Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9*

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

The human mineralocorticoid receptor (MR, 2 NR3C2), a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily, mediates most of the known effects of aldosterone (1,2). Besides its involvement in the regulation of electrolyte balance in epithelial cells, most notably in the distal collecting duct of the kidney and the colon, MR is also present in a variety of nonepithelial cells, such as cardiomyocytes and neurons (2)(3)(4). Two clinical trials, the Randomized Aldactone Evaluation Study (5) and Eplerenone Post-acute Myocardial Infarction Heart Failure Efficacy and Survival Study (6), shed new light on MR as an important pathogenic mediator of cardiac and vascular remodeling, because treatment with MR antagonist spironolactone or eplerenone was effective in significantly reducing the morbidity and mortality of patients with congestive heart failure. However, molecular mechanisms to account for the successful treatment remain to be elucidated. MR functions are directed by specific activation domains, designated as activation function 1 (AF-1), which resides in the N terminus, and activation function 2 (AF-2), which resides in the C-terminal ligand-binding domain. Regulation of gene transcription by MR requires the ligand-dependent recruitment of proteins characterized as coactivators (7)(8)(9)(10)(11)(12). Among these coactivators, three so-called p160 family coactivators, steroid receptor coactivator-1 (SRC-1) (13,14), SRC-2, and SRC-3, can interact with most nuclear receptors and are ubiquitously expressed in various tissues. These coactivators have histone acetyltransferase activity to overcome repressive effects of chromatin structure on transcription.
Besides these factors, several coregulators have various enzymatic activities, thus contributing to their abilities to enhance receptor-mediated transcription. The SUMO post-translationally modifies various proteins with roles in diverse processes, including regulation of transcription, chromatin structure, and DNA repair (15)(16)(17)(18)(19). Like ubiquitylation, the covalent attach-ment of SUMO to its targets (sumoylation) involves three enzymatic reactions as follows: the SUMO E1-activating, E2-conjugating enzymes, and E3 ligase. The SUMO E1 is a heterodimer of SAE1/SAE2 in mammals (Aos1/Uba2 in yeast). In contrast to the ubiquitin system where dozens of E2 enzymes have been identified, sumoylation uses only a single E2 enzyme, Ubc9. SUMO-1 is conjugated to target proteins at the consensus sequence KXE (where is any hydrophobic amino acid, and X is any amino acid). Three unrelated proteins have SUMO E3 ligase activity to promote transfer of SUMO from E2 to specific substrates as follows: the protein inhibitor of activated signal transducer and activator of transcription 1 (PIAS1) protein (20 -22), RanBP2 (23,24), and polycomb group protein Pc2 (25).

EXPERIMENTAL PROCEDURES
Antibodies-Goat anti-human MR (N-17) antibody, goat anti-human SRC-1 (M-341) antibody, and normal goat IgG were obtained from Santa Cruz Biotechnology. Mouse monoclonal anti-Xpress IgG was obtained from Invitrogen. Mouse anti-Ubc9 antibody was obtained from Pharmingen. Rabbit anti-HA antibody was obtained from Clontech. Mouse anti ␣-tubulin antibody was obtained from Oncogene.
Cell Culture, Transfections, and Luciferase Assays-COS-1 cells, COS-7 cells, and HEK293 cells were routinely maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Twenty four hours before transfection, 5 ϫ 10 4 cells per well of a 24-well dish were plated in the medium. All transfections were carried out by using Lipofectamine 2000 (Invitrogen) with 0.3 g/well of the luciferase reporter, 0.01 g/well of pRL-null internal control plasmids, and the indicated amounts of expression plasmids according to the manufacturer's instructions. After 18 -24 h, the medium was changed to DMEM with 10% fetal bovine serum and 10 Ϫ8 M aldosterone or vehicle. After an additional 24 h, 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 were expressed as the mean (ϮS.D.) of triplicate values obtained from a representative experiment that was independently repeated at least three times.
Generation of Human MR Expressing Cell Line, 293-MR-Human 293F embryonic kidney cells and transformants were routinely maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, UT). To establish stable transformants (293-MR cells), parent 293F cells were transfected with pNTAP-hMR (Stratagene) with Lipofectamine Plus reagents (Invitrogen) and cultured for 2 weeks in the presence of 700 g/ml G418 for transformant selection as described previously (46 -48). Individual colonies were selected and expanded for further analysis.
Yeast Two-hybrid Assay-Yeast two-hybrid assays were used to determine interaction of MR with Ubc9. Yeast Y187 cells were transformed with yeast expression plasmids encoding hMR and Ubc9. ␤-Galactosidase activity in liquid culture was determined with chlorophenol red ␤-D-galactopyranoside as described previously (49,50,52,53).
Glutathione S-transferase Pulldown Assay-Glutathione S-transferase (GST) (pGEX4T-1) protein, GST-Ubc9 (pGEX4T-1-Ubc9), and GST-Ubc9(C93S) (pGEX4T-1-Ubc9(C93S)) fusion proteins were expressed and extracted in Escherichia coli DH5␣ as described previously (49,52). GST pulldown assay was performed as described (49,52), with modifications: 50 l of glutathione-Sepharose beads 4B (Amersham Biosciences) stored in beads incubation buffer (50 mM potassium phosphate buffer (pH 7.4), 100 mM NaCl, 1 mM MgCl 2 , 10% glycerol, and 0.1% Tween 20) were incubated with bacterial extracts containing GST fusion proteins together with beads incubation buffer for 30 -60 min at room temperature. Preparation of bacteria extracts containing GST fusion protein was as described previ-ously (49,52). The supernatant was then removed, and the beads were washed three times with beads incubation buffer. In vitro-translated 35 S-labeled proteins were obtained by using TNT Coupled Reticulocyte Lysate Systems (Promega). Crude lysates were incubated with the beads in 200 l of beads incubation buffer for 60 min at 4°C with a circle rotator. Finally the beads were washed five times with 1 ml of beads incubation buffer, and the proteins were solubilized in SDS loading buffer and analyzed on SDS-PAGE (12.5% polyacrylamide gel). The input lanes contained 20% of the labeled protein used for binding.
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 (49,50). 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/l aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% Tween 20) and precleared with protein G plusagarose 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 anti-MR (Santa Cruz Biotechnology) or anti-Xpress (Invitrogen) antibodies, and antibodies used for the Western blots were anti-MR, anti-Xpress, anti-HA (Oncogene), and anti-SRC-1 (Santa Cruz Biotechnology) antibodies.
Immunohistochemical Staining-Tissues were isolated from 2-month-old wild type male mice, fixed in 4% paraformaldehyde overnight at 4°C, dehydrated in graded ethanol, and then processed for paraffin embedding. Sections (7 m) were incubated for 2 h in blocking buffer, which is constituted by 1% bovine serum albumin, 5% normal donkey serum, 20 g/ml donkey anti-mouse IgG Fab fragment (Jackson Immuno-Research, West Grove, PA), and 0.02% Triton X-100 in phosphate-buffered saline, and followed by incubation with the primary antibodies overnight at 4°C. Primary antibodies used in this experiment were as follows: mouse anti-MR, rabbit anti-Ubc9 (Santa Cruz Biotechnology), and rabbit anti-SRC-1 (Santa Cruz Biotechnology), and the optimized dilutions were 1:50 -1:100. After incubation with the combination of primary antibodies (MR and Ubc9, MR and SRC-1), slides were incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR) for 30 min at 4°C. 4Ј,6-Diamino-2-phenylindole (DAPI) was used for nuclear staining.
Fluorescence Imaging-The images of EGFP-tagged Ubc9 and YFP-tagged SRC-1 were described previously (49,54). HEK293 cells were transiently transfected with expression vectors of pDsRed-MR with pEGFP-Ubc9 or pYFP-SRC-1. Live cell microscopy of DsRed fusion, EGFP fusion, or YFP fusion proteins was performed on a confocal microscope (Axiovert 100 M, Carl Zeiss Co., Ltd.). Imaging for DsRed and EGFP or YFP was performed by excitation with 543 and 488 nm, respectively, from an argon laser, and the emissions were viewed through band passes ranging from 550 to 600 and 500 -550 nm, respectively, by band pass regulation with LSM510 (Carl Zeiss Co., Ltd.). All images were processed as tagged image file format (TIFF) files on Photoshop 7.0 using standard imageprocessing techniques.
Semiquantitative RT-PCR-The effect of endogenous Ubc9 on the endogenous Sgk, Ubc9, and GAPDH levels in the presence of 10 Ϫ8 M aldosterone was investigated by semiquantitative RT-PCR. For semiquantitative RT-PCR, total RNA was extracted from 293-MR cells and reverse-transcribed as described previously. Procedures of RT-PCR were performed as described previously elsewhere. Preliminary experiments were conducted to ensure linearity for the semiquantitative procedures. Hot start PCR was performed by heat-activating AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences) at 94°C for 4 min. Optimized cycling condition was 30 cycles (for Sgk) or 20 cycles (for Ubc9 and glyceraldehyde-3-phosphate dehydrogenase) for 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. Oligonucleotide primers were constructed from the published cDNA sequences of Sgk, Ubc9, and GAPDH cDNA. The sequences of the primers were as follows: Sgk sense,

Characterization of the Ubc9-MR Protein Interaction-We
have recently shown that nuclear orphan receptor chicken ovalbumin upstream promoter-transcription factor I activates human CYP11B2 gene transcription in cooperation with Ubc9 and PIAS1, thus resulting in aldosterone secretion in the adrenal zona glomerulosa cells (49,50). During the studies, we found that Ubc9 also markedly interacts with MR. We therefore described functional interaction of Ubc9 with MR in this study. Yeast ␤-galactosidase liquid assays were performed to examine interaction of MR with Ubc9. The full-length human MR encoding amino acids 1-984 interacted with Ubc9 in the absence of hormone, and the interaction was significantly enhanced in the presence of 10 Ϫ8 M aldosterone ( Fig. 1, lanes 3  and 4). The ligand-binding domain of MR encoding amino acids 671-984 did not interact with Ubc9 in the absence or presence of aldosterone ( Fig. 1, lanes 7 and 8). On the other hand, the N-terminal fragment of MR encoding amino acids 1-670 markedly interacted with Ubc9 ( Fig. 1, lanes 9 and 10).
These data indicate that Ubc9 interacts with the N-terminal MR in an aldosterone-sensitive manner.
To confirm this interaction biochemically, we performed GST-pulldown assays using GST-Ubc9 protein and in vitro translated [ 35 S]Met-labeled MR. The full-length MR-(1-984) interacted with GST-Ubc9 both in the absence and presence of aldosterone but not with GST alone (Fig. 2, upper panel).
Remarkably, the N-terminal MR-(1-670) strongly interacted with GST-Ubc9 ( Fig. 2, lower panel); however, the ligand-binding domain of MR-(671-984) showed no interaction with Ubc9 ( Fig. 2, middle panel). These in vitro interaction data are compatible with those in yeast cells. Furthermore, a sumoylationinactive mutant Ubc9(C93S) similarly interacted with the fulllength as well as N-terminal MR (Fig. 2, upper and lower panels), indicating that sumoylation activity is dispensable for interaction of Ubc9 with MR.
To analyze the physical interaction between MR and Ubc9 in mammalian cells, coimmunoprecipitation assays were performed. Mammalian expression plasmids of MR and Xpresstagged Ubc9 were transfected into HEK293 cells. Proteins precipitated with anti-MR antibody were resolved by SDS-PAGE, and Western blot using anti-Xpress antibody was conducted. The coimmunoprecipitation experiments clearly showed that both N-terminal (residues 1-670) and full-length MR were associated with Ubc9 ( Fig. 3, A, lane 2, and B, lane 2). In addition, both wild type-and sumoylation-inactive mutants of Ubc9 (C93S) were associated with MR at similar levels ( Fig. 3B, lanes  2 and 3). To investigate physiological relevance of these observations, we have generated MR stable transformants of 293-MR cells. Although HEK293F cells express no MR, a significant level of expression of MR protein was shown in 293-MR cells by Western blot and coimmunoprecipitations (Fig. 3C, upper  panel). We were able to show a significant interaction of endogenous Ubc9 with stably expressing MR in 293-MR cells (Fig. 3C, lower panel). Taken together with the interaction data in yeast cells, in vitro, and mammalian cells, Ubc9 interacts with the N-terminal region of MR.
Ubc9 Functions as a Coactivator of MR-mediated Transactivation in an Agonist-dependent Manner-To address functional effects of interaction between MR and Ubc9, transient transfection assays were performed. Transfection of human MR cDNA expression plasmid significantly activated three reporter constructs containing 3xMRE, ENaC, or MMTV in an aldosterone-dependent manner in COS-1 cells (Fig. 4A, lanes 6,  13, and 20). Coexpression of Ubc9 potentiated MR-mediated   transactivation of the reporter constructs in the presence of aldosterone (Fig. 4A, lanes 7, 14, and 21); however, the extent of potentiation by ectopic expression of Ubc9 varied depending on the reporter construct. Overexpression of Ubc9 alone had no significant effects on these reporter activities (Fig. 4A, lanes  5, 12, and 19), indicating that the ability of Ubc9 in enhancing MR-mediated transactivation is dependent on interaction with MR. In addition, increasing concentrations of aldosterone treatment activated MR-mediated transactivation, and coexpression of Ubc9 significantly enhanced its transactivation (Fig.  4B), indicating that Ubc9 potentiates MR-dependent transactivation in an aldosterone concentration-dependent manner. The increase in reporter activity was not because of increased cellular concentration of MR, because Western blot did not reveal alterations in immunoreactive MR protein content (Fig.  3B, 2nd and 3rd panels in lanes 1 and 2). These findings indicate that Ubc9 has transcriptional coactivator capacity of MR in vivo. We next tested effects of ligands on Ubc9's ability in MRmediated transcription. To examine the influence of the nature of the ligand on MR responses to Ubc9, experiments performed with cortisol but not spironolactone led to the same potentiating effects (data not shown). These results indicate that a particular ligand-binding domain conformation induced by the nature of the bound agonistic ligand is crucial for Ubc9 action on MR and thus is presumably dependent on the AF-2.
Endogenous Ubc9 Is Required for MR-mediated Transactivation-If Ubc9 functions as a coactivator of MR, reducing the endogenous level of Ubc9 should decrease the transcriptional activity by MR in transient transfection assays. As described previously (Fig. 4A), overexpression of MR activated 3xMRE-E1b-Luc reporter activity by 20-fold. Cotransfection of two sets of Ubc9 siRNA (siRNA-Ubc9#1 or siRNA-Ubc9#2), but not negative control (siRNA-Control), effectively reduced the endogenous levels of Ubc9 protein but had no effect on the ␣-tubulin protein level as shown in Western blot (Fig. 5B). Reduction of endogenous Ubc9 protein level decreased the MR-mediated transactivation by 63% (siRNA-Ubc9#1) (Fig.  5A, lane 7) and 37% (siRNA-Ubc9#2) (Fig. 5A, lane 8) in the presence of aldosterone but not in the absence (Fig. 5A, lanes  1-4). We also examined the effect of reducing endogenous Ubc9 on aldosterone-dependent activation of the Sgk gene in 293-MR cells. In a typical experiment siRNA against Ubc9 lowered endogenous levels of Ubc9 mRNA by about 90%, compared with cells receiving a control siRNA (Fig. 6, A, lower  panel, and B). The addition of the MR agonist aldosterone caused strong induction of Sgk mRNA levels by 3.5-fold, indicating that the stably expressing MR protein functions physiologically (Fig. 6, A and C). The siRNA against Ubc9 lowered the aldosterone-induced level of Sgk mRNA by 48%, as compared with the level by introduction of control siRNA (Fig. 6, A and C,  lanes 3 and 4). The Ubc9 and Sgk mRNA levels were normalized to GAPDH mRNA levels, thus demonstrating that the effects of the Ubc9-directed siRNA were gene-specific. Thus, although many different coactivators are involved in mediating transcriptional activation by MR, endogenous Ubc9 is necessary for efficient induction of the endogenous Sgk gene in response to hormones. These findings indicate that endogenous Ubc9 normally functions as a transcriptional coactivator for the MRmediated transactivation. Modification of MR by SUMO-1-SUMO-1 modification of transcription factors, including nuclear hormone receptors, is known to affect transcriptional activity, and we therefore examined whether MR is a substrate for SUMO-1 modification. MR displays several sumoylation consensus motifs, previously described as synergy control motifs (55). We generated a mutant MR construct in which four lysine residues within sumoylation consensus motifs were mutated into arginine, K89R/K399R/K494R/K953R, designated as KRmut MR by sitedirected mutagenesis. The ability of wild type and KRmut MR proteins to undergo SUMO-1 modifications was analyzed by in vivo sumoylation assays as described previously (38). The wild type-and KRmut-MR with or without HA-tagged SUMO-1 were transfected into HEK293 cells. Whole cell extracts from transfected HEK293 cells were immunoprecipitated with anti-MR antibody, followed by immunoblotting with anti-HA or anti-MR antibody (Fig. 7). The results showed that the slower migrating multiple bands were detected only when HA-SUMO-1 and wild type MR were coexpressed (Fig. 7, lane  2). Interestingly, sumoylation of MR appeared to be decreased in the presence of aldosterone than in the absence for unknown mechanisms (Fig. 7, top panel, lanes 2 and 4). When HA-SUMO-1 and KRmut MR were coexpressed, these high molecular weight protein bands were almost disrupted (Fig. 7,   Ubc9 Potentiates MR-mediated Transcription Independent of Sumoylation Activity-To elucidate whether E2 SUMO-1-conjugating enzyme activity of Ubc9 is required for coactivation of MR, transient transfection assays were performed utilizing the sumoylation-inactive mutant Ubc9 (C93S). Coexpression of Ubc9 (C93S) potentiated the transcriptional activity of wild type MR by 4-fold (Fig. 8A, lane 6 versus lane 4) in a similar manner to coexpression of wild type Ubc9 (Fig. 8A, lane 5 versus lane 4). The transcriptional properties of KRmut MR on 3xMRE-E1b-Luc reporter activity showed enhanced transcriptional activities by ϳ5-fold (Fig. 8A, lane 10 versus lane 4) compared with the wild type MR. This finding was also supported by data that overexpression of SENP1, a SUMO-1 isopeptidase, similarly potentiated MR-mediated transactivation (data not shown). Therefore, sumoylation of MR is likely to repress its transcriptional activities as shown previously (38). Coexpression of Ubc9 or Ubc9 (C93S) further potentiated the KRmut MR-mediated transactivation by 3-fold (Fig. 8A, lanes 10 -12). These findings suggest that sumoylation activity of Ubc9 is not necessary for enhancement of MR-mediated transcription. Because KRmut MR displayed stronger transactivation than wild type MR, it is tempting to test whether interaction of MR with Ubc9 was affected. The coimmunoprecipitation assays showed that wild type-as well as KRmut-MR similarly interacted with Xpress-tagged Ubc9 (Fig. 8B, top panel, lanes 2 and   3), indicating that interaction between MR and Ubc9 is not altered depending upon sumoylation status of MR. Similarly, both Ubc9 and Ubc9 (C93S) equally interacted with MR (Figs. 2 and 3B), indicating that interaction between MR and Ubc9 is not altered depending upon sumoylation activity of Ubc9.
Ubc9 and SRC-1 Synergistically Potentiated MR-mediated Transcription-Ubc9 is a strict ligand-dependent coactivator of MR, but Ubc9 does not directly interact with the ligandbinding domain of MR. Therefore, it is tempting to test that Ubc9 is associated with certain coactivators interacting with MR AF-2 domain. We investigated the combined effects of Ubc9 and several known AF-2 coactivators on MR-mediated transcription in transient transfection assays. Among several known AF-2 coactivators, we found that SRC-1, but not p/CIP or CBP/p300 (data not shown), synergistically potentiated MRmediated transactivation in cooperation with Ubc9. As shown in Fig. 9, overexpression of either SRC-1 (lanes 5-7) or Ubc9 (lanes 8 -10) in cells enhanced aldosterone-induced MR-dependent transcription by ϳ4-fold (Fig. 9, lanes 7 and 10), compared with untransfected cells (Fig. 9, lane 4) in a dose-dependent manner. When both Ubc9 and SRC-1 were overexpressed in cells, reporter gene activity was synergistically elevated to levels 12-13-fold higher than in untransfected cells (Fig. 9,  lanes 13 and 16 versus lane 4) in a manner that was more than additive of the effects of either protein alone. These results suggest that Ubc9 contributes to MR-dependent transcriptional activation at least partly by binding SRC-1 and recruiting it to cellular transcriptional machinery. Because recent reports (56) showed that relatively few coactivators, including SRC-1, strongly interact with MR in the presence of aldosterone, these findings are consistent with our data that Ubc9 and SRC-1 cooperatively function as coactivators of MR.
Next, to prove that an MR-Ubc9-SRC-1 complex contributes to MR-dependent transactivation in cells, we examined the physical association between MR, Ubc9, and SRC-1 using coimmunoprecipitation assays. First of all, endogenous Ubc9 was clearly associated with SRC-1 (Fig. 10A, lane 1), and the interaction was more pronounced when Ubc9 was ectopically overexpressed (Fig. 10A, lane 2). In addition, the N-terminal domain of MR-(1-670) was coimmunoprecipitated with SRC-1 (Fig. 10B, top panel, lanes 3 and 4), and the interaction was slightly enhanced when ectopic Ubc9 was overexpressed, which suggests Ubc9 may stabilize SRC-1 protein, thus increasing in MR-SRC-1 association as shown recently (57). Fulllength MR was coimmunoprecipitated with Ubc9 as well as SRC-1 in the presence of aldosterone (data not shown). Taken together with these coimmunoprecipitation data, both Ubc9 and SRC-1 were shown to form a complex with the N-terminal domain of MR-(1-670), thus transactivating MR-dependent transcription.
MR and Ubc9 Are Specifically Recruited to the MRE of Human ENaC Gene Promoter-As mentioned above, the MR-Ubc9 complex activated the human ENaC gene transcription (Fig. 4A). ChIP assays were used to test whether stably expressing MR and endogenous Ubc9 are recruited to the endogenous ENaC gene promoter in 293-MR cells. The cross-linked, sheared chromatin preparations were subjected to immunoprecipitation with various antibodies, and the precipitated DNA was analyzed by PCR amplification of the MRE (Ϫ1567/Ϫ1297) of the ENaC promoter. We have confirmed the size of the sonicated DNA was ϳ300 -600 bp, and these bands looked uniform (data not shown). When aldosterone is present in 293-MR cells, antibodies against MR, SRC-1, and Ubc9 efficiently immunoprecipitated the MRE (Ϫ1567/Ϫ1297) of the ENaC promoter only in the presence of aldosterone but not in the absence (Fig. 11). Normal IgG and no antibody (data not shown) failed to precipitate the ENaC promoter. In contrast to the MRE of the ENaC promoter, the control region (ϩ1/ ϩ265) of the ENaC gene was not detected in association with MR or Ubc9 (Fig. 11). These data highlight a finding that endogenous MR and Ubc9 are recruited to an aldosterone-sensitive gene promoter in a largely hormone-dependent manner in the context of chromatin in vivo.
Colocalization of MR with Ubc9 and SRC-1 in Cultured HEK293 Cells and the Collecting Duct Cells from Mouse Kidney-To determine whether MR, Ubc9, and SRC-1 could interact within a cellular environment, HEK293 cells were transfected with DsRed-tagged MR, EGFP-tagged Ubc9, YFP-tagged SRC-1, alone or in various combinations, and photographed using fluorescence microscopy (Fig. 12, A  and B). After cotransfection of HEK293 cells with pEGFP-Ubc9 and pDsRed-MR, EGFP-Ubc9 was localized in both nucleus and cytoplasm, whereas DsRed-MR was mainly localized in the cytoplasm in the presence of ethanol (vehicle). Treatment with 10 Ϫ8 M aldosterone resulted in nuclear accumulation of DsRed-MR, which was overlapped with EGFP-Ubc9 (Fig. 12A). Similarly, after cotransfection of HEK293 cells with YFP-SRC-1 and DsRed-MR, YFP-SRC-1 was localized in the nucleus and cytoplasm, whereas DsRed-MR was mainly localized in the cytoplasm in the presence of ethanol (vehicle). Treatment with 10 Ϫ8 M aldosterone resulted in nuclear accumulation of DsRed-MR and YFP-SRC-1 with occasional dot formation. These findings indicate that MR was colocalized with Ubc9 and SRC-1 in the nuclei only in the presence of aldosterone. The fluorescence imagings of ectopic expressing MR, Ubc9, and SRC-1 support colocalization of these proteins in cultured cells.
Finally, to examine whether the physical interaction between MR, Ubc9, and SRC-1 had a potentially physiological significance, we investigated whether endogenous MR, Ubc9, and SRC-1 were indeed colocalized within the mouse kidney as an aldosterone-sensitive tissue by immunohistochemical analysis (Fig. 13, A and B). As shown in Fig. 13, A and B, using specific  antibodies, we were able to demonstrate that MR interacted with Ubc9 as well as SRC-1 in the nuclei of cortical collecting duct cells of mouse kidney. The results from cell-based reporter assays, coimmunoprecipitation, ChIP, fluorescence imagings, and immunohistochemistry strongly support a physiological role of Ubc9 in MR transactivation in cooperation with SRC-1 in vivo.

DISCUSSION
In this study, we describe Ubc9, which interacts with the N-terminal transactivation domain of MR. Transient transfection assays together with small interfering RNA and RT-PCR indicated a physiological role of Ubc9 as a coactivator for the MRdependent transactivation. Furthermore, the coimmunoprecipitation and ChIP assays clearly showed that endogenous Ubc9, SRC-1, and MR form a ternary complex. Colocalization of these proteins was also observed in the nuclei of intact mouse tissues, indicating that our observation is relevant to physiological events.
MR is modified by SUMO-1 via three consecutive enzyme reactions, including E2 enzyme Ubc9, thus resulting in transcriptional repression based on the fact that KRmut MR, which is a sumoylationinactive mutant, disclosed enhanced aldosterone-mediated transactivation compared with wild type MR. Furthermore, desumoylation by overexpression of SENP1, a SUMO-1 isopeptidase, similarly potentiated MR-mediated transactivation (data not shown). These data indicate that sumoylation of MR leads to transcriptional repression. In fact, SUMO E3-ligase, PIAS1, was recently shown to function as a transcriptional corepressor of MR, the function of which is partly mediated by sumoylation of MR (38). The correlation between sumoylation and transcriptional repression is similarly shown in various nuclear receptors, such as glucocorticoid receptor (26 -30) and androgen receptor (21, 22, 31, 32, 34 -36, 58). Although sumoylation of MR results in transcriptional repression, this study shows a remarkable aspect of Ubc9 as a transcriptional coactivator for MR-mediated transactivation through formation of a complex with MR and SRC-1.
Ubc9 Functions as a Transcriptional Coactivator of MR-Ubc9 meets all the criteria for a transcriptional coactivator protein in the modulation of MR transcriptional properties. First, Ubc9 specifically interacted with MR in in vitro GST pulldown, yeast, and mammalian cells as shown in Figs. 1-3, and the interaction increased in the presence of aldosterone (Fig. 1). In addition, the interaction between the endogenous proteins was confirmed in 293-MR cells. All assays clearly showed that Ubc9 mainly interacted with the N-terminal MR-(1-670) containing AF-1 and DNA-binding domain but not with the C-terminal MR-(671-984) encompassing ligand-binding domain. Second, overexpression of Ubc9 had no effect on the reporter activities in the absence of transfected MR (Fig. 4A). However, Ubc9  potentiated the transactivation of three reporter constructs mediated by MR. Third, reduction of endogenous Ubc9 by small interfering RNA decreased MR-mediated transactivation of the reporter construct as well as an endogenous Sgk gene, indicating that endogenous Ubc9 normally contributes to MRmediated transactivation. Fourth, ChIP assays clearly showed that endogenous MR and Ubc9 were recruited to a native MRregulated ENaC promoter, demonstrating functional coupling between MR and Ubc9. Therefore, Ubc9 possesses all the characteristics expected for the transcriptional coactivator protein of MR.
To confirm further that Ubc9 is a coactivator of MR, we ruled out several possible ways in which the protein might enhance MR-mediated transactivation. First, as SUMO-1 conjugation plays an important role in protein modification, the effect of Ubc9 on MR transactivation might be the result of effects of Ubc9 on MR protein concentrations. Our preliminary experiments showed that overexpression of Ubc9 did not alter MR protein concentration in HEK293 cells (Fig. 3B). Second, it is also possible that overexpression of Ubc9 increases the concentrations of some coactivators or decreases the concentrations of some corepressors, which have been shown to interact with MR, but our preliminary results showed that overexpression of Ubc9 did not alter the protein concentration of SRC-1, GRIP1, or SMRT in the cells (data not shown). Because these experiments were performed by transient transfection, we are not able to conclude unequivocally that Ubc9 has no effects on the coregulator concentration, and further investigation is required. Third, another possibility is that overexpression of Ubc9 increases the DNA-binding affinity of MR. To exclude this possibility, we performed electrophoretic mobility shift assays to determine whether in vitro transcription-translated Ubc9 or extracts from cells overexpressing Ubc9 proteins affect the binding of MR to its response element DNA. The results showed that Ubc9 has no effect on MR binding to the response element (data not shown). Fourth, it has been proposed that SUMO-1 conjugation targets proteins to different cellular localizations. SF-1 can be directed into nuclear speckles and sequestered from the nucleolus in the presence of SUMO-1, thus resulting in transcriptional repression (43). It is therefore important to investigate whether and how MR activity and subcellular localization are functionally linked for further investigation. From these findings, Ubc9 clearly functions as a coactivator of MR in vivo.
Our data showed that both wild type and a sumoylationinactive mutant of Ubc9 (C93S) similarly enhanced a reporter activity mediated by MR, indicating that coactivation of MRmediated transcription by Ubc9 is likely to be independent of sumoylation activity. However, it is possible that ectopically produced Ubc9 regulate MR-mediated transactivation through  Mouse kidney was used to immunologically detect Ubc9, SRC-1, and MR. MR was colocalized with Ubc9 (A) as well as SRC-1 (B) in the nuclei of collecting duct cells but not glomerulus or mesangial cells. Arrows indicate representative nuclear colocalization of MR and Ubc9 or SRC-1. Panels of MR were incubated with mouse anti-MR antibody, followed by Alexa Fluor 488-conjugated anti-mouse IgG, whereas panels of SRC-1 and Ubc9 were incubated with rabbit anti-SRC-1 and anti-Ubc9, respectively, followed by Alexa Fluor 594-conjugated anti-rabbit IgG. Panels of MRϩUbc9 or MRϩSRC-1 were merged by each image. Nuclear staining was performed by 4Ј,6-diamino-2-phenylindole (DAPI). Scale bar, 10 m. not only sumoylation of MR but also conjugation of SUMO-1 to one or more other cellular factors involved in transcriptional regulation.
MR-Ubc9-SRC-1 Complexes Are Crucial for MR Transactivation-Although our data showed that Ubc9 interacted with the N-terminal but not with the ligand-binding domain of MR, Ubc9 functions as a strict ligand-dependent coactivator of MR. We therefore presumed that Ubc9 should be indirectly associated with the ligand-binding domain of MR through certain coactivator(s). We then found the combined effects of Ubc9 and SRC-1, but not GRIP-1, p/CIP, or CBP/p300 on MRdependent transactivation in transient transfection assays. Indeed, on aldosterone treatment, MR, Ubc9, and SRC-1 were recruited to an MR-responsive gene promoter in ChIP assays, indicating that these three proteins may form a complex. Because Ubc9 does not contain an autonomous activation domain when fused to the Gal4 DNA-binding domain, it seems likely that Ubc9 supports the transactivating effects of SRC-1 by acting as a scaffolding protein that stabilizes SRC-1 within the MR transcriptional complex or by stabilizing the SRC-1 protein level as shown recently (57). We showed that both Ubc9 and SRC-1 interacted with the N-terminal MR-(1-670) containing AF-1 domain. Ubc9 is also associated with SRC-1 (Fig. 10). A recent report (59) showed that SRC-1 is sumoylated, thus resulting in enhancing coactivation capacities for steroid receptors. Taken together with Fig. 10 and Ref. 59, it is likely that Ubc9 directly interacts with SRC-1. These results may provide a structural framework for the formation of a complex of these three proteins. Furthermore, fluorescence imagings and immunohistochemical analysis supported that MR was colocalized with Ubc9 and SRC-1 in the nuclei of cultured HEK293 cells and the collecting duct cells from mouse kidney, supporting the function of these complexes in vivo.
These results clearly showed that Ubc9 may have dual functions on MR. One function is sumoylation of MR through SUMO E2-conjugating enzyme activity, thus resulting in attenuation of ligand-dependent MR transactivation as shown previously (38). Another one is a new function as an MR coactivator by formation of a complex with SRC-1 and N-terminal MR beyond the SUMO E2-conjugating enzyme. Spatiotemporal regulation of these dual functions remains to be clarified.