Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription
2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m411820200
ISSN1083-351X
AutoresIsao Kurihara, Hirotaka Shibata, Sakiko Kobayashi, Noriko Suda, Yayoi Ikeda, Kenichi Yokota, Ayano Murai, Ikuo Saito, William E. Rainey, Takao Saruta,
Tópico(s)Protein Degradation and Inhibitors
ResumoAldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the –129/–114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription. Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the –129/–114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription. Aldosterone is exclusively produced in adrenal zona glomerulosa cells due to its unique expression of aldosterone synthase cytochrome P450 (CYP11B2), the enzyme required for the final steps of aldosterone biosynthesis. In aldosterone-producing adrenal cortical adenomas of patients with primary aldosteronism, overexpression of CYP11B2 is demonstrated at the transcriptional level (1Ogishima T. Shibata H. Shimada H. Mitani F. Suzuki H. Saruta T. Ishimura Y. J. Biol. Chem. 1991; 266: 10731-10734Abstract Full Text PDF PubMed Google Scholar, 2Shibata H. Suzuki H. Ogishima T. Ishimura Y. Saruta T. Acta Endocrinol. (Copenh.). 1993; 128: 235-242Crossref PubMed Scopus (38) Google Scholar). Although the reason for aberrant expression of CYP11B2 in these adenomas is not known, mutations in the CYP11B2 gene do not appear to be the cause (3Takeda Y. Furukawa K. Inaba S. Miyamori I. Mabuchi H. J. Clin. Endocrinol. Metab. 1999; 84: 1633-1637Crossref PubMed Scopus (53) Google Scholar, 4Pilon C. Mulatero P. Barzon L. Veglio F. Garrone C. Boscaro M. Sonino N. Fallo F. J. Clin. Endocrinol. Metab. 1999; 84: 4228-4231Crossref PubMed Google Scholar). We therefore postulated that transcription factors and/or coregulators may play important roles in CYP11B2 overexpression in the tumors. The trans-acting factors that regulate CYP11B2 expression remain poorly defined. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), 1The abbreviations used are: SF-1, steroidogenic factor-1; STAT, signal transducers and activators of transcription; PIAS1, protein inhibitors of activated STAT 1; COUP-TFI, chicken ovalbumin upstream promoter-transcription factor I; SUMO, small ubiquitin-related modifier; NBRE, NGFIB response element; CREB, cAMP response element-binding protein; EMSA, electrophoretic mobility shift assay; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; E1, ubiquitin-activating enzyme; E2, SUMO carrier protein; E3, SUMO-protein isopeptide ligase; CMV, cytomegalovirus; IP, immunoprecipitation; GFP, green fluorescent protein; EGFP, enhanced GFP; RSV, Rous sarcoma virus; GR, glucocorticoid receptor; Ang II, angiotensin II; DsRed, Discosoma sp. Red. is shown to play a crucial regulator of most steroid hydroxylase genes, including CYP17 and CYP11B1 (5Val P. Lefrancois-Martinez A.M. Veyssiere G. Martinez A. Nucleic Recept. 2003; 1: 508-518Crossref Scopus (207) Google Scholar, 6Sasano H. Shizawa S. Suzuki T. Takayama K. Fukaya T. Morohashi K. Nagura H. J. Clin. Endocrinol. Metab. 1995; 80: 2378-2380Crossref PubMed Google Scholar). However, SF-1 actually represses rather than activates expression of hCYP11B2 (7Bassett M.H. Zhang Y. Clyne C. White P.C. Rainey W.E. J. Mol. Endocrinol. 2002; 28: 125-135Crossref PubMed Scopus (132) Google Scholar, 8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar, 9Bassett M.H. White P.C. Rainey W.E. Mol. Cell. Endocrinol. 2004; 217: 67-74Crossref PubMed Scopus (164) Google Scholar). In addition, other transcription factors that are expressed in the adrenal cortex include the NGFI-B family of orphan nuclear receptors, such as Nurr1, NGFI-B, and NOR-1. The NGFI-B family receptors are highly expressed in the adrenal zona glomerulosa cells as well as in aldosterone-producing adenomas (8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar, 10Lu L. Suzuki T. Yoshikawa Y. Murakami O. Miki Y. Moriya T. Bassett M.H. Rainey W.E. Hayashi Y. Sasano H. J. Clin. Endocrinol. Metab. 2004; 89: 4113-4118Crossref PubMed Scopus (41) Google Scholar, 11Bassett M.H. Suzuki T. Sasano H. De Vries C.J. Jimenez P.T. Carr B.R. Rainey W.E. J. Biol. Chem. 2004; 279: 37622-37630Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These three nuclear receptors are rapidly induced early response genes that enhance transcription by binding to a consensus sequence, named NBRE-1, as well as an Ad5 element of the hCYP11B2 promoter. In addition, CREB and ATF-1 enhance transcription of the hCYP11B2 gene by binding to a CRE (9Bassett M.H. White P.C. Rainey W.E. Mol. Cell. Endocrinol. 2004; 217: 67-74Crossref PubMed Scopus (164) Google Scholar, 12Clyne C.D. Zhang Y. Slutsker L. Mathis J.M. White P.C. Rainey W.E. Mol. Endocrinol. 1997; 11: 638-649Crossref PubMed Scopus (0) Google Scholar, 13Condon J.C. Pezzi V. Drummond B.M. Yin S. Rainey W.E. Endocrinology. 2002; 143: 3651-3657Crossref PubMed Scopus (104) Google Scholar). Our previous data (12Clyne C.D. Zhang Y. Slutsker L. Mathis J.M. White P.C. Rainey W.E. Mol. Endocrinol. 1997; 11: 638-649Crossref PubMed Scopus (0) Google Scholar) showed that human adrenocortical H295R nuclear proteins containing chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) were bound to the –129/–114 sequence, designated as the Ad5 element of the hCYP11B2 promoter by electrophoretic mobility shift assays. The COUP-TFI was originally identified as an activator of the chicken ovalbumin gene (14Tsai S.Y. Tsai M.J. Endocr. Rev. 1997; 18: 229-240Crossref PubMed Scopus (302) Google Scholar, 15Wang L.H. Tsai S.Y. Cook R.G. Beattie W.G. Tsai M.J. O'Malley B.W. Nature. 1989; 340: 163-166Crossref PubMed Scopus (388) Google Scholar); however, COUP-TFs mostly function as transcriptional repressor of many target genes. COUP-TFs inhibit the transcription of other nuclear receptor such as retinoic acid receptor and thyroid hormone receptor (14Tsai S.Y. Tsai M.J. Endocr. Rev. 1997; 18: 229-240Crossref PubMed Scopus (302) Google Scholar). Furthermore, COUP-TFI represses basal transcriptional activity by active repression utilizing transcriptional corepressors, such as N-CoR and SMRT (16Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar). We and other investigators have previously demonstrated that COUP-TFI and SF-1 regulate the bovine CYP17 expression in a mutually exclusive manner (17Bakke M. Lund J. Mol. Endocrinol. 1995; 9: 327-339Crossref PubMed Google Scholar, 18Shibata H. Kurihara I. Kobayashi S. Yokota K. Suda N. Saito I. Saruta T. J. Steroid Biochem. Mol. Biol. 2003; 85: 449-456Crossref PubMed Scopus (31) Google Scholar). We have previously reported that COUP-TFI is expressed in the normal adrenal cortex and that expression levels of COUP-TFI is inversely correlated with those of CYP17, but correlated with those of N-CoR in adrenal cortical adenomas (18Shibata H. Kurihara I. Kobayashi S. Yokota K. Suda N. Saito I. Saruta T. J. Steroid Biochem. Mol. Biol. 2003; 85: 449-456Crossref PubMed Scopus (31) Google Scholar, 19Shibata H. Ando T. Suzuki T. Kurihara I. Hayashi K. Hayashi M. Saito I. Kawabe H. Tsujioka M. Mural M. Saruta T. Endocr. Res. 1998; 24: 881-885Crossref PubMed Scopus (13) Google Scholar, 20Shibata H. Ando T. Suzuki T. Kurihara I. Hayashi K. Hayashi M. Saito I. Murai M. Saruta T. J. Clin. Endocrinol. Metab. 1998; 83: 4520-4523Crossref PubMed Scopus (24) Google Scholar, 21Shibata H. Ikeda Y. Morohashi K. Mukai T. Kurihara I. Ando T. Suzuki T. Kobayashi S. Hayashi K. Hayashi M. Saito I. Saruta T. Endocr. Res. 2000; 26: 1039-1044Crossref PubMed Scopus (12) Google Scholar, 22Shibata H. Ikeda Y. Mukai T. Morohashi K. Kurihara I. Ando T. Suzuki T. Kobayashi S. Murai M. Saito I. Saruta T. Mol. Genet. Metab. 2001; 74: 206-216Crossref PubMed Scopus (60) Google Scholar, 23Shibata H. Kobayashi S. Kurihara I. Saito I. Saruta T. Horm. Res. 2003; 59: 85-93Crossref PubMed Scopus (12) Google Scholar). We, therefore, have screened COUP-TFI-interacting proteins from a human adrenocortical adenoma cDNA library using a yeast two-hybrid system and identified Ubc9 (24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar) and PIAS1, which are small ubiquitin-related modifier-1 (SUMO-1)-conjugating enzyme and SUMO-1 ligase, respectively. The SUMO post-translationally modifies many proteins with roles in diverse processes, including regulation of transcription, chromatin structure, and DNA repair (25Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (652) Google Scholar, 26Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1390) Google Scholar, 27Verger A. Perdomo J. Crossley M. EMBO Rep. 2003; 4: 137-142Crossref PubMed Scopus (376) Google Scholar, 28Gill G. Curr. Opin. Genet. Dev. 2003; 13: 108-113Crossref PubMed Scopus (193) Google Scholar, 29Gill G. Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (625) Google Scholar, 30Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (581) Google Scholar). The SUMO modification has not been generally associated with increased protein degradation. Rather, similar to non-proteolytic roles of ubiquitin, SUMO modification regulates protein localization and activity. The SUMO E1-activating, E2-conjugating enzymes, and E3-ligase are involved in the sumoylation machinery. In contrast to the ubiquitin system where dozens of E2 enzymes have been identified, Ubc9 is the only known SUMO-E2-conjugating enzyme. Several SUMO-E3 ligases have been identified that promote transfer of SUMO from E2 to specific substrates. To date, three unrelated proteins have been suggested to have SUMO-E3 ligase activity; the protein inhibitors of activated STAT1 (PIAS1) proteins (31Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 32Kotaja N. Aittomaki S. Silvennoinen O. Palvimo J.J. Janne O.A. Mol. Endocrinol. 2000; 14: 1986-2000Crossref PubMed Scopus (145) Google Scholar, 33Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), RanBP2 (34Kirsh O. Seeler J.S. Pichler A. Gast A. Muller S. Miska E. Mathieu M. Harel-Bellan A. Kouzarides T. Melchior F. Dejean A. EMBO J. 2002; 21: 2682-2691Crossref PubMed Scopus (266) Google Scholar, 35Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar), and polycomb group protein Pc2 (36Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). The present study described that both Ubc9 and PIAS1 can function as transcriptional coactivators of COUP-TFI for the hCYP11B2 gene transcription in a sumoylation-independent manner. These proteins are shown to form a complex in the nucleus and exhibit a very unique localization in the adrenal zona glomerulosa cells. We demonstrated here that COUP-TFI, Ubc9, and PIAS1 are recruited to an endogenous CYP11B2 promoter, thus contributing to aldosterone biosynthesis in adrenal zona glomerulosa cells. Plasmid Constructs—Several COUP-TF constructs, such as pG-BKT7-COUP-TFI, pGBKT7-COUP-TFI-(55–315), pGBKT7-COUP-TFI-(86–183), pGBKT7-COUP-TFI-(150–183), pGBKT7-COUP-TFI-(315–423), pGBKT7-COUP-TFI-(55–423), pRSV-COUP-TFI, pRSV-COUP-TFIΔ35, pDsRed-COUP-TFI, and pGBKT7-COUP-TFII were described previously (16Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar, 24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar) (Fig. 1A). Several other constructs, including pG-BKT7-Ubc9, pGADT7-Ubc9, pcDNA3.1/His-Ubc9, pcDNA3.1/His-Ubc9 (C93S), pcDNA3.1/His-Ubc9-(1–58), and pAS1cyh2-TRβ-(168–456) were described previously (16Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar, 24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar). pGBT9-Ad4BP/SF-1, pGBT9-DAX-1, and p3xFLAG-CMV10-PIAS1 were generous gifts by Professor Ken-ichirou Morohashi (National Institute for Basic Biology, Japan). pGL3-Basic-human CYP11B2 (–1521/+2), pGL3-Basic-human CYP11B2 mutAd5, pGL2-wtAd5, pGL2-m5Ad5, and pGL2-m7Ad5 were described previously (7Bassett M.H. Zhang Y. Clyne C. White P.C. Rainey W.E. J. Mol. Endocrinol. 2002; 28: 125-135Crossref PubMed Scopus (132) Google Scholar, 8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar). pGADT7-PIAS1-(5–651) was first identified as a COUP-TFI-interacting protein from human adrenocortical adenoma cDNA library. Several PIAS1 fragments, such as PIAS1-(1–651), -(1–150), -(1–300), -(1–405), -(301–651), -(406–651), and -(5–73/564–651), were subcloned into pGADT7 vector using a PCR amplification with primers containing oligonucleotide linkers of restriction enzyme sites (Fig. 1B). Mutagenesis of PIAS1 was performed with the QuikChange site-directed mutagenesis kit (Stratagene) and the mutant PIAS1 (C351S) was generated. pEGFP-PIAS1 and pEGFP-PIAS1 (C351S) were generated utilizing PCR amplifications from pGADT7-PIAS1 and pGADT7-PIAS1 (C351S), respectively. Cloning of Ubc9 and PIAS1 by a Yeast Two-hybrid System—Yeast two-hybrid screening was conducted with a MATCHMAKER Two-Hybrid System 3 kit (Clontech) and COUP-TFI (amino acids 55–423) as bait. A human adrenocortical adenoma cDNA library was prepared as shown previously (24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar). Yeast strain AH109 containing pGBKT7-COUP-TFI-(55–423) was transformed with a human adrenocortical adenoma cDNA library in pGADT7 (Clontech) and plated on synthetic complete medium lacking tryptophan, adenine, leucine, and histidine. His+ and Ade+ colonies exhibiting β-galactosidase activity by filter lift assay were further characterized according to the manufacturer's protocol (Clontech). β-Galactosidase activity was determined with chlorophenol red β-d-galactopyranoside as described previously (16Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar, 24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar). To recover the library plasmids, total DNA from the yeast was isolated with a Zymoprep™ yeast plasmid Miniprep kit (Zymo Research, Orange, CA) and used to transform Escherichia coli (HB101) in the presence of ampicillin. To ensure that the correct cDNAs were identified, the library plasmids isolated were transformed into Y187 containing pGBKT7-COUP-TFI-(55–423), and β-galactosidase activity was determined. The specificity of the interaction of #2–3 (PIAS1-(5–651)) and #2–4 (Ubc9-(1–158)), both part of the 20 positive clones, with COUP-TFI was determined by mating with Y187, which contains pGBKT7-lamin (Clontech). The β-galactosidase activities of these diploids were examined by the filter lift and chlorophenol red β-d-galactopyranoside methods. The sequence of the #2–3 and #2–4 clones was identical to the GenBank™-submitted sequence of PIAS1 and Ubc9, respectively. The yeast two-hybrid system was also used to determine protein-protein interaction between COUP-TFI/COUP-TFII/Ad4BP/DAX-1/TRβ and these clones. Western Blot Analysis and Coimmunoprecipitation—The cells were lysed with lysis buffer (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 2 mm phenylmethylsulfonyl fluoride), and Western blots were performed before the immunoprecipitation (IP) steps to confirm protein expression by corresponding antibodies as described previously (24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar). The same samples for the Western blots were diluted to 1 ml in IP buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10 mm dithiothreitol, 5 ng/μl aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 0.1% Tween 20) and precleared with protein G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA), and antibodies were added for 1 h. Immune complexes were adsorbed to protein G plus-agarose beads and washed four times in IP buffer. Proteins were then separated on 12.5% polyacrylamide gels and transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences). The primary antibodies used for immunoprecipitation were rabbit polyclonal anti-COUP-TFI antibody (generous gift by Dr. Ming-Jer Tsai) (15Wang L.H. Tsai S.Y. Cook R.G. Beattie W.G. Tsai M.J. O'Malley B.W. Nature. 1989; 340: 163-166Crossref PubMed Scopus (388) Google Scholar), and the antibodies used for the Western blots were anti-COUP-TFI, anti-Xpress (Invitrogen), anti-FLAG (Sigma), anti-Ubc9 (BD Biosciences Pharmingen), anti-PIAS1 (Santa Cruz Biotechnology), and anti-α-tubulin (Oncogene Research Product) antibodies. Fluorescence Imaging—The images of EGFP-tagged Ubc9 and DsRed-COUP-TFI were described previously (24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar). COS-1 cells were transiently transfected with expression vectors of pEGFP-PIAS1, and pDsRed-COUP-TFI. Live cell microscopy of GFP fusion and DsRed fusion proteins was performed on a confocal microscope (Axiovert 100M, Carl Zeiss Co., Ltd.). Imaging for GFP and DsRed was performed by excitation with 488 and 543 nm, respectively, from an argon laser, and the emissions were viewed through band passes ranging from 500 to 550 nm, and 550 to 600 nm, respectively, by band pass regulation with LSM510 (Carl Zeiss Co., Ltd.). All images were processed as TIFF (tagged image file format) files on Photoshop 7.0 using standard image-processing techniques. Northern Blot Analysis—The human tissue Northern blots (Clontech) were hybridized at 42 °C overnight with 32P-labeled cDNA probes of the full-length 1.1-kb hUbc9, 1.9-kb hPIAS-1, full-length 1.3-kb hCOUP-TFI, or 1.1-kb glyceraldehyde-3-phosphate dehydrogenase (Clontech) cDNAs according to the manufacturer's protocol. The membranes were washed at a final stringency of 0.1 × SSC-0.1% SDS at 50 °C and analyzed with a BAS 3000 image scanner (Fuji Film Co.). The mRNA levels were determined by comparison with glyceraldehyde-3-phosphate dehydrogenase mRNA levels. Immunohistochemistry—Formalin-fixed tissues were embedded in paraffin, sectioned at 6 μm, and mounted on silane-coated slides. For immunohistochemistry, sections were dewaxed, rehydrated, followed by blocking endogenous peroxidase using 3% (v/v) hydrogen peroxidase in phosphate-buffered saline, which were then subjected to microwave antigen retrieval in 0.01 m citrate buffer. Thereafter, they were washed in phosphate-buffered saline and blocked with a blocking solution containing 5% bovine serum albumin in phosphate-buffered saline for 30 min. They were subsequently incubated overnight at 4 °C with primary antibodies diluted appropriately with the blocking solution. Primary antibodies for immunohistochemistry included rabbit anti-COUP-TFI, anti-Ubc9 (BD Biosciences, Pharmingen), anti-PIAS1 (Santa Cruz Biotechnology). After two washes in phosphate-buffered saline, immunoreactivities were detected using a Vectastatin ABC Elite kit (Vector Laboratories, CA) and a Vecta DAB substrate kit (Vector Laboratories). As negative controls, sections were incubated with the preimmune or control serum in place of the primary antibody. Electrophoretic Mobility Shift Analysis—Nuclear extracts were prepared as described previously (8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar, 11Bassett M.H. Suzuki T. Sasano H. De Vries C.J. Jimenez P.T. Carr B.R. Rainey W.E. J. Biol. Chem. 2004; 279: 37622-37630Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 12Clyne C.D. Zhang Y. Slutsker L. Mathis J.M. White P.C. Rainey W.E. Mol. Endocrinol. 1997; 11: 638-649Crossref PubMed Scopus (0) Google Scholar). For in vitro transcription/translation, 0.5 μg of pGBT9-Ad4BP/SF-1 and pFL-COUP-TFI was used in conjunction with the TnT-coupled reticulocyte lysate system (Promega), as directed by the manufacturer. EMSA conditions were as described previously (8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar, 11Bassett M.H. Suzuki T. Sasano H. De Vries C.J. Jimenez P.T. Carr B.R. Rainey W.E. J. Biol. Chem. 2004; 279: 37622-37630Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 12Clyne C.D. Zhang Y. Slutsker L. Mathis J.M. White P.C. Rainey W.E. Mol. Endocrinol. 1997; 11: 638-649Crossref PubMed Scopus (0) Google Scholar) using 5 μg of H295R nuclear extract or 0.5–5.0 μl of reticulocyte extract. Protein-DNA complexes were separated from free probe by electrophoresis (2 h) on a 4% polyacrylamide, 2.5% glycerol gel using 1× TGE as running buffer (50 mm Tris-Cl, 38 mm glycine, 2.7 mm EDTA, pH 8.5). For detection of supershift complex, anti-SF-1 (a generous gift of Dr. Ken-ichirou Morohashi) and anti-COUP-TFI (a generous gift of Dr. Ming-Jer Tsai) antibodies were used. Mammalian Cell Culture, Transient Transfections, and Luciferase Assays—H295R cells, derived from human adrenocortical carcinoma cells (8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar, 12Clyne C.D. Zhang Y. Slutsker L. Mathis J.M. White P.C. Rainey W.E. Mol. Endocrinol. 1997; 11: 638-649Crossref PubMed Scopus (0) Google Scholar, 37Rainey W.E. Mol. Cell. Endocrinol. 1999; 151: 151-160Crossref PubMed Scopus (95) Google Scholar), were used for luciferase assays. H295R cells were routinely maintained in Dulbecco's modified Eagle's medium/F-12 (Invitrogen) supplemented with 2.5% NuSerum (Collaborative Bio, Bedford, NA) and 1% ITS (insulin-transferrin-selenium) culture supplement (Invitrogen). Twenty-four hours before transfection, 1 × 105 cells per well of a 24-well dish were plated in the medium. All transfections into H295R cells were carried out using Lipofectamine 2000 (Invitrogen) with indicated amounts of expression plasmids, according to the manufacturer's protocol. Cells were harvested 48 h after transfection, and cell extracts were assayed for both Firefly and Renilla luciferase activities with a Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was determined as ratio of Firefly/Renilla luciferase activities, and data are expressed as the mean (±S.D.) of triplicate values obtained from a representative experiment that was independently repeated at least three times. RNA Interference—H295R cells transfection with siRNAs, and luciferase assays were performed as described previously (8Bassett M.H. Suzuki T. Sasano H. White P.C. Rainey W.E. Mol. Endocrinol. 2004; 18: 279-290Crossref PubMed Scopus (165) Google Scholar, 12Clyne C.D. Zhang Y. Slutsker L. Mathis J.M. White P.C. Rainey W.E. Mol. Endocrinol. 1997; 11: 638-649Crossref PubMed Scopus (0) Google Scholar, 37Rainey W.E. Mol. Cell. Endocrinol. 1999; 151: 151-160Crossref PubMed Scopus (95) Google Scholar). H295R cells were plated into 24-well plates, grown until reaching 70–80% confluence, and transfected with 30 pmol of negative control sequence, Ubc9-, or PIAS1-specific siRNA duplex using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Whole cell extracts were prepared as described previously as follows: siRNA Ubc9a sense, 5′-GGC CAG CCA UCA CAA UCA ATT-3′; siRNA Ubc9a antisense, 5′-UUG AUU GUG AUG GCU GGC CTC-3′; siRNA Ubc9b sense, 5′-GGA ACU UCU AAA UGA ACC ATT-3′; siRNA Ubc9b antisense, 5′-UGG UUC AUU UAG AAG UUC CTG-3′; siRNA PIAS1a sense, 5′-GGU CCA GUU AAG GUU UUG UTT-3′; siRNA PIAS1a antisense, 5′-ACA AAA CCU UAA CUG GAC CTG-3′; siRNA PIAS1b sense, 5′-GGU UAC CUU CCA CCU ACA ATT-3′; siRNA PIAS1b antisense, 5′-UUG UAG GUG GAA GGU AAC CTG-3′; and Silencer Negative Control #1 siRNA (Ambion) were used. Chromatin Immunoprecipitation—ChIP assay was performed as described previously (38Shiio Y. Eisenman R.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13225-13230Crossref PubMed Scopus (529) Google Scholar). The cross-linked, sheared chromatin solution was used for immunoprecipitation with 3 μg of anti-COUP-TFI, anti-Xpress antibody, anti-FLAG antibody, or normal IgG. The immunoprecipitated DNAs were purified by phenol-chloroform extraction, precipitated by ethanol, and amplified by PCR using primers flanking the human CYP11B2 Ad5 region (–335 to –52 from the transcription initiation site) or 3′-untranslated region (1939–2198 from the transcription initiation site): CYP11B2 Ad5 sense primer: 5′-CCT CTC ATC TCA CGA-3′ (–335/–321) and CYP11B2 Ad5 antisense primer: 5′-AAC CTG CTC TGG AAA-3′ (–66/–52); CYP11B2 control sense primer: 5′-CAT TAA GCG GGA TCC-3′ (1939/1953) and CYP11B2 control antisense primer: 5′-CAA GAC CTG GTC CAT-3′ (2184/2198). DNA samples with serial dilution were amplified by PCR to determine the linear range for the amplification (data not shown). Statistics—All experiments were performed in triplicate several times. The error bars in the graphs of individual experiments correspond to the S.D. of the triplicate values. Identification of Ubc9 and PIAS1 as COUP-TFI-interacting Proteins by Yeast Two-hybrid System—To search for proteins that might regulate the activity of the COUP-TFI, we performed a yeast two-hybrid screen with COUP-TFI encoding amino acids 55–423 as bait and a cDNA library prepared from a human adrenocortical adenoma as described previously (24Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar). In this manner, we identified a full-length
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