SUMO-3 Enhances Androgen Receptor Transcriptional Activity through a Sumoylation-independent Mechanism in Prostate Cancer Cells

Androgens are important for male sexual development, which depend on the cognate receptor, the androgen receptor. The transcriptional activity of the androgen receptor, like other nuclear receptors, is regulated by accessory proteins that can have either positive or negative effects. Through a yeast functional screen, we have identified SUMO-3 as a regulator of androgen receptor activity in prostate cancer cells. SUMO-3 is one of three eukaryotic proteins that become post-translationally conjugated to their target proteins in a manner analogous to the attachment of ubiquitin. In primary prostate epithelial cells, PrEC, and the prostate cancer cells, PC-3, SUMO-3 has a weak negative effect on androgen receptor transcriptional activity. In contrast, SUMO-3 and it close relative SUMO-2 strongly enhance transactivation by endogenous androgen receptor in LNCaP cells. This positive effect is observed in both androgen-dependent and androgen-independent LNCaP cells. Interestingly, SUMO-1, unlike SUMO-3 and SUMO-2, can inhibit, but not stimulate, androgen receptor activity. Mutational analysis of the androgen receptor and SUMO-3 demonstrates that the SUMO-3-positive activity does not depend on either the sumoylation sites of the androgen receptor or the sumoylation function of SUMO-3. Stable overexpression of SUMO-3 in LNCaP cells significantly enhances the androgen-dependent proliferation of these cells. Additionally, siRNA-mediated repression of SUMO-2 significantly inhibits the growth of both androgen-dependent and -independent LNCaP cells. Collectively, these results suggest (i) a novel mechanism for elevating AR activity through the switch of SUMO-3 from a weak negative regulator in normal prostate cells to a strong positive regulator in prostate cancer cells and (ii) a proliferative role for SUMO-3 and SUMO-2 in the growth of prostate cancer cells that is independent of sumoylation of the androgen receptor. Androgens are important for male sexual development, which depend on the cognate receptor, the androgen receptor. The transcriptional activity of the androgen receptor, like other nuclear receptors, is regulated by accessory proteins that can have either positive or negative effects. Through a yeast functional screen, we have identified SUMO-3 as a regulator of androgen receptor activity in prostate cancer cells. SUMO-3 is one of three eukaryotic proteins that become post-translationally conjugated to their target proteins in a manner analogous to the attachment of ubiquitin. In primary prostate epithelial cells, PrEC, and the prostate cancer cells, PC-3, SUMO-3 has a weak negative effect on androgen receptor transcriptional activity. In contrast, SUMO-3 and it close relative SUMO-2 strongly enhance transactivation by endogenous androgen receptor in LNCaP cells. This positive effect is observed in both androgen-dependent and androgen-independent LNCaP cells. Interestingly, SUMO-1, unlike SUMO-3 and SUMO-2, can inhibit, but not stimulate, androgen receptor activity. Mutational analysis of the androgen receptor and SUMO-3 demonstrates that the SUMO-3-positive activity does not depend on either the sumoylation sites of the androgen receptor or the sumoylation function of SUMO-3. Stable overexpression of SUMO-3 in LNCaP cells significantly enhances the androgen-dependent proliferation of these cells. Additionally, siRNA-mediated repression of SUMO-2 significantly inhibits the growth of both androgen-dependent and -independent LNCaP cells. Collectively, these results suggest (i) a novel mechanism for elevating AR activity through the switch of SUMO-3 from a weak negative regulator in normal prostate cells to a strong positive regulator in prostate cancer cells and (ii) a proliferative role for SUMO-3 and SUMO-2 in the growth of prostate cancer cells that is independent of sumoylation of the androgen receptor. The physiological functions of the androgens testosterone and 5α-dihydrotestosterone (DHT) 4The abbreviations used are: DHT, dihydrotestosterone; SUMO, small ubiquitin-like modifier 1; AR, human androgen receptor; MMTV, mouse mammary tumor virus; CAT, chloramphenicol acetyltransferase; SC, synergy control; GR, glucocorticoid receptor; PIAS, protein inhibitor of activated STAT; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; siRNA, small interfering RNA; RT, reverse transcription; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; STAT, signal transducers and activators of transcription. are mediated by the androgen receptor (AR) (reviewed in Ref. 1.Kokontis J.M. Liao S. Vitam. Horm. 1999; 55: 219-307Crossref PubMed Scopus (87) Google Scholar), a member of the nuclear receptor superfamily (reviewed in Refs. 2.Mangelsdorf D.J. 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Crossley M. EMBO Rep. 2003; 4: 137-142Crossref PubMed Scopus (376) Google Scholar). The biological functions of sumoylation include protein subcellular translocation, subnuclear structure formation, and modulation of transcriptional activity (reviewed in Ref. 25.Hochstrasser M. Cell. 2001; 107: 5-8Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Sumoylation depends upon the activity of small ubiquitin-related modifier (SUMO), a protein moiety that is conjugated to a specific lysine residue on target proteins (reviewed in Ref. 23.Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar). Three SUMO family members exist, SUMO-1/Smt3C, SUMO-2/Smt3A, and SUMO-3/Smt3B, and all are ubiquitously expressed in mammals (27.Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar, 28.Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (652) Google Scholar). At the amino acid level, SUMO-2 and SUMO-3 are 87% identical but only ∼50% identical to SUMO-1 (27.Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). Although they exhibit low homology in amino acid sequence, SUMO-1 and ubiquitin are structurally related and share significant similarity in secondary and tertiary structures (29.Hay R.T. Trends Biochem. Sci. 2001; 26: 332-334Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Therefore, it is not surprising that the processes of sumoylation and ubiquitination are mechanistically similar (reviewed in Ref. 24.Verger A. Perdomo J. Crossley M. EMBO Rep. 2003; 4: 137-142Crossref PubMed Scopus (376) Google Scholar). Like ubiquitination, the conjugation of SUMO is mediated by a series of enzymatic reactions catalyzed by E1, E2, and E3 enzymes that are distinct from those enzymes that catalyze ubiquitination (27.Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). The SUMO E1 enzymes SAE1 (SUMO-activating enzyme) and SAE2 activate SUMO and transfer it to the E2 enzyme Ubc9, which then directs the conjugated SUMO to its target substrates (27.Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). In vitro evidence has indicated that Ubc9 is sufficient for binding to the SUMO acceptor site and efficiently transferring SUMO to selected targets (27.Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). However, recent evidence shows that a specific E3 ligase might be required for efficient sumoylation in vivo. Three classes of proteins have been identified to have SUMO E3 ligase activity: the protein inhibitor of activated STAT (PIAS) family proteins (30.Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (466) Google Scholar, 31.Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), the polycomb protein Pc2 (32.Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar), and RanBP2 (Ran-binding protein 2) (33.Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). The PIAS proteins are reported to act as SUMO-E3 ligases for the SUMO-1 conjugation to AR in vivo and in vitro (34.Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), resulting in inhibition of AR transcriptional activity (22.Poukka H. Karvonen U. Janne O.A. Palvimo J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (371) Google Scholar). Interestingly, several recent studies have shown that the PIAS proteins can have multiple effects on AR activity, depending on the type of PIAS protein, promoter, and cells (reviewed in Ref. 35.Heinlein C.A. Chang C. Endocr. Rev. 2002; 23: 175-200Crossref PubMed Scopus (631) Google Scholar). The PIAS family is composed of several homologous proteins, including PIAS1, PIAS3, PIASxα, PIASxβ, and PIASy. Nishida et al. (34.Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) showed that AR-dependent transcription is either repressed by PIAS1 and PIASxα in the presence of exogenous SUMO-1 and PIAS RING finger-like domain or enhanced in the absence of sumoylation. PIAS3 inhibits AR transactivation in LNCaP and HeLa cells but enhances AR activity in HepG2 and AR-overexpressing LNCaP cells (36.Gross M. Liu B. Tan J. French F.S. Carey M. Shuai K. Oncogene. 2001; 20: 3880-3887Crossref PubMed Scopus (151) Google Scholar, 37.Kotaja N. Aittomaki S. Silvennoinen O. Palvimo J.J. Janne O.A. Mol. Endocrinol. 2000; 14: 1986-2000Crossref PubMed Scopus (145) Google Scholar, 38.Junicho A. Matsuda T. Yamamoto T. Kishi H. Korkmaz K. Saatcioglu F. Fuse H. Muraguchi A. Biochem. Biophys. Res. Commun. 2000; 278: 9-13Crossref PubMed Scopus (73) Google Scholar). Moreover, Ubc9, the SUMO E2 enzyme, can stimulate AR transcriptional activity that is independent of its ability to catalyze SUMO-1 conjugation (39.Poukka H. Aarnisalo P. Karvonen U. Palvimo J.J. Janne O.A. J. Biol. Chem. 1999; 274: 19441-19446Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). These results demonstrate that the enzymes of the sumoylation pathway can have diverse effects on AR activity. We add to this diversity with the current study, in which we show that SUMO-3 can have a negative or strongly positive effect on AR, depending on the type of prostate cancer cells. Further, the positive effect does not depend on either the sumoylation sites of AR or the sumoylation function of SUMO-3. Finally, SUMO-1 is different from either SUMO-3 or SUMO-2, because it is unable to enhance AR activity. Plasmids—For mammalian expression, AR and c-Jun in pSG-5 have been described (40.Shemshedini L. Knauthe R. Sassone-Corsi P. Pornon A. Gronemeyer H. EMBO J. 1991; 10: 3839-38349Crossref PubMed Scopus (172) Google Scholar). SUMO-3, SUMO-2, and SUMO-1 (generous gifts from Dr. T. Nishimoto) were expressed from the mammalian expression plasmid pCMV-FLAG (41.Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). p5HB-AR and a mutant of p5HB-AR (K385E/K519E) were generous gifts from Dr. J. Iñiguez-Lluhí (42.Iniguiez-Lluhi J.A. Pearce D. Mol. Cell. Biol. 2000; 20: 6040-6050Crossref PubMed Scopus (179) Google Scholar). pSRC-1 and pSRC-3 were generous gifts from Dr. B. Rowan (43.Rowan B.G. Weigel N.L. O'Malley B.W. J. Biol. Chem. 2000; 275: 4475-4483Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). TIF-2 (kindly provided by Dr. P. Chambon) has been described (44.Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (953) Google Scholar). FLAG-SUMO3/pCI-Neo was constructed by first excising SUMO-3 from FLAG-SUMO3/pCMV with EcoRI/SmaI and inserting it into the pCI-Neo vector digested with EcoRI/SmaI. Then the FLAG segment was inserted into SUMO-3/pCI-Neo digested with EcoRI/XbaI. The FLAG segment was the annealed oligonucleotides 5′-AATTCATGGACTACAAAGACGATGACGACAAG-3′ and 5′-CTAGACTTGTCGTCATCGTCTTTGTAGTCCAT-3′. SUMO-3(ΔGG) was constructed by annealing the oligonucleotides 5′-AATTGATGTGTTCCAACAGCAGACGTGACCC-3′ and 5′-GGGTCACGTCTGCTGTTGGAACACATC-3′ (from Integrated DNA Technology) and inserting into FLAG-SUMO-3 digested with SmaI and MfeI. The reporter plasmids used in mammalian cells have the gene for chloramphenicol acetyltransferase (CAT) driven by different promoters. The AR-inducible reporter plasmid MMTV-CAT (40.Shemshedini L. Knauthe R. Sassone-Corsi P. Pornon A. Gronemeyer H. EMBO J. 1991; 10: 3839-38349Crossref PubMed Scopus (172) Google Scholar) and ARE3-E1B-CAT (generous gift from Dr. E. Sanchez) (45.Sanchez E.R. Hu J.L. Zhong S. Shen P. Greene M.J. Housley P.R. Mol. Endocr. 1994; 8: 408-421Crossref PubMed Scopus (67) Google Scholar) are described previously. For the yeast functional screen, LexA-AR(AB) was constructed by digestion of the AB region out of AR(AB)/pTL1NLS (46.Bubulya A. Wise S.C. Shen X.Q. Burmeister L.A. Shemshedini L. Endocrine. 2000; 13: 55-62Crossref PubMed Scopus (18) Google Scholar) with EcoRI and BglI and inserting into the EcoRI and BamHI sites of pEG202 (47.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1324) Google Scholar). All newly generated constructs were confirmed by DNA sequencing (done by the Ohio State University Plant Microbe Genomics Facility). Cell Transfection and CAT Assays—PC-3 cells were maintained in F12K medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Hyclone). LNCaP (androgen-dependent and -independent cells) and DU145 cells were maintained in RPMI1640 medium (Sigma) supplemented with 10% FBS. PrEC cells were maintained in the PrEGM medium (Clonetics). C33 and C81 cells (kindly provided by Dr. Lin) (48.Igawa T. Lin F.F. Lee M-S. Karan D. Batra S.K. Lin M-F. Prostate. 2002; 50: 222-235Crossref PubMed Scopus (165) Google Scholar) were grown in RPMI1640 medium with 5% FBS, 1% glutamine, and 0.5% gentamycin. All cells were cultured with 5% CO2 at 37 °C. These cell lines were grown in RPMI1640 medium with 10% FBS and 0.1 mg/ml neomycin. β-Galactosidase assay and CAT assay were described previously (40.Shemshedini L. Knauthe R. Sassone-Corsi P. Pornon A. Gronemeyer H. EMBO J. 1991; 10: 3839-38349Crossref PubMed Scopus (172) Google Scholar). For transient transfection of LNCaP and DU145 cells, cells were grown to 80–90% confluence in RPMI1640 complete medium. Four hours before transfection, medium was changed to RPMI1640 with 5% FBS (dextran-coated charcoal-treated). Transient transfection was performed with FuGene6 reagent (Roche Applied Science). DHT was added 24 h after transfection. LNCaP and DU145 cells were incubated for 24 h in RPMI1640 with 5% FBS (dextran-coated charcoal-treated) with or without DHT, respectively. Reporter analysis (β-galactosidase assay and CAT assay) were done after the incubation. For PC-3 cells, transient transfection was performed with the calcium phosphate precipitation (CaPO4) method (49.Shenk J.L. Fisher C.J. Chen S.Y. Zhou X.F. Tillman K. Shemshedini L. J. Biol. Chem. 2001; 276: 38472-38479Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In PrEC cells, transient transfection was performed with FuGene6 in PrEGM medium. Transfection efficiency was standardized by measuring β-galactosidase activity, originating from the co-transfected plasmid pCH110 (2 μg). Note that for all transfections, empty expression plasmid was used to bring the final plasmid amount to 9 μg/transfection. CAT activity was quantified by scanning with the Bio-Rad Molecular Imager FX of autoradiograms of three independent replicates for each transfection. Thus, each CAT value represents the average of three repetitions plus the S.D. siRNA Transfections—LNCaP and C81 cells were transfected with a SUMO-2 siRNA (5′-GGGAUGAAUCUGUAACUUAtt-3′ and anti-sense oligonucleotide) (purchased from Ambion). A luciferase siRNA with 42% GC content was used as control siRNA (GL3 siRNA from Dharmacon). The X-tremeGENE siRNA transfection reagent was used to transfect siRNA into cells following the prescribed protocol (Roche Applied Science). Generation of Stable Cell Lines—LNCaP cells were grown in 100-mm dishes with 10 ml of RPMI1640 complete medium until 60–70% confluence, and then cells were transfected with 2 mg of FLAG-SUMO3/pCI-Neo. The transfection was done with FuGene6 described above. After a 48-h incubation, the LNCaP cells were selected in RPMI1640 complete medium containing 0.9 mg/ml neomycin. The medium was refreshed every 4 days until individual colonies appeared. The generation of stable LNCaP cell lines C14 and AJ6 and PC-3 lines C-3, A-103, and V-28 has been described (50.Chen S.Y. The Role of Human Androgen Receptor in the Growth and Survival of Prostate Cancer Cells. Ph.D. dissertation, University of Toledo, Toledo, OH2002Google Scholar). The AJ6 LNCaP cells stably express antisense c-Jun. C14 is a control LNCaP cell line stably transfected with an empty pCI-Neo vector. A-103 cells stably express AR, V-28 cells express a fusion protein containing AR and the VP16 activation domain (51.Chasman D.I. Leatherwood J. Carey M. Ptashne M. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 4746-4749Crossref PubMed Scopus (142) Google Scholar), and C-3 cells were transfected with empty PCI-neo vector. Cellular Proliferation Assay—8 × 104 LNCaP cells were seeded in 6-well plates with 5 ml of RPMI1640 medium containing 2% dextran-coated charcoal-treated FBS. After a 2-day incubation, either ethanol (vehicle control) or 100 nm DHT was added into the wells. After an additional 2, 4, or 6 days of incubation, the MTT assay was done according to the manufacturer's instructions (Sigma). Cell number was quantified by measuring absorbance at a wavelength of 570 nm. Note that bar graphs represent the averages of thee independent experiments plus S.D. Western Blot Analysis—For Western blot analysis, COS and LNCaP cells were grown in 100-mm dishes and subjected to transfection. Whole-cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis as described (52.Bubulya A. Chen S.Y. Fisher C.J. Zheng Z. Shen X.Q. Shemshedini L. J. Biol. Chem. 2001; 276: 44704-44711Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The nitrocellulose blots were probed with the anti-FLAG antibody M2 (Sigma), anti-AR antibody PA1-111A (Affinity Bioreagents), or anti-β-actin AC-15 (Abcam). The ECL chemiluminescence detection kit (Amersham Biosciences) was used to develop the Western blots. Northern Blot Analysis—A multiple human tissue blot was obtained from Clontech (catalog no. 7761-1) and probed for SUMO-3 mRNA according to the manufacturer's instructions. Semiquantitative Reverse Transcription (RT)-PCR—To prepare RNA for the RT reaction, total RNA from LNCaP, PC-3, or PrEC cultured cells was extracted using the Trizol reagent (Invitrogen). For the RT-PCR (reverse transcription-PCR) assays, cDNA was prepared from the isolated RNA using the Moloney murine leukemia virus reverse transcriptase, according to the manufacturer's instructions (Fisher). The PCRs were carried out using primers specific for the mRNA: the upstream primer 5′-GGGCAACCAATCAATGAAAC-3′ and the downstream primer 5′-AGTCAGGATGTGGTGGAACC-3′ (SUMO-3), the upstream primer 5′-CTGGCCCTCAAGCATGTAAC-3′ and the downstream primer 5′-AAATCTGAGGCCACAACACC-3′ (SUMO-2), the upstream primer 5′-ACCGTCATCATGTCTGACCA-3′ and the downstream primer 5′-TGGAACACCCTGTCTTTGAC-3′ (SUMO-1); the upstream primer 5′-TCATAAGCAGCGACCTTGTG-3′ and the downstream primer 5′-ACCGAAGGAAGAGACCCTGT-3′ (Ubc9); the upstream primer 5′-CTTCTTGTCGGCTTGAAAGG-3′ and the downstream primer 5′-ACCATGGGGTTGAGATTCTG-3′ (SAE1); the upstream primer 5′-GACAGAGCTGACCCTGAAGC-3′ and the downstream primer 5′-TTTTCCGCCATAGTTTGTCC-3′ (SAE2). Glyceraldehyde-3-phosphate dehydrogenase-specific primers (upstream primer 5′-CGACCACTTTGTCAAGCTCA-3′ and the downstream primer 5′-AGGGGAGATTCAGTGTGGTG-3′) were used in the RT-PCR as a control. RT-PCR was carried out for 30 cycles using the following conditions: denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. The PCR products were electrophoretically separated on a 2% agarose gel and stained with ethidium bromide. Yeast Transformation for the Functional Screen—The yeast transformation protocol was described previously (53.Shen X.Q. Bubulya A. Zhou X.F. Khazak V. Golemis E.A. Shemshedini L. Endocrine. 1999; 10: 281-289Crossref PubMed Google Scholar). A 20-ml culture of YPH499/pSH18–34 (47.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1324) Google Scholar) was grown and transformed with AR(AB)/pEG202 in Glu/CM–Ura–His liquid dropout medium overnight at 30 °C. The culture was diluted into a 300-ml Glu/CM–Ura–His liquid dropout medium with 2 × 106 cells/ml and incubated at 30 °C until A600 reading reached 0.5, after which it was centrifuged for 5 min at 1000–1500 × g. The liquid was discarded, and the cells were resuspended in 1.5 ml of TE buffer, 0.1 m lithium acetate. 1 μg of P19 library (54.Howell B.W. Afar D.E. Lew J. Douville E.M. Icely P.L. Gray D.A. Bell J.C. Mol. Cell. Biol. 1991; 11: 568-572Crossref PubMed Scopus (96) Google Scholar) and 50 μg of high quality sheared salmon sperm carrier DNA were added to each of 30 sterile 1.5-ml microcentrifuge tubes. 50 μl of the resuspended yeast cells were added to each tube, and they were incubated for 30 min at 30 °C, and Me2SO was then added to 10%. The samples were heat-shocked for 10 min at 42 °C. For 28 tubes, the complete content of one tube was added per 24 × 24-cm Glu/CM–Ura–His–Leu/X-Gal dropout plate and incubated at 30 °C. For the remaining two tubes, 360 μl of each tube was spread on 24 × 24-cm Glu/CM–Ura–His–Leu/X-Gal dropout plate. The remaining 40 μl from each tube was used to make a series of 1:10 dilution in sterile water. Dilutions were plated on 100-mm Glu/CM–Ura–His–Leu dropout plates. All plates were incubated at 30 °C until colonies appeared (2–3 days). Colonies were monitored for color (blue or white). Among 300,000 transformed colonies, nine white colonies appeared on the X-gal medium. Plasmid was isolated from each of the white colonies and used for retransformation. Only four of the isolated plasmids were able to cause a white phenotype upon retransformation. DNA sequencing showed that one of the four plasmids matched the open reading frame of mouse SUMO-3, as well as the part of the 5′- and 3′-untranslated region. Yeast Liquid β-Galactosidase Assay—A single yeast colony was inoculated in 3 ml of YPD (or appropriate selective) medium and incubated overnight at 30 °C. 20–50 μl of each overnight culture was inoculated in 4 ml of YPD medium (or appropriate selective medium and/or inducing conditions) and grown to middle or late log phase. This was subjected to a liquid β-galactosidase assay using 2-nitrophenyl β-d-galactopyranoside as described (55.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1998; 2 (John Wiley and Sons, Inc., New York)Google Scholar). DNA Sequencing Analysis—cDNA fragment was isolated from the positive clones using the Yeastmaker™ Yeast Plasmid Isolation Kit (BD Biosciences) and amplified using the upstream oligonucleotide 5′-GTTTTTCAAGTTCTTAGATG-3′ and the downstream oligonucleotide 5′-CTGGCAATTCCTTACCTTCC-3′ (Integrated DNA Technology). DNA sequencing for the nine putative cDNA clones was carried out using the same primers (sequencing done by Ohio State University Plant Microbe Genomics Facility). SUMO-3 Was Identified Using a Yeast Functional Screen—In an effort to identify novel repressors of AR, we developed a modified yeast two-hybrid system that we call a "yeast functional screen." Like the yeast two-hybrid system, the yeast functional screen depends on a fusion protein containing the LexA DNA-binding domain fused to a protein of interest and the LexA-responsive LacZ reporter (47.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1324) Google Scholar). Unlike the yeast two-hybrid system, the protein of interest must have strong transcriptional activity so that all yeast colonies expressing this fusion protein will become blue when grown on X-gal plates. Expression of a protein that blocks the transcriptional activity of the protein of interest will cause yeast colonies to remain white (Fig. 1A). In the present study, the AR(AB) region acted as a bait protein and exhibited strong transcriptional activity in yeast, compared with the