OTU Domain-containing Ubiquitin Aldehyde-binding Protein 1 (OTUB1) Deubiquitinates Estrogen Receptor (ER) α and Affects ERα Transcriptional Activity*

Estrogen receptor (ER) α is an essential component in human physiology and is a key factor involved in the development of breast and endometrial cancers. ERα protein levels and transcriptional activity are tightly controlled by the ubiquitin proteasome system. Deubiquitinating enzymes, a class of proteases capable of removing ubiquitin from proteins, are increasingly being seen as key modulators of the ubiquitin proteasome system, regulating protein stability and other functions by countering the actions of ubiquitin ligases. Using mass spectrometry analysis of an ERα protein complex, we identified OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) as a novel ERα-interacting protein capable of deubiquitinating ERα in cells and in vitro. We show that OTUB1 negatively regulates transcription mediated by ERα in transient reporter gene assays and transcription mediated by endogenous ERα in Ishikawa endometrial cancer cells. We also show that OTUB1 regulates the availability and functional activity of ERα in Ishikawa cells by affecting the transcription of the ERα gene and by stabilizing the ERα protein in the chromatin.

In mammals the effects of the steroid hormone 17␤-estradiol (E2) 2 are mediated by estrogen receptor (ER) ␣ and estrogen receptor ␤. ER␣ is a member of the nuclear hormone receptor superfamily of transcription factors (1). In the cell ER␣ exists in either an inactive, unliganded form bound to the HSP90 chaperone or in an active liganded state in which ER␣ recruits coactivator protein complexes to gene promoters and initiates transcription of ER␣ target genes (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). The ubiquitin proteasome system plays crucial and multifaceted roles in the regulation of both receptor pools (7, 16 -24). The ubiquitin proteasome system is a multicomponent complex that regulates protein stability and degradation through the interplay of ubiquitin, ubiquitinating enzymes, deubiquitinating enzymes (DUBs), and 26 S proteasome (25)(26)(27). In the case of unliganded and inactive ER␣, the E3 ligase CHIP maintains appropriate steady state receptor levels and is responsible for the clearance of misfolded ER␣ (18,19). The effect that the ubiquitin proteasome system has on active, liganded ER␣ is complex because its activity is intimately tied to its stability. Our group and others have shown that inhibition of ER␣ degradation inhibits its function and that inhibition of ER␣ transcriptional activity stabilizes the receptor protein (21-23, 28 -30). Métivier et al. (31) indicated that the initial steps of transcription are linked to monoubiquitination of the liganded ER␣ and that monoubiquitination may enhance receptor interaction with DNA or coactivators. As transcription progresses, ER␣ and chromatin surrounding the promoter are sequentially modified by successive coactivator complexes (8,28,29,31) and degraded as a consequence of the recruitment of several ubiquitin E3 ligases: BRCA1/BARD1, MDM2, E6-AP, and EFP (16,17,(32)(33)(34)(35). In addition, Zhang et al. (24) showed that the LMP2 subunit of the 26 S proteasome is recruited to the gene by SRC coactivators and that its presence is necessary for ER␣-mediated transcription and cycling on the promoter of the estrogenresponsive pS2 gene.
DUBs are cysteine proteases (with the exception of JAMM family DUBs, which are metalloproteases) that catalyze the removal of ubiquitin (Ub) from Ub-modified proteins and for the processing of tandemly linked nascently translated Ub precursors (36 -41). Based on the structure of the active site and the mechanism of catalysis, DUBs are divided into five groups: UCHs, USPs, MJDs, OTUs, and JAMMs. Deconjugation of Ubprotein substrates is achieved either by removal of the entire Ub chain from the protein or by removal of individual or multiple ubiquitins from the chain in a process termed "editing." Different DUBs exhibit preferences for mono and poly-Ub chains or for K48 or K63-based Ub-Ub linkages (40). DUBs also specifically target a distinct and wide range of ubiquitinated proteins, displaying a diverse array of DUB-specific biological functions (37).
Three DUBs have been reported to interact with steroid hormone receptors. Two of these, 2A-DUB and USP22, are part of histone acetyltransferase complexes (pCAF complex and STAGA, respectively), and both enhance AR transcription by removing histone H2A monoubiquitination (41,42). USP10 has also been shown to coactivate AR-mediated transcription (43). However, in all these cases, the receptor itself has not been observed to be deubiquitinated.
In this study we have identified OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) as an ER␣-interacting DUB. OTUB1 is a deubiquitinating enzyme that has known in vitro deubiquitinating activity and a preference for K48-linked polyubiquitin chains (44 -46). However, no protein substrates that are deubiquitinated by OTUB1 have been identified in living cells. Here, we show that OTUB1 interacts with ER␣, deubiquitinates the receptor in cells and in vitro, and represses its transcriptional activity in transient reporter assays as part of its negative regulation of ER␣ activity. OTUB1 also regulates endogenous ER␣ both by regulating the transcription of ER␣ gene in Ishikawa cells and by stabilizing ER␣ protein in the chromatin.
siRNA Treatment, Transient Transfections, Immunoprecipitations, and Immunoblot Assays-All of the cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum or 10% charcoal-stripped serum for hormone treatment. For transient transfections, HeLa and HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen). For the transfection of Ishikawa cells, we used FuGENE 6 (Roche Applied Science). For siRNA knockdown, the cells were transfected for 4 days with 50 nM of siRNA using TransientTKO (Mirus). Luciferase assays were performed with substrate purchased from Promega and used according to the manufacturer's instructions. Immunoprecipitations and Western blots were performed as described before using anti-FLAG or protein A beads (Invitrogen) and antibodies F10 and D12 (Santa Cruz) for ER␣, anti-HA (Sigma), anti-FLAG (Affinity BioReagents), and rabbit and mouse control IgG (Santa Cruz). Antibody against human OTUB1 was custom produced and assayed by Genemed Synthesis, Inc. in rabbits using two synthetic peptides corresponding to the N-terminal variable domain of human OTUB1 (QQKIKDLHKKYSYIRKT and KQEPLGSDSEGVNC).
RNA Isolation and Real Time Quantitative PCR-Ishikawa cells total RNA was extracted using Aurum Total RNA Mini Kit (Bio-Rad). The PR, GREB1, OTUB1, ER␣ mRNA, and the cyclophilin mRNA (as an internal control) were quantitated as in Yi et al. (47) by TaqMan-based reverse transcriptase PCR using the AIB1 Prism 7700 sequence detection system (Applied Biosystems). TaqMan primer-probe sets for PR, GREB1, and OTUB1 were purchased from Applied Biosystems. The primers for the ER␣ mRNA are as follows: forward, 5Ј-GACAGG-GAGCTGGTTCACATG; reverse, 5Ј-GGAGGGTCAAATC-CACAAAGC; and probe, 5Ј-FAM-TGGCACCCTCTTCGC-CCA-TAMRA. The primers for the cyclophilin mRNA were described previously (47).
Ubiquitin-AFC Analysis of Immunoprecipitated ER␣ Complex-For the purpose of analyzing deubiquitinating activity associated with ER␣, ER␣ was immunoprecipitated from HEK293T cells transfected with pCR3.1-hER␣ or empty vector for 2 days or from MCF-7 cells. The cells were lysed in a lysis buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 5% glycerol, and 1 mM DTT) without protease inhibitors at 4°C. The lysates were sonicated at low power (10% duty cycle, output 2, using a Branson sonifier 250) and spun down, and the supernatants precleared with protein A beads for 30 min. ER␣ antibody (2.5 g of each F10 and D12) or normal mouse IgG (5 g) was then added to supernatants and incubated for 2 h. Protein A beads (Invitrogen) were added for 1 h. Protein A beads were spun down and washed twice with 0.2% TBS with Tween 20 detergent and twice with TBS. The beads were then resuspended in 100 l of ubiquitin-AFC reaction buffer (48). 50 mM HEPES, pH 8.0, 0.5 mM EDTA, 0.5 mM DTT, and 1 mg bovine serum albumin. Finally, ubiquitin-AFC (Boston Biochem) was added at a concentration of 0.5 M. A control reaction was pretreated for 5 min with 2 M of ubiquitin-aldehyde (Boston Biochem) at 37°C before the addition of ubiquitin-AFC. The reactions were incubated in the dark at room temperature with shaking for 30 min. Measurement of AFC release was performed by excitation at 405 nm and measured at an emission wavelength of 505 nm using a fluorescent plate reader. Statistical analysis was performed using Student's t test.
In Vitro Deubiquitination Assay-Recombinant ER␣ (300 ng/reaction) from Invitrogen was in vitro ubiquitinated using Boston Biochem ubiquitination kit (catalog number K-960) and FLAG-ubiquitin (catalog number U-120). The ubiquitination reaction was then diluted to 400 l with lysis buffer without protease inhibitors and ER␣ antibodies (2.5 g of each F10 and D12; Santa Cruz) were added for 2 h. Protein A beads were then added for 1 h and washed afterward twice with 0.2% TBS with Tween 20 detergent and twice with TBS. The beads were then resuspended in deubiquitination reaction buffer: 20 mM Tris-Cl, pH 8.0, 0.5 mM DTT, and 10% energy solution (Boston Biochem from K-960). Finally, 2 g of recombinant purified GST-OTUB1 (Abgent) was added. The reaction was incubated at room temperature for 4 h or overnight. Following incubation, 5ϫ SDS loading buffer was added to the reaction and analyzed by Western blotting using anti-FLAG and Anti-ER␣ antibodies and chemiluminscence.
Alkaline Phosphatase Assay-Alkaline phosphatase assays were performed using an alkaline phosphatase kit from AnaSpec (71230). Ishikawa cells were transfected with siOTUB1 or a control siRNA pool as described above. E2 (100 nM) or ethanol was added to the cells 2 days after transfection for 48 h. The cells were then harvested and processed according to the manufacturer's instructions.
Absorbance was measured at 405 nm. Absorbance values were then normalized with a protein concentration that was determined by Bradford analysis (Bio-Rad). Statistical analysis was performed using the Student's t test.
Nuclear and Chromatin Fractionation-Nuclear and chromatin fractionations were performed according to the protocol published by Wysocka et al. (49). In short, cells were washed in phosphate-buffered saline and resuspended in solution A (without Triton X-100). Triton X-100 0.1% (final) was then added to cells for 5 min on ice. The cells were spun down at 1300 ϫ g for 4 min. Precipitate (P1) was washed with solution A without Triton X-100 and lysed using solution B for 10 min on ice. Lysate was then spun down at 1700 ϫ g for 4 min, precipitate-washed (chromatin fraction) with solution B once, and then centrifuged at 10,000 ϫ g for 1 min. The remaining chromatin was then resuspended in SDS loading buffer, sonicated, and analyzed using SDS-PAGE. Solution A contained 10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 1 mM DTT, 10 mM NaF, 1 mM Na 2 VO 3 , and protease inhibitor mixture (Roche Applied Science). Solution B contained 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 10 mM NaF, 1 mM Na 2 VO 3 , and protease inhibitors (Roche Applied Science).
Mass Spectrometry Analysis of the ER␣ Protein Complex-HEK293T cells were grown in Dulbecco's modified Eagle's medium without phenol red supplemented with 10% charcoalstripped fetal calf serum. The cells were transfected with ER␣ for 48 h and treated with E2 (1 nM) where indicated for 2 h. The cells were lysed in radioimmune precipitation assay buffer, and ER␣ was immunoprecipitated using ER␣ antibodies (F10 and D12; Santa Cruz) for 2 h and protein A beads (Invitrogen). The lysates were then run on SDS-PAGE. The whole gel was cut in five parts, trypsinized, and prepared for electrospray ionization-liquid chromatography/liquid chromatography (ESI-LTQ) mass spectrometry. The peptides were identified using SeQuest software and the human RefSeq library.

RESULTS
Deubiquitinating Enzymes Are Part of ER␣ Complex-To determine whether DUBs are associated with ER␣ protein, complexes were immunoprecipitated from 293T cells transiently transfected with an ER␣ expression vector and incubated with a recombinant DUB substrate, ubiquitin-AFC. Fluorometry-detected release of the AFC moiety revealed that the ER␣ complex contains DUB activity (Fig. 1A). Next, we repeated this experiment using an endogenous ER␣ protein complex immunoprecipitated from MCF-7 cells, indicating that DUBs are associated with the endogenous receptor as well (Fig. 1B).
To identify the ER␣-interacting DUB(s), ER␣ immunoprecipitated from the 293T cells was analyzed by mass spectrometry. Mass spectrometric analysis revealed that ER␣ interacts in its unliganded state with OTUB1 (supplemental Fig. S1 and supplemental Table S1). Subsequent immunoprecipitations and reciprocal immunoprecipitation experiments of OTUB1 and ER␣ confirmed the mass spectrometry analysis, finding that OTUB1 binds ER␣ (Fig. 2, A  and B). Next, we investigated whether ER␣ interaction with OTUB1 is dependent on the presence of E2. Because the receptor interaction with OTUB1 did not change in the presence of E2 (Fig.  2C), we concluded that the ER␣-OTUB1 interaction is hormoneindependent. Additionally, we tested whether OTUB1 would bind progesterone receptor B (PR-B). Under the same experimental conditions no observable binding of OTUB1 to PR-B was detected (Fig. 2D).
OTUB1 Deubiquitinates ER␣ in HEK 293T Cells and in Vitro-Because OTUB1 is a DUB with confirmed in vitro deubiquitinating activity toward polyubiquitin chains (44 -46, 50), we investigated whether OTUB1 can deubiquitinate ER␣. To study this process we used wild type OTUB1 and two OTUB1 mutant molecules: a catalytically inactive OTUB1 C/S and an OTUB1 Ubmut that cannot bind ubiquitin (Fig.  3A). A catalytically inactive OTUB1 (OTUB1-C/S) expression vector was constructed by substituting active site cysteine 91 to serine (44) (Fig. 3A). Balakirev et al. (44) identified two conserved OTUB1 domains capable of binding ubiquitin: an UIM domain located immediately next to the active site and an UBA-like domain located toward the C terminus of the molecule. In the UIM domain we substituted conserved alanine, serine, and glutamate residues to glycine, alanine, and alanine, respectively. In the same molecule we deleted the other UBA-like domain to create an OTUB1 protein that cannot interact with ubiquitin (OTUB1 Ubmut) (Fig. 3A).
We examined ER␣ ubiquitination in HEK293T cells in the presence of overexpressed wild type OTUB1 or OTUB1 mutants. As shown in Fig. 3B (first two lanes) overexpression of OTUB1 substantially reduced slower migrating ubiquitinated ER␣ species. The expression of catalytically inactive OTUB1 (OTUB1-C/S) restored ER␣ ubiquitin levels (Fig. 3B, third lane), indicating that deubiquitination of ER␣ by OTUB1 is FIGURE 1. ER␣ is associated with DUBs. A, HEK293T cells were transfected with empty vector (no ER␣ control) or ER␣. ER␣ was immunoprecipitated form the cells and exposed to recombinant DUB substrate ubiquitin-AFC. Ubiquitin aldehyde (0.5 M) was used to demonstrate the specificity of deubiquitinating activity (ER␣ and DUB inhibitor). 5% of cellular lysate was used as a positive control for deubiquitinating activity. Release of the fluorescent AFC moiety was measured at an excitation level of 405 nm and an emission level of 505 nm. B, endogenous ER␣ complexes were immunoprecipitated with either nonspecific IgG (IgG) or with ER␣ antibody (ER␣) from MCF7 cells and exposed to ubiquitin-AFC as described previously. dependent on OTUB1 catalytic activity. The expression of the OTUB1 Ubmut that is incapable of ubiquitin binding further increased the amount of ubiquitinated ER␣ (Fig. 3B, fourth lane), suggesting that the OTUB1 Ubmut may act as a dominant negative with respect to receptor deubiquitination.
Interestingly, we observed that mutation of a single conserved residue in the catalytic site of OTUB1 significantly decreased the interaction of OTUB1 with ER␣ (Fig. 3B, compare second lane and third lane), indicating that the active site cysteine plays an essential role as a substrate-interacting surface. More surprisingly, we found that the ubiquitin-binding mutant OTUB1 Ubmut bound ER␣ substantially more strongly than the wild type OTUB1 (Fig. 3B, compare second and fourth  lane). This finding is significant because it shows that ubiquitin binding is not necessary for a deubiquitinating enzyme to recognize and bind its substrate. Analysis of the crystal structure of human OTUB1 (45) suggests that OTUB1 is catalytically inactive in an unliganded state (in the absence of K48-linked chains). We speculate that because the OTUB1 Ubmut cannot recognize ubiquitin chains, it remains auto-inhibited and unable to release ER␣.
To show that OTUB1 is capable of deubiquitinating ER␣ in vitro, we ubiquitinated recombinant ER␣ under cell-free conditions using an in vitro ubiquitin kit consisting of ubiquitinactivating, conjugating, and E3 ligase enzymes (Boston Biochem). This ubiquitinated ER␣ was then incubated with recombinant purified GST-OTUB1. As shown in Fig. 3C recombinant GST-OTUB1 is capable of deubiquitinating ER␣ in vitro.
OTUB1 Attenuates ER␣-mediated Transcription in HeLa and HEK 293T Cell Lines-To examine the potential role of OTUB1 in ER␣-mediated transcription, we performed ERE-LUC reporter assays in HeLa cells. Expression of OTUB1 significantly attenuated ER␣ transcriptional activity even in the presence of coactivator (Fig. 4, A and B). In addition, OTUB1 affected the transcriptional activity of ER␤ (Fig. 4C). However, OTUB1 did not have an effect on PR-B mediated transcription in the same assay (Fig. 4D), suggesting that OTUB1 transcriptional repression is specific for ER␣. Furthermore, OTUB1 failed to suppress the activity of the activation function 2 (AF2) domain of ER␣ fused to GAL4 DNA-binding domain (Fig. 4E). To examine the potential role of activation function 1 (AF1) in OTUB1 repression of ER␣, we evaluated the effect of OTUB1 expression on the ER␣179C construct lacking the AF1 region of the ER␣ molecule. Fig. 4F indicates that the absence of AF1 does not contribute substantially to the ability of OTUB1 to repress ER␣ and that this effect is most likely conveyed by the DNA-binding domain and hinge regions of ER␣. However, the AF1 may still play a role in OTUB1 repression, given that ER␤ is repressed less than ER␣, and it has low AF1 activity (13).
OTUB1 is a deubiquitinating enzyme, and its effect on protein targets is presumably mediated by its ability to bind and cleave ubiquitin chains. Therefore, we assessed the impact of OTUB1 C/S on the activity of ER␣ in HeLa cells. Surprisingly, the expression of the OTUB1 catalytic mutant (OTUB1 C/S) also suppressed ER␣ mediated transcription (5A). Because we were unable to express the OTUB1 ubiquitin-binding mutant (OTUB1 Ubmut) in HeLa cells, possibly because of its increased protein degradation, we evaluated the effect of OTUB1 and the OTUB1 Ubmut on transcription in 293T cells. As indicated in Fig. 5B, the expression of the OTUB1 Ubmut restored ER␣mediated transcription and even increased ER␣ estradiol-dependent coactivation. These results indicate that OTUB1 is capable of inhibiting ER␣-mediated transcription and that its inhibitory effect is dependent on ubiquitin binding.
Loss of Endogenous OTUB1 Increases Production of PR mRNA and Activity of Placental Alkaline Phosphatase-To evaluate the effect of the loss of OTUB1 on transcription mediated by endogenous ER␣, we knocked down OTUB1 in the endometrial Ishikawa cancer cell line using siRNA (Fig. 6A). Partial loss of OTUB1 protein in Ishikawa cells caused a significant increase in E2-dependent ER␣-mediated transcription of PR mRNA (Fig. 6B). This effect is specific to the PR gene because no observable effect was seen on transcription of the E2-responsive GREB1 gene (Fig. 6C). In addition to increasing PR mRNA, the loss of endogenous OTUB1 also increased placental alkaline phosphatase activity (Fig. 6D).
OTUB1 Mediates ER␣ Levels by Regulating the Transcription of ER␣ Gene-While examining the effects that OTUB1 has on the expression of endogenous ER␣ target genes in Ishikawa cells, we noticed that the loss of OTUB1 caused increased production of ER␣ mRNA (Fig. 7A). This observation prompted us to investigate the potential role of OTUB1 in the regulation of the ER␣ protein level. Consistent with our previous finding, we observed that knockdown of OTUB1 increased, and overexpression of OTUB1 decreased the level of endogenous ER␣ in the cells (Fig. 7, B and C). To confirm that the observed change in the ER␣ protein level is transcriptional rather than posttranslational, we performed knockdown of OTUB1 in the presence of the translation inhibitor cycloheximide. Knockdown of OTUB1 did not change the ER␣ protein level in the presence of cycloheximide, indicating that OTUB1 affects ER␣ at the transcriptional level (Fig. 7D). Similarly, knockdown of OTUB1 in the presence of MG132 resulted in an overall increase in ER␣ protein, further suggesting that this increase is likely caused by an increase in ER␣ mRNA (Fig. 7E).
Expression of OTUB1 in Ishikawa Cells Increases ER␣ Retention in the Insoluble Nuclear Fraction-To gain an insight into the mechanism by which OTUB1-mediated deubiquitination of ER␣ is affecting ER␣ biology and to differentiate between the effect of OTUB1 on ER␣ function and its protein stability, we investigated the cellular localization of OTUB1. OTUB1 contains a bona fide nuclear localization signal and resides in the nucleus of porcine kidney cells (44,46). However, the precise localization of OTUB1 within the nucleus has not been examined. We used a nuclear fractionation protocol to find out

. OTUB1 deubiquitinates ER␣ in cells and in vitro.
A, schematic representation of wild type OTUB1, catalytically inactive OTUB1 C/S, and OTUB1 that cannot bind ubiquitin (OTUB1 Ubmut). B, HEK293T cells were transfected with the indicated vectors or empty vector controls. FLAG-ER␣ was immunoprecipitated and precipitates were analyzed using antibodies against ER␣, HA tag, and OTUB1. The lower panels represent Western analysis of inputs. C, OTUB1 deubiquitinates ER␣ in vitro. Purified recombinant ER␣ protein was in vitro ubiquitinated with FLAG-ubiquitin and then exposed to purified recombinant GST-OTUB1 as described under "Experimental Procedures." Western blot analysis was done using antibodies against ER␣ and FLAG-ubiquitin.
whether OTUB1 is present in the chromatin (Fig. 8A). OTUB1 was determined to reside in the insoluble nuclear fraction. Interestingly, overexpression of OTUB1 caused an increase in unliganded ER␣ presence in the insoluble chromatin fraction (Fig. 8B, lanes 1 and 5). This increase is particularly intriguing given that overexpression of OTUB1 decreased the overall level of ER␣ (Fig. 7C). The increased presence of ER␣ in the chromatin can be due to ER␣ retention in the nuclear matrix and the reduction in ER␣ turnover (16,29). We evaluated the ER␣ presence in the chromatin fraction in the presence of the proteasome inhibitor MG132. As shown in the Fig. 8B (lanes 3 and 7), the addition of MG132 did not cause further stabilization of ER␣, indicating that OTUB1 protects chromatin-bound ER␣ from proteasome-mediated degradation and that overexpres-sion of OTUB1 abrogates the ability of MG132 to illicit any further increase in ER␣ association with chromatin. These data, together with the finding that OTUB1 acts through the ER␣ DNA-binding domain, are consistent with results by Reid et al. (29) showing that MG132 stabilization of ER␣ represses its activity and increases its residence in the nuclear matrix.

DISCUSSION
Using the recombinant DUB substrate ubiquitin-AFC, we show for the first time that ER␣ can be isolated in a complex with deubiquitinating enzymes (Fig. 1). Furthermore, utilizing mass spectrometry analysis, we identified OTUB1 as a novel ER␣-interacting protein capable of deubiquitinating ER␣ in cells and in vitro (supplemental Fig. S1 and Fig. 3). We also show that OTUB1 binding to ER␣ is dependent on an intact OTUB1 active site, because OTUB1 C/S has diminished binding affinity. On the other hand, binding of OTUB1 Ubmut to ER␣ is enhanced, indicating that OTUB1 likely recognizes ER␣ independent of the ubiquitination status of the receptor (Fig. 3). We also show that OTUB1 negatively affects ER␣-mediated transcription in transient reporter assays and from an endogenous ER␣ target gene (Figs. 4 -6). Finally, we show that OTUB1 regulates ER␣ levels in the Ishikawa endometrial cancer cell line containing endogenous ER␣, both at the level of the expression of ER␣ gene and by increasing the residence of the ER␣ protein in the chromatin (Figs. 7 and 8).
ER␣ Interacts with Deubiquitinating Enzymes-Because ER␣ is an ubiquitinated protein (2, 21-24), we hypothesized that ER␣ may also be regulated by deubiquitinating enzymes. Although nuclear receptors have been shown to recruit DUBs to regulate histone H2A and H2B ubiquitination as in the case of androgen receptor and 2A-DUB, USP22, and USP10 (41-43), DUBs have not been shown to target nuclear receptors themselves.
Our initial step in this study was to coimmunoprecipitate ER␣ complexes from ER␣ expressed 293T cells and incubate them with the recombinant DUB substrate ubiquitin-AFC. This novel approach that utilizes ubiquitin-AFC probe enabled us to detect a robust deubiquitinating activity associated with ER␣ complex, indicating that one or more DUBs are being recruited to ER␣ both in its unliganded and liganded state (data not shown). We further extended this by performing mass spectrometry analysis of ER␣ complexes, identifying OTUB1 as an ER␣-interacting DUB. Furthermore, we showed that both intact OTUB1 active site and ubiquitin recognition are important modulators of OTUB1-ER␣ interaction. The observation that OTUB1 C/S has decreased affinity for ER␣ is in accordance with findings by Edelmann et al. (45) that showed that the OTUB1 active site undergoes a conformational change upon binding to protein substrate. Point mutations in the active site may therefore interfere with this conformational change and decrease the binding of ubiquitin. Based on our findings with OTUB1 Ubmut, we conclude that presence of an ubiquitin chain is not necessary for OTUB1 to recognize and bind ER␣, but that it may be necessary for OTUB1 to release ER␣ after deubiquitination. In light of this interpretation and the fact that OTUB1 binds ER␣ in both the unliganded and liganded states, it is tempting to speculate that OTUB1 resides on ER␣ prior to ubiquitination and that it leaves ER␣ after deubiquitination together with the cleaved polyubiquitin chain.
OTUB1 Deubiquitinates ER␣-OTUB1 is a DUB with an in vitro affinity for K48-linked (and to the lesser extent to K63linked) polyubiquitin chains (44 -46, 50). Although OTUB1 deubiquitinating activity has been confirmed in vitro, no protein substrates have been identified to undergo deubiquitination by OTUB1. Soares et al. (50) implicated OTUB1 involvement in T cell anergy where OTUB1 binds and destabilizes the E3 ligase GRAIL through a mechanism that does not directly involve the DUB activity of OTUB1. In addition, in the proteomic study by Edelmann et al. (45), two more OTUB1 interactors have been identified: FUS/TLS and Rack1, both of which are involved in RNA splicing. However, the significance of these interactions and whether FUS/TLS and Rack1 are deubiquitinated by OTUB1 remains unknown at this time. In this study, we identify ER␣ as a protein substrate that is deubiquitinated by OTUB1 in vitro and in vivo. We show that the wild type OTUB1, but neither its catalytic (OTUB1 C/S) nor ubiquitin binding (OTUB1 Ubmut) mutant, is capable of efficiently removing polyubiquitin aggregates from ER␣ (Fig. 3).
OTUB1 Negatively Regulates ER␣-mediated Transcription-Our data indicate that OTUB1 has a negative effect on ER␣-mediated transcription in transient reporter assays. We also show that the OTUB1 effect on transcription is specific for ER␣ because OTUB1 does not repress PR-B-or glucocorticoid receptor-mediated ( Fig. 4 and data not shown) transcription. We also show that OTUB1 repression is not mediated by the AF2 domain of ER␣ because GAL4-AF2 (encompassing amino acids 302-595) fails to be repressed. In the subsequent experiment using ER␣179C that lacks the AF1 domain, we determined that the majority of OTUB1 repression is mediated by the ER␣ DNA-binding domain and possibly by the ER␣ hinge region (Fig. 4).
Although the OTUB1 C/S catalytic mutant does not reverse the inhibitory effect on transcription, we speculate that it is still able to inhibit ER␣-mediated transcription because of its continued ability to interact with the receptor and interfere with the dynamic exchange of ubiquitin required for efficient transcription to ensue. To this end, Balakirev et al. (44) and others (45,46,50) have shown that expression of OTUB1 slightly reduces the cellular ubiquitin pool and that expression of OTUB1 C/S does not completely reverse this phenotype (data not shown). On the other hand, expression of an OTUB1 Ubmut that cannot bind ubiquitin completely restores ER␣ transcription, a result that is in concordance with its effect on ER␣ ubiquitination. While performing these experiments we noticed that transient expression of both OTUB1 and OTUB1 C/S causes a spindle like morphology in HeLa but not in HEK293T cells and Ishikawa cells. This occurrence in HeLa cells was separate from our observation that OTUB1 represses ER␣ because we were able to detect this repression in all of the cell lines tested. Finally, transient reported data are consistent with our finding that the loss of OTUB1 in Ishikawa cells containing endogenous ER␣ up-regulates ER␣-mediated transcription of PR gene promoter and increases the activity of estrogen-dependent placental alkaline phosphatase.
OTUB1 Regulates ER␣ Stability Both at the Transcriptional and Post-translational Level-Using overexpression and knockdown of OTUB1 in Ishikawa cells, we show that OTUB1 regulates transcription of ER␣ gene (Fig. 7). We find that expression of OTUB1 decreases, and knockdown increases overall levels of ER␣ in Ishikawa cells, further implicating OTUB1 in regulating the biological response to estrogens. Although we hypothesize that OTUB1 is directly affecting the transcription of the ER␣ gene in Ishikawa cells, we cannot exclude the possibility that OTUB1 may have an effect on ER␣ mRNA stability. Regardless, this coordinated event FIGURE 7. OTUB1 changes ER␣ protein levels by affecting expression of ER␣ mRNA. A, loss of OTUB1 in Ishikawa cells increases levels of ER␣ mRNA. Cells were treated with siOTUB1 or siRNA control for 4 days and then collected for analysis by Q-PCR. B, cells were transfected with siOTUB1 or siRNA control for 4 days and treated with E2 or ethanol (EtOH) for 2 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. The protein bands were quantified by Image J and normalized to actin. C, Ishikawa cells were transfected with HA-OTUB1 or empty vector and treated for 2 h with E2 or ethanol vehicle. The lysates were analyzed as in B. D, Ishikawa cells were transfected with siOTUB1 or siRNA control for 4 days. Cycloheximide (15 M) was then added for indicated time points. The lysates were analyzed as in B. E, Ishikawa cells were treated with siOTUB1 or siRNA as described previously. The cells were then treated with 4 M MG132 for 4 h, and EtOH or E2 for 2 h as indicated. The cell lysates were analyzed as described in B. FIGURE 8. OTUB1 stabilizes ER␣ in the chromatin. A, OTUB1 is located in the insoluble chromatin fraction in Ishikawa cells. The nuclear fraction was isolated from untreated Ishikawa cells and subfractioned to insoluble chromatin fraction and a soluble nuclear fraction as described under "Experimental Procedures." The fractions were analyzed by Western blot with indicated antibodies. B, expression of OTUB1 in Ishikawa cells causes retention of ER␣ in the chromatin. Ishikawa cells were transfected with pCMV6 or HA-OTUB1 and treated as indicated with MG132 (4 M) for 4 h and with estradiol (E2) or ethanol (EtOH) for 2 h. The chromatin fractions were isolated as in A and analyzed using indicated antibodies. The protein bands were quantified using Image J and normalized to histone. IB, immunoblot. on both the transcriptional and post-translational level specifically down-regulates estrogen action because it does not impact PR-B activity. Furthermore, utilizing a chromatin fractionation protocol, we show that OTUB1 resides in the chromatin and that its overexpression increases ER␣ stability and residency in the chromatin. In light of our result that OTUB1 may interfere with ER␣ DNA binding, we conclude that OTUB1 stabilizes ER␣ and causes its immobilization to the nuclear matrix. This is consistent with the effect of MG132 on ER␣ stabilization, activity, and localization to the nuclear matrix (29).
Overall, our data indicate that OTUB1 affects ER␣ both at the level of expression of the ER␣ gene and at the level of protein stability in the chromatin. Our group and others (2, 7, 21-24, 28, 29, 31) have shown that ER␣ protein turnover is necessary for the ER␣ function and that stabilization of ER␣ protein abrogates its transcriptional activity. Here we find that OTUB1 stabilizes ER␣ protein in the insoluble nuclear fraction, thereby reducing ER␣ transcriptional activity. This negative effect that OTUB1 has on ER␣ functional output is further reinforced by OTUB1-mediated reduction in transcription of the ER␣ gene itself and consequently, the reduction in availability of the overall ER␣ pool in the cell. Because ER␣ is known to regulate its own promoter (51), it is possible that OTUB1 reduces ER␣ mRNA levels by inhibiting ER␣ transcription of its own promoter, thereby creating an inhibitory feedback loop. In conclusion, OTUB1 has evolved as a specific negative regulator of ER␣-mediated transcription by decreasing the overall amount of ER␣ present in the cell and by preventing ER␣ turnover in the chromatin.
ER␣ transcriptional activity is regulated by numerous post-translational modifications including phosphorylation, SUMOylation, glycosylation, methylation, acetylation, and ubiquitination. For most of these modes of post-translational modification of ER␣, processes that reverse these modifications also exist. Here, we see that receptor deubiquitination also exists and plays a critical role in ER␣ function as a transcription factor.