Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation*

Background: Progesterone regulates the cyclic function of the human endometrium through its receptors and coregulatory proteins. Results: Primate-specific melanoma antigen-A11 (MAGE-11) interacts with the human progesterone receptor-B (PR-B) unique NH2-terminal region to coregulate progesterone-dependent gene activation. Conclusion: MAGE-11 is an isoform-specific coregulator of human PR-B. Significance: PR-B and progesterone regulation of human endometrium requires a primate-specific steroid receptor coregulator. Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH2-terminal 110LLXXVLXXLL119 motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.

Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoformspecific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH 2 -terminal 110 LLXXVLXXLL 119 motif that interacts with the MAGE-11 F-box region in a phosphorylation-and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.
Estrogen and progesterone are essential steroid hormones that prepare the human uterine endometrium for embryo implantation and pregnancy (1). Estrogen promotes cell replication in the proliferative endometrium early in the menstrual cycle, whereas progesterone is anti-proliferative. The cumulative effect of progesterone over the early and mid-secretory phases results in a mid-secretory state of receptivity for embryo implantation. The decrease in progesterone near the end of the late secretory phase in the absence of pregnancy causes endometrial shedding and menstruation. Progesterone acts on the late secretory endometrium to prevent shedding and pregnancy loss in a conception cycle. Progesterone also regulates the uterine myometrium during pregnancy by maintaining normal gestational myometrial quiescence (2,3). Alterations in critical actions of progesterone result in infertility (2)(3)(4).
Actions of progesterone are mediated through classical genomic and nongenomic pathways. Genomic regulation involves progesterone receptor (PR) 2 -A and -B (5,6), two identical isoforms except that PR-B has 164 extra NH 2 -terminal amino acid residues. PR-C is a more recently reported third isoform that lacks the NH 2terminal and DNA binding regions and may inhibit PR-B action; however, the existence of PR-C in vivo has been questioned (7,8). Although PR-A and -B interact with many coregulators, none has yet explained the greater transcriptional activity of PR-B relative to PR-A that derives from activation function 3 in the unique PR-B NH 2 -terminal region (4, 9 -14). Rapid nongenomic actions of progesterone may require PR-B in addition to G-protein-coupled receptors (15,16).
Unlike other mammals, endometrium of human and some nonhuman primates (Old World monkeys and great apes) undergoes spontaneous cyclic decidualization in response to progesterone and menstruation in response to progesterone * The work was supported in whole or part by National Institutes of Health (NIH) Grant HD16910 (a United States Public Health Service grant from NICHD); NIH Eunice Kennedy Shriver NICHD Grant HD067721; Cooperative Agreement U54-HD35041 from the Eunice Kennedy Shriver NICHD as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research; and NCI, NIH, Center Grant P01-CA77739. 1  withdrawal (17,18). A predominance of PR-A function in the mouse uterus was shown by normal mouse uterine morphology in PR-B knock-out mice (9,19). Mouse uterus expresses predominantly one PR isoform, whereas human uterine cells can express PR-A and -B at similar levels. A notable exception is the predominant expression of PR-B in the mid-secretory human glandular epithelium (20 -22). A recent study suggests that PR-B may be an important mediator of human fertility in the mid-secretory phase (23). Differences in isoform-specific PR functions between primates and rodents suggest evolutionary divergence among PR coregulatory proteins. MAGE-11 (melanoma antigen-A11) is a primate-specific coregulator involved in steroid hormone receptor function. MAGE-11 was first identified as a cancer-testis antigen and human androgen receptor (AR) coregulator. MAGE-11 increases AR transcriptional activity by interacting with the human AR NH 2 -terminal FXXLF motif, the same motif that mediates the androgen-dependent AR NH 2 -and COOH-terminal interaction important for AR transactivation (24 -26). Transcriptional enhancing effects of MAGE-11 involve interactions with p160 coactivators and p300 histone acetyltransferase (27,28). Consistent with its cancer-testis antigen classification, MAGE-11 levels increase during prostate cancer progression to the castration-resistant phenotype, where it amplifies AR signaling and promotes tumor growth in an environment of low circulating androgen (29,30). MAGE-11 is also expressed at very low levels in normal tissues of the male and female human reproductive tracts. Most notable is the menstrual cycle-dependent expression of MAGE-11 in the early and mid-secretory normal human endometrium (31).
Although androgens are known to influence human endometrial function (32)(33)(34), the regulated expression of MAGE-11 in the early and mid-secretory human endometrium during the menstrual cycle suggests that MAGE-11 may influence the activity of PR. It is noteworthy that progesterone action in human endometrium is closely linked to cyclic AMP (34), a second messenger signaling molecule that acutely stimulates the expression of MAGE-11 (31). Additionally, the PR-B NH 2 -terminal region absent in PR-A interacts with the PR ligand-binding domain in a hormone-dependent NH 2 -and COOH-terminal interaction similar to AR (24,35,36). The primate-specific expression of MAGE-11, the up-regulation of MAGE-11 in the secretory human endometrium, the close evolutionary relationship between AR and PR, and the importance of AR and PR-B NH 2 -terminal domains in transactivation all suggest that MAGE-11 could be an important coregulator of human PR-B.
In this report, we show that MAGE-11 interacts with the unique NH 2 -terminal region of human PR-B and is responsible for isoform-specific PR-B up-regulation of FKBP5 but not the RASD1 gene that is primarily regulated by the PR-A/B heterodimer. The unique PR-B NH 2 -teminal 110 LLXXVLXXLL 119 motif region interacts with the F-box region of MAGE-11 and is required for the transcriptional effects of MAGE-11 and p300. The studies suggest that synergy between MAGE-11 and p300 explains the greater transcriptional activity and gene specificity of PR-B relative to PR-A in humans.

EXPERIMENTAL PROCEDURES
Human Endometrial Tissues and Cell Lines-Endometrial tissue was obtained with informed consent and approval by the institutional review board of the University of North Carolina (Chapel Hill, NC). Tissue was from the proliferative, early, middle, and late secretory phases of 18 -35-year-old women with normal 25-35-day intermenstrual intervals. Subjects were excluded who used hormone contraception or medications that affect reproductive hormone levels. Menstrual cycle phase was assigned as described (31).
Quantitative Real-time RT-PCR-Ishikawa cells (1.2 ϫ 10 6 cells/6-cm dish) were transferred to medium containing charcoal-stripped serum the day after plating. After 2 days, cells were treated with progesterone or estradiol, and total RNA was harvested in 1 ml of TRIzol reagent (Invitrogen)/6-cm dish. RNA was extracted from endometrial tissues using TRIzol and analyzed by RT-PCR (31).
For lentivirus short hairpin RNA (shRNA) knockdown of MAGE-11, Ishikawa cells (8 ϫ 10 5 /well in 6-well plates) were cultured for 24 h in 2 ml of serum-containing medium and incubated without virus or with 125 l of HEK293 cell medium containing ϳ10 6 lentivirus particles/ml. Lentivirus expressing MAGE-11 shRNA-827, -947, and -964, empty vector, and 18-bp spacer nonspecific shRNA were prepared from the Open Biosystems TRC1 shRNA library using standard protocols. After a 48-h virus incubation at 37°C, cells from each well were passaged into four 6-cm dishes in the presence (shRNA) and absence (no virus) of 3 g/ml puromycin dihydrochloride (Cellgro) for selection. After 4 days of culture, cells were transferred to charcoal-stripped serum-containing medium and treated the next day with and without progesterone.
For quantitative RT-PCR of RNA, first strand complementary DNA was prepared using SuperScript II reverse transcriptase (Invitrogen). Real-time RT-PCR of FKBP5 and RASD1 was performed using an Eppendorf Realplex4 Mastercycler in 20-l reactions containing 4 l of cDNA (20 ng), 10 l of SYBR Green Master Mix (Qiagen) in the QuantiTect SYBR Green PCR kit (Qiagen), 2 l of 2 M FKBP5 forward primer (5Ј-AAAGGC-CAAGGAGCACAAC-3Ј) and 2 M FKBP5 reverse primer (5Ј-TTGAGGAGGGGCCGAGTTC-3Ј) or 2 l of 2 M RASD1 forward primer (5Ј-ACTCCTTCGAGGAGGTGCAGCGG-3Ј) and 2 M RASD1 reverse primer (5Ј-TCGCGGTAGA-AGTCGCGGTCAC-3Ј), and 2 l of RNase-free water. PCR was performed at 94°C for 20 min for one cycle followed by 50 cycles of 94°C for 40 s, 57°C for 40 s, and 72°C for 40 s. Standard curves were generated by amplifying 10-fold serial dilutions of cDNA. mRNA levels were calculated based on standard curves. Ct values were normalized to peptidylprolyl isomerase A mRNA using forward primer 5Ј-ATCTTGTCCATGGCAAA-TGC-3Ј and reverse primer 5Ј-GCCTCCACAATATTCATGCC-3Ј. DNA primers were from Integrated DNA Technologies.
Chromatin Immunoprecipitation (ChIP)-ChIP assays were performed as described (47) with some modifications. Ishikawa cells (5 ϫ 10 6 cells/10-cm dish, 2 dishes/group) plated in medium containing 10% fetal bovine serum were grown for 3 days to ϳ80% confluence and transferred to 6 ml of medium containing 10% charcoal-stripped fetal bovine serum (Atlanta Biologicals, Inc.). The next day, cells were treated with and without 50 nM R5020 and 0.1 g/ml EGF from 3 to 24 h and fixed using 1% formaldehyde by rocking for 10 min at room temperature. Reactions were stopped with 0.125 M glycine for 5 min at room temperature. Cells were washed, harvested, and washed twice with cold phosphate-buffered saline by centrifugation. Cell pellets stored at Ϫ80°C were lysed for 20 min at 4°C in 350 l of ChIP lysis buffer containing 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.15 M NaCl, 50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 50 mM NaF, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and Roche Applied Science protease inhibitor mixture. Samples were sonicated twice on ice for 20 s total (0.5 s on, 2 s off) at 18% amplitude with 1-min intervals using a Branson Digital model 450 sonifier. An equal volume (350 l) of ChIP lysis buffer was added to supernatants after centrifugation. Samples were precleared for 30 min at 4°C with rotation by adding 60 l of a 50% suspension of equal parts Protein A-agarose (Gold Biotechnology) and Protein G-PLUSagarose beads (Santa Cruz Biotechnology, Inc.) in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE buffer) containing 0.2 mg/ml sonicated salmon sperm DNA. After centrifugation for 5 min at 6000 rpm, supernatants were transferred to new tubes and incubated for 3 h at 4°C with rotation with 10 g of mouse immunoglobulin G (Santa Cruz Biotechnology, Inc.) or 10 g of mouse monoclonal PR antibody 1294 provided by Dean P. Edwards (Baylor College of Medicine). Protein A/G beads (30 l) in a 50% suspension in TE buffer containing 0.2 mg/ml sonicated salmon sperm DNA were added and incubated over-night at 4°C with rotation. Samples were sequentially washed for 5 min with 1 ml of 50 mM HEPES, pH 7.8, 0.14 M NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS; 1 ml of 50 mM HEPES, pH 7.8, 0.5 M NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS; and 1 ml of 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.25 M LiCl, 0.5% Triton X-100, 0.5% sodium deoxycholate and twice with TE buffer. Beads were eluted twice with 75 l of TE buffer containing 1% SDS for 10 min at 65°C. Cross-links were reversed using 0.23 M NaCl overnight at 65°C. DNA was isolated in 30 l of elution buffer with the QIAquick gel extraction kit 250 (Qiagen). Quantitative PCR was performed in duplicate using 4 l of sample, 10 l of SYBR Green 1 (Qiagen or Bio-Rad SsoAdvanced SYBR Green Supermix), 2 l of 10 M forward and reverse primers, and 2 l of water. FKBP5 fifth intron first progesterone response element sequence GGTACACACT-GTTCT was amplified using forward primer 5Ј-TGCTGGAA-AGGAGAGGAA-3Ј and reverse primer 5Ј-CCCTGATGATT-GCTGCA-3Ј. FKBP5 fifth intron second progesterone response element sequence AGAACAGGGTGTTCT was amplified using forward primer 5Ј-GGTTTAGGGGTTCTTGCA-3Ј and reverse primer 5Ј-CATCAAGCGAGCTGCAA-3Ј. Amplification of a control FKBP5 intron 5 region that lacks a progesterone response region (PRE) used forward primer TGAGAC-CAGTCTGGCCAA and reverse primer ACCTCCCAGGAT-CAAGCA to amplify a fragment located at intron 5 positions 5660 -5796. The RASD1 progesterone response sequence GGAACAATGTGTACC in the second intron was amplified using forward primer 5Ј-GCAGGCTTAATTCGTCCA-3Ј and reverse primer 5Ј-TCCTGGAGGCTGCAGA-3Ј. DNA in 20-l reactions was amplified by PCR for 94°C for 20 min followed by 50 cycles at 94°C for 40 s, 57°C for 40 s, and 72°C for 40 s and then 94°C for 15 s, 60°C for 15 s, 94°C for 15 s, and 10°C.
Tissue sections immunostained for FKBP5 and MAGE-11 using the Vectastain Elite kit were blocked for 20 min with 1.5% normal goat serum, incubated with primary antibody overnight at 4°C in a humidified chamber, blocked again for 10 min with 1.5% goat serum, and incubated at room temperature with biotinylated anti-mouse (FKBP5) or anti-rabbit secondary anti-body (MAGE-11) (Vector Laboratories) (1:200 dilution) for 30 min. Sections were incubated for 30 min at room temperature with avidin DH-biotinylated horseradish peroxidase H complex (1:50 dilution) followed by a 10-min incubation with the DAB (3,3Ј-diaminobenzidine) Peroxidase Substrate Kit SK-4100 (Vector Laboratories).
PR was immunostained using the Vectastain ABC Standard kit after antigen retrieval using 0.01 M sodium citrate and 0.01 M citric acid, pH 6.0, for 15 min at the high setting in a microwave (49). p300 was immunostained using the Vectastain ABC Standard kit without treatment with citrate buffer. Sections were blocked for 10 min with 2% normal goat serum, incubated with primary antibody overnight at 4°C in a humidified chamber, blocked again for 10 min with 2% goat serum, and incubated for 1 h at room temperature with biotinylated anti-rabbit secondary antibody (1:200 dilution) (Vector Laboratories).
Sections were incubated for 1 h at room temperature with avidin DH-biotinylated horseradish peroxidase H complex (1:400) followed by a 10-min incubation with the DAB Peroxidase Substrate Kit SK-4100 (Vector Laboratories). Sections were counterstained with 0.05% toluidine blue in 30% ethanol and photographed using a SPOT-4 Insight FireWire Digital Camera (Diagnostic Instruments) as described (31).

MAGE-11 and FKBP5 Increase in Early and Mid-secretory
Endometrium-Human endometrium undergoes cyclic changes in response to progesterone in preparation for embryo implantation and pregnancy (1). To investigate a function for MAGE-11 in progesterone-dependent gene regulation, the menstrual cycle-dependent expression of MAGE-11 was compared with progesterone-regulated immunophilin FKBP5 (known also as FKBP51) (31,50). Endometrial expression of MAGE-11 during the menstrual cycle was also compared with PR and p300, a transcriptional regulator that interacts with MAGE-11. RNA levels were assayed using quantitative real-time RT-PCR, and protein was assessed by immunohistochemistry.
FKBP5 and MAGE-11 mRNA was low in the proliferative phase endometrium between menstrual cycle days 5 and 10 but increased up to ϳ30-fold in the mid-secretory phase (Fig. 1, A  and B). In contrast, PR mRNA was higher in the proliferative phase (Fig. 1C), whereas p300 mRNA was variable and independent of the menstrual cycle (Fig. 1D). RASD1 is a member of the Ras family of G-proteins. RASD1 mRNA is also up-regulated by progesterone (see below), but like p300, RASD1 mRNA was independent of the menstrual cycle (data not shown).
Immunostaining serial sections of human endometrium through the menstrual cycle showed low MAGE-11 and FKBP5 in the proliferative phase that increased in the early and midsecretory endometrium (Fig. 2). MAGE-11 localized in nuclei of glandular epithelial and stromal cells, whereas FKBP5 was in cytoplasmic granules in the mid-secretory phase. However, MAGE-11 and FKBP5 colocalized in glandular epithelial cell nuclei at LHϩ12, cycle day 24 of the menstrual cycle (Fig. 2, M  and N). p300 was evident in nuclei throughout the menstrual cycle. PR was prominent in stromal and epithelial cell nuclei of the early and mid-secretory phase endometrium. The intensity of PR staining declined in glandular epithelial cells in the late secretory phase (Fig. 2P).
Isoform-specific PR Up-regulation of FKBP5 and RASD1-A role for MAGE-11 in isoform-specific PR gene regulation was investigated in human endometrial Ishikawa cells that stably express similar levels of PR-A, PR-B, and both PR-A and -B (Fig.  3A) (38). Progesterone increased endogenous FKBP5 mRNA in IKPRB and IKPRAB cells but had no effect in parental IKLV or IKPRA cells (Fig. 3B). In contrast, progesterone strongly increased endogenous RASD1 mRNA in IKPRAB cells, with a
Time and dose-response studies showed an increase in FKBP5 mRNA with 0.1 nM progesterone in IKPRB cells (Fig.  4A) that was greater than IKPRAB cells (Fig. 4E). The highest levels of FKBP5 mRNA were between 6 and 12 h of progesterone treatment in both cell lines (Fig. 4, B and F). Progesterone-dependent PR-B up-regulation of FKPB5 protein was evident in IKPRB cells 8 and 24 h after treatment with R5020, a synthetic progestin (Fig. 5A, top). Induction of endogenous RASD1 mRNA required higher concentrations of progesterone in both cell lines (Fig. 4, C and G), with maximal levels seen at 12 h (Fig. 4, D and H).
Requirement for MAGE-11 in PR-B Up-regulation of FKBP5-MAGE-11 is a low abundance transcriptional regulator in nor-mal human tissues. Accordingly, MAGE-11 protein was weakly detected in IKPRB cells compared with LAPC4 prostate cancer cells (Fig. 5A, bottom) (29). PR-B was detected in IKPRB cells but not LAPC-4 cells.
The results suggest that MAGE-11 is an isoform-and genespecific coregulator of PR-B. MAGE-11 was required for progesterone up-regulation of FKBP5 by PR-B but not the PR-A/B heterodimer and was inhibitory to PR-B up-regulation of RASD1, which was regulated primarily by the PR-A/B heterodimer.
Recruitment of PR to Progesterone Response Elements-Intron 5 of the FKBP5 gene contains two previously reported progesterone response regions, PRE-1 and PRE-2 (Fig. 6A) (50 -52). Use of mouse monoclonal antibody 1294 that recognizes human PR in ChIP assays (53) demonstrated recruitment of PR in a time-and progestin-dependent manner to FKBP5 PRE-1 and PRE-2 in IKPRB cells (Fig. 6, B and C). Maximal recruitment of PR was 3 and 12 h after treatment with R5020.
In contrast to strong recruitment of PR to FKBP5 PRE-1 in IKPRB cells, PR was not recruited to the FKBP5 PRE-1 enhancer in IKPRA cells (Fig. 6D). No PCR amplification was observed using FKBP5 intron 5 primers for a region not expected to contain a PRE. However, PR was recruited to the  RASD1 intron 2 PRE region (Fig. 7A) in IKPRB (Fig. 7B) and IKPRAB cells (Fig. 7C).
The ChIP assay results provide further evidence for isoformspecific PR-B activation of the FKBP5 gene. However, the low levels of MAGE-11 in IKPRB cells were prohibitive for detection by ChIP.
Coregulatory Effects of MAGE-11, p300, and p160 Coactivators-A requirement for MAGE-11 in isoform-specific PR-B up-regulation of endogenous FKBP5 was investigated further in interaction and reporter gene assays. PR-B coimmunoprecipitated with FLAG-MAGE in a progesterone-dependent manner to a greater extent than PR-A (Fig. 8A). The weak progesterone-independent association between FLAG-MAGE and PR-A probably results from MAGE-11 association with other factors in the complex.
The ability of MAGE-11 to increase PR-B transactivation of FKBP5 was shown by expressing similar levels of PR-B or PR-A ( Fig. 8B) with pIE2-Luc, a luciferase reporter gene that contains the intron 5 PRE-2 region of the FKBP5 gene. PR-B was more effective than PR-A in progesterone-dependent transactivation of pIE2-Luc (Fig. 8C) over a dose-response range between 0.01 and 10 nM progesterone (Fig. 8D).
Activities of PR-B and -A were compared in the presence of MAGE-11 and TIF2, a p160 coactivator that interacts with the PR ligand-binding domain through its LXXLL motifs. PR-A and -B transactivation of pIE2-Luc was similar with the coexpression of TIF2 with and without MAGE-11 (Fig. 8E). In contrast, PR-B was more active than PR-A in FKBP5 activation when expressed with p300 with or without MAGE-11 (Fig. 8F).
The results provide support for the isoform-specific PR-B coregulator activity of MAGE-11. Transcriptional equivalence between PR-A and -B with increased p160 coactivator expression probably results from a direct interaction between MAGE-11 and TIF2 (27). Isoform-specific transcriptional superiority of PR-B relative to PR-A depended on synergistic effects of MAGE-11 and p300.
Synergy between MAGE-11 and p300-The greater transcriptional activity of PR-B relative to PR-A with MAGE-11 and p300 suggested a role for PR-B NH 2 -terminal 164 amino acid residues not present in PR-A. This was demonstrated by the transcriptional enhancing effects of MAGE-11 and p300 on GAL-PR-B- (1-164), a GAL4 DNA-binding domain fusion with the unique PR-B NH 2 -terminal region. Increasing amounts of MAGE-11 (Fig. 9A) or p300 (Fig. 9B) increased GAL-PR-B-(1-164) activity. Dependence on MAGE-11 for the transcriptional effects of p300 was demonstrated using MAGE-11 siRNA. MAGE-11 siRNA-2 that inhibits MAGE-11 expression (27,28) inhibited the transcriptional effects of p300 on GAL-PR-B-(1-164) (Fig. 9C). Specificity of inhibition was suggested by lack of  . PR recruitment to progesterone response region of human RASD1 gene. A, the structure of the human RASD1 gene contains two coding exons. Shown is the initiating ATG in exon 1, TGA stop codon in exon 2, and PRE sequence in intron 2. B, IKPRB cells were incubated with and without 50 nM R5020 and 0.1 g/ml EGF from 3 to 24 h. DNA was cross-linked and extracted for ChIP as described under "Experimental Procedures." Protein-DNA complexes were immunoprecipitated using 10 g of mouse monoclonal PR antibody 1294 and assayed for RASD1 PRE as described under "Experimental Procedures." C, IKPRAB cells were incubated for 2 h with and without 50 nM R5020. DNA was cross-linked and extracted for ChIP, and protein-DNA complexes were immunoprecipitated using 5 g of control mouse monoclonal IgG or 5 g of mouse monoclonal PR antibody 1294. Error bars, S.E. inhibition by nonspecific siRNA or MAGE-11 siRNA-3, neither of which decrease MAGE-11 expression (27,28).
To determine whether the unique PR-B NH 2 -terminal region is sufficient for the synergistic effects of MAGE-11 and p300, GAL4 DNA-binding domain fusion proteins with different regions of the PR-B NH 2 -terminal domain were expressed. The absence of cooperativity between MAGE-11 and p300 with GAL-PR-B-(1-164) (Fig. 9D) suggests that the unique PR-B NH 2 -terminal region, although activated by p300 or MAGE-11 (Fig. 9, A and B), is not sufficient for synergism between these coregulators. GAL-PR-B-(456 -546) contains PR activation function 1 and was activated by p300 but not MAGE-11, although MAGE-11 increased the response to p300. GAL-PR-B-(1-559) contains the entire PR-B NH 2 -terminal region and was synergistically activated by MAGE-11 and p300.
The results suggest that the unique PR-B NH 2 -terminal 164amino acid region facilitates increased transactivation by MAGE-11 and p300. Transcriptional synergy between MAGE-11 and p300 depends on PR NH 2 (Fig. 10A) (52). To investigate a requirement for the 110 LLXXVLXXLL 119 motif in the transcriptional effects of MAGE-11, mutagenesis was performed at hydrophobic residues. Down-regulation of PR-B by progesterone was not seen for PR-A or PR-B V114A,L115A and L118A,L119A mutants (Fig. 10B). MAGE-11 also down-regu-   (Fig. 10C). Mutations in the PR-B 110 LLXXVLXXLL 119 motif disrupted the ability of MAGE-11 (Fig. 10D) and p300 (Fig. 10E) to activate the FKBP5 progesterone response region.
The results suggest that the PR-B NH 2 -terminal 110 LLXXVLXXLL 119 motif is involved in PR-B down-regulation by progesterone and MAGE-11 and is required for the coregulatory effects of MAGE-11 and p300.
The results show that MAGE-11 interaction with PR-B involves the unique PR-B NH 2 -terminal 110 LLXXVLXXLL 119 motif and the F-box region of MAGE-11. The data suggest similar requirements for MAGE-11 interaction with the PR-B NH 2 -terminal 110 LLXXVLXXLL 119 motif and the AR NH 2 -terminal FXXLF motif (27).

Effects of PR-B NH 2 -terminal Phosphorylation and
110 LLXXVLXXLL 119 Motif-The PR-B NH 2 -terminal region is extensively phosphorylated (41). To test the impact of phosphorylation on PR-B activation by MAGE-11, mutations were introduced into PR-B and the PR-B upstream DNA-binding domain fragment, PR-BUS-DBD (Fig. 12A). PR-BUS-DBD migrated as multiple bands (Fig. 12B, lane 2) that were shifted to a faster migrating band by a series of serine phosphorylation site mutations (Fig. 12B, lane 6). Some of the 110 LLXXVLXXLL 119 motif mutants slowed the migration of PR-BUS-DBD (Fig. 12C) (41) even after treatment with -phosphatase (Fig. 12D). However, the multiple serine phosphorylation site mutations in PR-B did not alter the ability of MAGE-11 to increase transactivation of the FKBP5 progesterone response region (Fig. 12E). The results suggest that increased PR-B transactivation by MAGE-11 that depends on the PR-B NH 2 -terminal 110 LLXXVLXXLL 119 motif is independent of PR-B NH 2terminal phosphorylation.

MAGE-11 Stabilization of PR-B Complex with FKBP5-
The ability of MAGE-11 to increase progesterone-dependent PR-B transactivation of the FKBP5 gene suggested that MAGE-11 and FKBP5 may interact in a common mechanism to increase progesterone-dependent gene transcription. An interaction between MAGE-11 and FKBP5 was suggested by the coimmunoprecipitation of MAGE-11 with FLAG-FKBP5 (Fig. 13A,  lane 2). A weak interaction between FLAG-FKBP5 and PR-B (Fig. 13A, lanes 5 and 6) was strongly increased by MAGE-11 in the absence and presence of progesterone (Fig. 13A, lanes 7  and 8).  (Fig. 13D). Interaction between GAL-FKBP5 and VP-MAGE in mammalian two-hybrid assays was unaffected by MAGE-11 Lys-240 and -245 monoubiquitinylation site and Thr-360 phosphorylation site mutations but was inhibited by MAGE-11 F-box mutations (Fig. 13E). The results suggest that MAGE-11 interacts with FKBP5 and stabilizes a complex with PR-B, as summarized in Fig. 14.

Primate-and Isoform-specific Coregulation of Human PR-B-
Differences among placental mammals in the hormonal control of uterine function suggest that human and nonhuman primates evolved new mechanisms for steroid hormone regulation of the cyclic development of endometrium required for implanta-tion and pregnancy (14,24,54,55). Species-specific differences in uterine function involve the two PR isoforms, PR-A and -B, that are transcribed from the same gene using different promoters (14). Although PR-A is the predominant functional isoform in the mouse uterus, a prominent role for PR-B in human endometrium was suggested by promoter hypermethylation studies that caused progesterone resistance (56).
MAGE-11 is a primate-specific transcriptional coregulator characterized initially for its ability to amplify human AR signaling through its interaction with the AR NH 2 -terminal FXXLF motif region (25,39). The MAGE-11 gene is located at Xq28 on the human X chromosome and is part of a MAGE-A subfamily of cancer-testis antigens. The entire MAGE family of ϳ60 members contain retroposed insertions that diverged during mammalian evolution to provide new functions important for fertilization in primates (57)(58)(59)(60)(61)(62)(63)(64)(65). Although some MAGE genes are conserved between mice and humans, MAGE-11 arose by species-specific gene duplication in primates (57, 66 -70).
The menstrual cycle-dependent expression of MAGE-11 in the endometrium of normal cycling women and its localization in nuclei of human uterine epithelial, stromal, and endothelial cells suggest an expanded role in steroid hormone signaling. In this report, we provide evidence that MAGE-11 is an isoform-

Isoform-specific Human PR-B Coregulator MAGE-A11
OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 specific coregulator of human PR-B. MAGE-11 interacts with the NH 2 -terminal region of PR-B not present in PR-A and increases progesterone-dependent gene activation through synergistic effects with the p300 acetyltransferase. The actions of MAGE-11 and p300 appear to explain the greater transcriptional activity of PR-B relative to PR-A. The transcriptional enhancing effects of MAGE-11 on progesterone-dependent gene expression mimic the effects of progesterone to downregulate PR-B. The studies suggest that MAGE-11 mediates the greater transcriptional activity of PR-B relative to PR-A in progesterone regulation of the human endometrium.
MAGE-11 as PR-B and AR Coregulator-The mechanisms by which MAGE-11 functions as a PR-B coregulator have striking similarities to its regulation of human AR. MAGE-11 increases human PR-B and AR transcriptional activity through an interaction with NH 2 -terminal ␣-helical motifs involved in ligand-dependent NH 2 -and COOH-terminal interactions (24,35,36). MAGE-11 regulates the single full-length form of human AR through an interaction with the AR NH 2 -terminal FXXLF motif region and has isoform-specific coregulator activity with PR-B through an interaction with the NH 2 -terminal 110 LLXXVLXXLL 119 motif unique to PR-B. The same monoubiquitinylation and F-box regions in MAGE-11 are required to interact with PR-B and AR, and MAGE-11 functions synergistically with p300 and p160 coactivators to increase the transcriptional activity of both receptors (27,28). A difference was noted, however, in that MAGE-11 down-regulates PR-B in the absence of progesterone but stabilizes AR in the absence of androgen (25). The lack of the NH 2 -terminal 110 LLXXVLXXLL 119 motif in PR-A required to interact efficiently with MAGE-11 suggests that MAGE-11 acting with p300 accounts for the transcriptional superiority of PR-B relative to PR-A.
The similar increase in transactivation of the FKBP5 progesterone response region by PR-A and -B with the expression of TIF2 suggests that higher levels of p160 coactivators render PR-A transcriptionally similar to PR-B. We suggested previously that mammalian evolution has favored a shift in activation domain usage away from AF2 in the ligand-binding domain toward the NH 2 -terminal activation domains of steroid receptors (71). The ability of MAGE-11 to amplify the effects of TIF2 similarly for PR-A and -B reflects the general ability of MAGE-11 to interact and function synergistically with TIF2 to increase receptor transcriptional activity (27,72). In contrast, only PR-B and AR functioned synergistically with MAGE-11 and p300. The coevolution of MAGE-11 expression in primates with the human AR NH 2 -terminal sequence required to interact with MAGE-11 (26) is consistent with their interrelationship. The predominance of PR-A in mouse uterine function supports the idea that steroid receptor transactivation in less evolved mammals is more dependent on AF2 activation by p160 coactivators, whereas human and nonhuman primates make use of more variable sequence in the NH 2 -terminal activation domains.
The importance of MAGE-11 in maximizing transcriptional output in normal human physiology is suggested by the naturally occurring AR NH 2 -terminal mutation R405S, which created a new phosphorylation site and caused partial androgen insensitivity in a newborn genetic male (73). The AR R405S mutation disrupted the ability of MAGE-11 to increase AR and serial mutants containing S20A,S25A with and without S99A,S100A,S101A,S102A with and without S130A with and without S162A (10 g 8 , containing S20A,S25A,S99A,S100A,S101A,S102A,S130A,S162A mutations (6 g) were expressed in COS cells. Cell extracts (10 g of protein/lane) were probed on the transblot using PR antibody. Bottom, p5M-PR-B WT and p5M-PR-B-(SA) 8 (25 ng) were expressed in CV1 cells with 3 g of pIE2-Luc FKBP5 luciferase reporter vector and 0.5 g of pSG5 empty vector (Ϫ) or 0.5 g of pSG5-MAGE. Luciferase activity was determined after incubation with and without 1 nM progesterone. Error bars, S.E. transcriptional activity associated with the AR NH 2 -and COOH-terminal interaction. An increase in MAGE-11 during prostate cancer progression amplifies AR signaling and promotes prostate cancer progression (29,30). The menstrual cycle-dependent increase in MAGE-11 and its up-regulation by cyclic AMP provide a mechanism to amplify PR-B signaling in the mid-secretory human endometrium and establish endometrial receptivity for implantation of the embryo.
Feed-forward Progesterone-dependent PR-B Gene Regulation by MAGE-11 and FKBP5-FKBP5 (known also as FKBP51) is a 51-kDa immunophilin up-regulated by progesterone, androgen, and glucocorticoids in different tissues and cell lines (50,74,75). FKBP5 is a peptidylprolyl isomerase (14,76) inhibited by the immunosuppressive drugs FK506, rapamycin, and cyclosporine-A (77). FKPB5 functions as a cochaperone of steroid hormone receptors (48, 78 -80); interacts with hsp70 and hsp90; and is involved in apoptosis (81), microtubule stabilization (82), and negative regulation of Akt (83). The progesterone-dependent increase in FKBP5 by PR-B and MAGE-11 suggests a feed-forward mechanism in human endometrium. However, previous studies showed transient overexpression of FKBP5 decreased progesterone sensitivity in reporter gene assays (50), and FKBP5 association with the unliganded glucocorticoid receptor caused glucocorticoid resistance in a New World monkey (84). It may be that MAGE-11 expression in primates and Old World monkeys establishes a positive role for FKBP5 in progesterone action. In agreement with this, the FK506 inhibitor of FKBP5 peptidylprolyl isomerase activity inhibited progesterone-induced transcription in human breast cancer cells (85).
The similar increase in MAGE-11 and FKBP5 mRNA and protein in the early and mid-secretory normal cycling human endometrium (31) suggests that MAGE-11 is involved in mech-  anisms to promote progesterone gene regulation. This was substantiated by a dependence on MAGE-11 for progesterone-dependent up-regulation of endogenous FKBP5 in endometrial cells and in reporter gene assays where MAGE-11 preferentially increased PR-B activation of the FKBP5 intron 5 progesterone response region. In contrast, MAGE-11 did not increase progesterone up-regulation of RASD1, which was regulated primarily by the PR-A/B heterodimer. MAGE-11 stabilized a complex between FKBP5 and PR-B through its interaction with FKBP5 and the PR-B NH 2 -terminal region. Progesterone and PR-B up-regulation of FKBP5, which depends on MAGE-11, and MAGE-11 interaction with PR-B and FKBP5 suggest a mechanism to enhance progesterone regulation of the mid-secretory human endometrium.