is supposed to be essential for the repressive activity and for the binding of HDAC6 [30]. On the other hand, effects of sumoylation on other proteins are subtle. It is

Fig. 6. Expression of SUMO-1 and PIAS enhances the transcriptional repressive ability of RFP. (A) HEK293 cells were transiently transfected with a GAL4tethered luciferase reporter plasmid containing SRE as an enhancer and an SV40 promoter, together with a GAL4-BD RFP expression plasmid and the plasmids containing SUMO-1 and PIAS, as indicated. Luciferase activity in cells transfected with the plasmid containing the GAL4-binding domain alone was set at 100%, and luciferase activities of cells transfected with the indicated plasmids were expressed as percentages of a control value. The values represent means + standard deviations (mean + SD) from at least three independent experiments. The amounts of plasmids used are given in Materials and methods. (B) The PIAS1 and PIASy plasmids were transfected into HEK293 cells in the absence or presence of the SUMO-1 plasmid. Luciferase assay was performed as described above. (C) Effects of PIAS1 C350A and PIASy C342A mutants on the transcriptional repressive activity of RFP.

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possible to speculate that, in addition to the nuclear sequestration, sumoylation of other components of the transcriptional machinery may modulate the transcriptional activity of RFP.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 04.022.

The role of PIAS and RFP in spermatogenesis In rat spermatogenesis, PIASx and PIAS1 mRNA are highly expressed in spermatocytes and Sertoli cells [54]. PIAS1 is also expressed in human and mouse testis and is associated with the androgen receptor in Sertoli and Leydig cells [55]. Several studies have indicated that PIAS1 and PIASxa are involved in the SUMO modification of the androgen receptor and change the transcriptional activity [32]. RFP is highly expressed in male germ cells and forms a characteristic nuclear structure in primary spermatocytes [38]. Interestingly, our immunohistochemical staining results of mouse testis suggest that all of anti-RFP, antiSUMO-1 and anti-PIASy antibodies recognize the same nuclear structure in primary spermatocytes. It was recently reported that SUMO localizes to the XY body of pachytene spermatocytes that is an intra-nuclear structure juxtaposed with the inner nuclear membrane and comprises the transcriptionally repressed sex chromosomes [45]. Formation of the XY body involves condensation of the X and Y chromosomes and is accompanied by repression of many genes encoded by the X and Y chromosomes [46]. As it is known that sumoylation of target proteins is associated with transcription repression, we suspected that PIAS proteins conjugate SUMO to RFP in the XY body of primary spermatocytes, thereby contributing to gene silencing. Although how the formation of the XY body is regulated is unknown, our findings suggest that PIAS and RFP proteins may be cooperatively involved in normal spermatogenesis. Further investigation will provide new insight into roles of these proteins in the XY body. In summary, we revealed the association of RFP and PIAS proteins that are SUMO E3 ligases. RFP is sumoylated, and PIAS proteins enhance the sumoylation of RFP through an association between the coiled-coil domain of RFP and the acidic region of the PIAS proteins. In addition, PIAS proteins clearly enhance the translocation and the transcriptional repressive ability of RFP. Finally, immunohistochemical staining results suggest a physiological role for the association of RFP and PIAS proteins in spermatogenesis.

Acknowledgments We thank K. Imaizumi, K. Uchiyama and S. Kawai for excellent technical assistance. This work was supported by Grants-in-Aid for Center of Excellence (COE) Research, Scientific Research on Priority Areas Cancer and Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.T.).

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