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Endocrinology, doi:10.1210/en.2007-0326
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Endocrinology Vol. 148, No. 11 5209-5219
Copyright © 2007 by The Endocrine Society

Prostaglandin F2{alpha} Suppresses Rat Steroidogenic Acute Regulatory Protein Expression via Induction of Yin Yang 1 Protein and Recruitment of Histone Deacetylase 1 Protein

Qiyuan Liu, Kathleen A. Merkler, Xiaohui Zhang and Mark P. McLean

Departments of Obstetrics and Gynecology (Q.L., K.A.M., X.Z., M.P.M.) and Molecular Pharmacology and Physiology (M.P.M.), University of South Florida, Tampa, Florida 33612

Address all correspondence and requests for reprints to: Mark P. McLean, Ph.D., University of South Florida, 12901 Bruce B. Downs Boulevard, MDC037, Tampa, Florida 33612. E-mail: mmclean{at}health.usf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin F2{alpha} (PGF2{alpha}) plays a pivotal role in ovarian luteolysis by inhibiting the expression of steroidogenic acute regulatory (StAR) protein, leading to a decrease in intracellular cholesterol transport and luteal steroid production. Previously we have demonstrated that the transcription factor Yin Yang 1 (YY1) bound to three regions in the StAR promoter in vitro and repressed promoter activity. This study further defined the YY1-mediated PGF2{alpha} effect on the inhibition of StAR protein expression through YY1 interaction with a single region in the StAR promoter in vivo. PGF2{alpha} consistently suppressed StAR mRNA and protein expression in cultured luteal cells in a dose-dependent manner. PGF2{alpha} also enhanced YY1 protein expression and binding to its cis-element in a time-dependent pattern that preceded the decline in StAR protein levels. The StAR promoter region bound by YY1 was also associated with histone deacetylase 1 (HDAC1). PGF2{alpha} treatment promoted HDAC1 binding to and suppressed the histone H3 acetylation in this region. On the contrary, YY1 knockdown decreased HDAC1 binding, increased histone H3 acetylation, enhanced StAR protein expression, and negated PGF2{alpha} effect on StAR protein expression. Luciferase assays showed that YY1 overexpression inhibited StAR promoter activity and the addition of a HDAC inhibitor, trichostatin A, abrogated the effect of YY1. Trichostatin A-treated luteal cells displayed increased StAR protein expression. These data indicate that PGF2{alpha} enhances a direct YY1/StAR promoter interaction and the recruitment of HDAC1 to the promoter, thereby preventing transcriptional activation of the StAR gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RAT, AS well as in human and other mammals, the processes of growth (follicular maturation), differentiation (luteogenesis), and degeneration (luteolysis) characterize ovarian development. These events are associated with alterations in the type, pattern, and quantity of steroid hormone secreted. A significant component in each of these processes is cholesterol, because this molecule is both a universal constituent of cell membranes and an essential precursor in the synthesis of steroid hormones. Recent investigations suggest that the delivery of cholesterol from cellular stores to the inner mitochondrial membrane, where cholesterol is converted to pregnenolone by cytochrome P450 side-chain cleavage enzyme, represents a key hormone-mediated step in the corpus luteum during luteogenesis and luteolysis. Clark et al. (1) isolated and cloned a 30-kDa luteinizing hormone-induced mitochondrial protein from MA-10 cells named steroidogenic acute regulatory (StAR) protein that controls the rate-limiting step in steroidogenesis: the transport of cholesterol from the outer to the inner mitochondrial membrane (2). The significance of the role of StAR in steroid production is evident in the human disorder congenital lipoid adrenal hyperplasia, in which patients present with a loss of steroid hormone production due to the lack of functional StAR protein (3). A similar phenotype is evident in StAR knockout mice, in which cholesterol accumulates within steroidogenic tissues leading to termination of steroid output (4, 5). Therefore, regulation of acute steroidogenesis is dependent on controlling StAR protein expression and function. The presence of StAR mRNA transcripts in adrenal and gonadal cells directly correlates with the response of steroidogenic cells to hormone action, establishing the functional importance of transcriptional regulation of the StAR gene in steroidogenic cells (6, 7). Transcriptional activators of the StAR gene (8), such as sterol regulatory element binding protein-1a (SREBP-1a), steroidogenic factor 1, nuclear factor Y (NF-Y), and Sp-1, have been studied in great detail. Notably, overexpression of SREBP-1a increases StAR promoter activity in both human bladder carcinoma HTB9 cells (8) and human granulosa-lutein cells (9). However, transcriptional factors that repress StAR expression have received less attention.

Previous studies suggested that Yin Yang 1 (YY1) was one of the candidates that suppressed StAR transcriptional activity (9, 10). YY1 is a transcription factor that contains zinc finger domains and has been shown to activate, repress, or initiate transcription (11). YY1 may be associated with and modulated by adenovirus-derived E1A, a protein that activates the AAV P5 promoter (12). The presence of E1A induces YY1-mediated activation of transcription. In its absence, the role of YY1 is reversed, converting YY1 to a transcriptional repressor (13, 14). YY1 functioning as a transcriptional repressor has been reported for a variety of genes including skeletal {alpha}-actin (15), human {alpha}-myosin heavy chain (16), serum amyloid (17), β-casein (18, 19), human {gamma}-interferon (10, 20, 21), murine β-interferon (22), {epsilon}-globin (23), 25-hydroxyvitamin D3-24-hydroxylase (24), and StAR (10, 21). The wide-ranging effects of YY1 agree with its ubiquitous expression in tissues (11).

The activity of YY1 as a transcription factor could be regulated by gene expression, protein cellular localization, and the discriminatory binding of cofactors. YY1 protein levels are regulated in many situations. The expression of YY1 in NIH3T3 cells is affected by cell density and growth factors such as IGF-1 (25). YY1 is down-regulated in F9 cells after long-term treatment with retinoic acid (26). The changes in YY1 expression are observed during myoblast differentiation (15), in the process of aging (27), and in human heart failure (16). A recent study by Joshi et al. (28) found that prohibitin reduced YY1 promoter activity through the E2F binding site. The availability of YY1 to the promoter of targeted genes is also dependent on its cellular localization. The COOH-terminal domain (257–341 amino acids) of YY1 is necessary for nuclear matrix association (29). YY1 nuclear localization, followed by increased DNA-binding activity, is observed during the onset of the G1/S phase in the cell cycle (30). Inside the nucleus, YY1 often requires the help of cofactors that interact with its repression domains to facilitate repression. Sufficient evidence suggests that the recruitment of histone deacetylases (HDAC), which maintain chromatin in a state of transcription inertness, contributes to the suppressive effect of YY1 on various promoters (11). YY1 represses Hoxa11-mediated downstream target gene transcription via YY1 recruitment of HDACs to form a complex (31). YY1 is a repressor of transcription in differentiated H9C2 cells via its interaction with HDAC5 (32). Overexpression of YY1 increases occupancy by HDAC1 at a positioned nucleosome near the transcription start site of the integrated HIV-1 long terminal repeat, thereby down-regulating its expression (33). YY1 might also recruit HDAC through other adaptors such as FK506-binding protein 25 (34). The cooperative silencing effect of Myc and YY1 on the integrin {alpha}-3 gene involves deacetylation activity in the promoter as well (35).

Although in vitro studies suggest that YY1 is a negative regulatory element for StAR expression (9, 10), there is little evidence to establish their relationship under physiological conditions. Regression of the corpus luteum is an example of a normal degenerative cellular function, which is an intrinsic step in the reproductive cycle. In pregnant and pseudopregnant rats, a high correlation between luteal prostaglandin F2{alpha} (PGF2{alpha}) concentration and the demise of luteal function is reported, demonstrating a role for luteal PGF2{alpha} in luteolysis (36, 37, 38). Treatment of rats with PGF2{alpha} has been found to induce YY1 expression in whole ovary tissue (10). In a separate study, PGF2{alpha} was identified to suppress StAR mRNA level in rat ovaries after animal ovulation (39). In the present study, we demonstrate that YY1 mediates the inhibitory function of PGF2{alpha} on StAR expression in luteal cells and, using this cellular model, characterize the mechanisms involved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal model
Female Sprague Dawley rats were purchased from Harlan Industries (Madison, WI). Throughout the experiments, animals had free access to food and water and were housed under a 12-h dark, 12-h light cycle. Follicular development and ovulation were induced in 28-d-old rats by injection of 15 IU pregnant mare horse serum (Sigma, St. Louis, MO) followed by 15 IU human chorionic gonadotropin (Sigma) 48 h later. All procedures for treatment and methods for tissue sampling were approved by the University of South Florida Animal Care Committee.

Rat luteal cell isolation and culture
Corpora lutea obtained from at least 15 rats were pooled for each luteal cell dispersion. The rats were euthanized 9 d after human chorionic gonadotropin treatment, and the ovaries were dissected out. Luteal cells were obtained according to the procedure used by Nelson et al. (40). The ovaries were placed in a sterile dissecting dish containing Hank’s balanced salt solution (without calcium and magnesium) supplemented with 2% BSA and 25 mM HEPES, pH 7.4 (dissection media), and the whole corpora lutea were dissected out. Care was taken to remove any follicular contamination and to not tear the corpora lutea. Dissected corpora lutea were maintained in a water-jacketed cellstir spinner flask (Wheaton Scientific, Millville, NJ) at 37 C and stirred at 100 rpm until all corpora lutea were dissociated. Intact corpora lutea were incubated at 37 C with 50 U/ml collagenase type IV (Worthington Biochemical, Lakewood, NJ), 2.4 U/ml dispase II (Roche Applied Sciences, Indianapolis, IN), and 200 U/ml DNase I (Roche Applied Sciences) in four consecutive 30-min incubations with stirring at 100 rpm. The tissue was allowed to settle, the supernatant was discarded, and fresh enzyme solution was added after each incubation. At the end of the 2-h period, corpora lutea that still appeared intact were treated for 15 min in 10 ml PBS solution containing 25 mM HEPES, pH 7.4, 2% BSA, and 0.02% EDTA (wt/vol) at 37 C with stirring at 100 rpm. The fraction containing the whole corpora lutea and some loosened cells was centrifuged at 200 x g. The cycle was repeated twice more. This results in the disintegration of corpora lutea and total dispersion of the luteal cells. The total luteal cell dispersion was filtered through a 100 µm nylon cell strainer (BD Falcon, Bedford, MA). Cells were washed twice in PBS to remove BSA. The cell viability was judged by trypan blue exclusion. The cells were plated out in phenol red-free DMEM/Ham’s F12 containing 10% FBS.

SDS-PAGE and Western blot
Luteal cells were cultured for 3 d then treated with 1 µM PGF2{alpha}. Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0 mM EDTA, 1.0% Nonidet P-40, 0.25% sodium-deoxycholate) containing protease inhibitor cocktail (Roche Applied Sciences). A total of 50 µg extracted protein was loaded on a SDS-PAGE, transferred to nitrocellulose, and immunoblotted as previously described (10). The blots were probed with a rabbit polyclonal antibody against YY1 (Geneka Biotechnologies, Montreal, Quebec, Canada), rabbit polyclonal antibody against StAR (Abcam, Cambridge, MA), rabbit polyclonal antibody against SREBP-1a (Santa Cruz Biotechnology, Santa Cruz, CA), and goat polyclonal antibody against β-actin (Santa Cruz Biotechnology).

RNA extract and Northern blot
Total RNA was prepared from cultured luteal cells according to previous reports (39, 41). Briefly, cells were lysed in TriReagent (Molecular Research Center, Cincinnati, OH) for 5 min at room temperature, and the aqueous phase was separated from the homogenate by addition of chloroform, incubation at room temperature for 10 min, and centrifugation at 12,000 x g for 15 min at 4 C. RNA was precipitated from the aqueous phase by addition of 4 ml isopropanol and incubation at room temperature for 10 min. After centrifugation at 12,000 x g for 10 min at room temperature, the RNA pellet was washed with 8 ml 75% ethanol and dissolved in diethyl pyrocarbonate-treated deionized water. RNA was quantified by absorbance at 260 nm using a GeneQuant II RNA/DNA calculator (Pharmacia Biotech, Piscataway, NJ).

Twenty micrograms of total RNA were treated with denaturing buffer (1x MOPS, 7.4% formaldehyde, 50% formamide) containing xylene cyanol and bromophenol blue at 65 C for 15 min. The denatured RNA was loaded into a 1.2% agarose gel containing 1.1% formaldehyde. After size fractionation, the RNA was blotted onto a BrightStar-Plus positively charge nylon membrane (Ambion, Austin, TX) by capillary transfer, and the RNA was fixed to the membrane by UV cross-linking (0.3 J/cm2). Ethidium bromide staining of the gel to visualize the 28S and 18S rRNA was used to confirm the integrity and equal loading of RNA in each lane.

Northern blot hybridization was performed using an 867-bp rat StAR cDNA probe. The probes were labeled with [{alpha}-32P]dCTP by using the Prime-It II random primer labeling kit (Stratagene, Cedar Creek, TX). The Northern blot was prehybridized at 42 C for 30 min in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). Hybridization was completed in the same hybridization buffer containing the 32P-labeled probe at 42 C for at least 16 h. Blots were washed three times for 10 min each at room temperature with 1x SCC (150 mM NaCl, 25 mM sodium citrate, pH 7.0)/0.1% SDS and three times for 10 min each at room temperature with 0.1x SSC/0.1% SDS. RNA:cDNA hybrids were visualized on BioMax MR Film (Kodak, Rochester, NY) using two intensifying screens and a 12- to 48-h exposure period. The RNA transcript size was determined by comparison to a RNA molecular weight marker (Promega Corp., Madison, WI) run in parallel to the sample RNA lanes.

Chromatin immunoprecipitation (ChIP)
A modified technique described by Bennett and Osborne (42) was used. Chromatin from 2 x 106 luteal cells was cross-linked by treating with 1.0% formaldehyde for 10 min at 37 C. The reaction was stopped by the addition of glycine (0.125 M final concentration). The cells were washed twice with cold PBS containing protease inhibitor cocktail (Roche Applied Sciences) and collected in 0.4 ml lysis buffer (50 mM Tris-HCl, pH 8.1; 10 mM EDTA; 1% SDS) containing 0.5 mM PMSF (Sigma) and protease inhibitor cocktail after incubation on ice for 10 min. The lysates were sonicated three times for 10 sec each with a Sonic Dismembrator Model 100 (Fisher Scientific, Pittsburgh, PA) (output power = 10 W) resulting in DNA fragment sizes of 0.2–0.5 kb. Samples were immediately processed or stored at –80 C for ChIP analysis. The samples were centrifuged at 12,000 x g for 15 min, then 10 µl of the supernatant was used as input and the remainder was diluted 10-fold in dilution buffer (16.7 mM Tris-HCl, pH 8.1; 167 mM NaCl; 1.2 mM EDTA; 1.1% Triton X-100; 0.01% SDS) containing protease inhibitor cocktail. The diluted fraction was subjected to 2 h of preclearing at 4 C with 50 µl protein A agarose/salmon sperm DNA (Upstate USA, Charlottesville, VA) then immunoprecipitated overnight with mouse monoclonal antibody against SREBP-1a (Santa Cruz Biotechnology), rabbit polyclonal antibody against YY1 (Active Motif, Carlsbad, CA), polyclonal antibody against HDAC1 (Upstate USA), or rabbit polyclonal antibody against acetyl histone H3 (Upstate USA). The complexes were recovered by a 2-h incubation at 4 C with 50 µl protein A agarose/salmon sperm DNA. Precipitates were serially washed with 2 ml of Washing Buffer I (20 mM Tris-HCl, pH 8.1; 150 mM NaCl; 2 mM EDTA; 0.1% SDS; 1.0% Triton X-100), 2 ml of Washing Buffer II (20 mM Tris-HCl; pH 8.1, 500 mM NaCl; 2 mM EDTA; 0.1% SDS; 1.0% Triton X-100), 2 ml of Washing Buffer III (10 mM Tris, pH 8.1; 0.25 mM LiCl; 1 mM EDTA; 1.0% Nonidet P-40; 1% sodium deoxycholate), and twice with 2 ml of Washing Buffer IV (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The precipitated chromatin complexes were removed twice from the beads, each through a 30-min incubation in 250 µl freshly prepared elution buffer (0.1 M NaHCO3, 1.0% SDS) with gentle stirring at room temperature. Cross-linking was reversed by a 4-h incubation with 0.25 M NaCl (final concentration) at 65 C, and the proteins were digested for 1 h using 40 µg/ml proteinase K (Roche Applied Sciences). DNA was purified by phenol/chloroform extraction and ethanol precipitation and used as the template in PCR for 30–35 cycles to achieve the optimal in-group differentiation. GoTaq DNA polymerase (Promega Corp.) was used in the PCR amplification under the following conditions: denaturing at 95 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec.

Knockdown by small interfering RNA (siRNA)
Luteal cells were isolated and plated out as previously described. YY1-specific siRNA or control siRNA (Dharmacon, Lafayette, CO) was transfected into the cells using DharmaFECT 4 Reagent according to the protocol provided by the manufacturer. Forty-eight hours after transfection, cells were either lysed in RIPA buffer for SDS-PAGE or cross-linked in 1.0% formaldehyde for ChIP assay.

Transfection and luciferase assay
The human embryonic kidney cell line, HEK293T, plated in six-well plates was transfected with the specific StAR promoter-luciferase reporter gene construct either in the presence or absence of YY1-pCMV5 (10) and/or SREBP-1a-pCMV5 (8) using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The luciferase assay was performed using the Dual Luciferase Reporter Assay System (Promega). Briefly, 20 µl of lysate were added to 100 µl luciferase substrate, and luciferase activity was measured using a Femtomaster FB12 luminometer (Zylux Corp., Maryville, TN). Cotransfection of a plasmid containing the renilla luciferase gene in the pGL3 vector was used as an internal control to correct for differences in transfection efficiency in individual wells. Trichostatin A (TSA) treatment, when required, was started 24 h before cell lysis.

Statistical analysis
The statistical data are shown as the average value ± SEM of three independent experiments. The significance of change (P < 0.05) was analyzed as appropriate by one-way ANOVA followed by the Tukey post hoc test or Student’s t test using SPSS software (SPSS Inc., Chicago, IL) as indicated in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression patterns of StAR, SREBP-1a, and YY1 proteins in PGF2{alpha}-treated rat luteal cells
Primary cultures of rat luteal cells were treated with increasing concentrations of PGF2{alpha} over a 24-h period. As shown in Fig. 1Go, 24 h treatment of the cells with 0.1 µM PGF2{alpha} suppressed StAR protein expression compared with vehicle-treated control, and StAR protein was further decreased when the PGF2{alpha} concentration was increased. A 2- to 9-fold decrease in StAR protein levels was observed in the range of 0.1–1.0 µM PGF2{alpha}.


Figure 1
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  		FIG. 1. Concentration-dependent effects of PGF2{alpha} on the expression of StAR, YY1, and SREBP-1a proteins. Rat luteal cells were obtained and cultured. Three days later, cells were treated with PGF2{alpha} at different concentrations for 24 h. Cell lysates were prepared for and subjected to SDS-PAGE then transferred to nitrocellulose and immunoblotted with the indicated antibodies. The detection of β-actin protein expression served as a lane loading control. Results shown are representative of three independent experiments.

 
Although we did not observe a significant change in SREBP-1a protein expression in PGF2{alpha}-treated cells, PGF2{alpha} treatment enhanced YY1 protein expression in a concentration-dependent manner. The change in YY1 protein expression was inversely related to that of StAR. An 8-fold induction of YY1 expression was observed in cells treated with 1.0 µM PGF2{alpha}.

To determine how quickly PGF2{alpha} treatment changes the expression of StAR and YY1 and whether it might momentarily affect SREBP-1a protein expression, we measured the protein levels of these three molecules in luteal cells after treating them with 1.0 µM PGF2{alpha} and collecting protein lysates at different times over a 24-h period (Fig. 2Go). We found that StAR protein expression began to decrease slightly by 15 ± 7% (P = 0.11) as early as 1 h after treatment and continued to decline up to 24 h. A significant decrease of 75 ± 2% (P < 0.001) in StAR protein level was observed up to 24 h after treatment. Interestingly, YY1 protein induction was increased by 70 ± 9% (P = 0.007) after just 1 h of treatment and continued to increase up to 6.9 ± 1.0-fold (P = 0.004) over a 24-h period. SREBP-1a expression was constant over the same time period. Both StAR and YY1 proteins were unchanged in vehicle-treated cells except that the YY1 level slightly decreased after incubation for 24 h, confirming that the reduction of StAR and induction of YY1 in PGF2{alpha}-treated cells were not due to cellular growth and differentiation over the time period or other nonspecific effects.


Figure 2
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  		FIG. 2. Time-dependent effects of PGF2{alpha} on the expression of StAR, YY1, and SREBP-1a protein. Rat luteal cells were obtained and cultured. Three days later, cells were treated with 1.0 µM PGF2{alpha} and collected at different time intervals. Cell lysates were prepared for and subjected to SDS-PAGE then transferred to nitrocellulose and immunoblotted with the indicated antibodies. The detection of β-actin protein expression served as a lane loading control. A, Immunoblots. Results shown are representative of three independent experiments. B, Densitometry results of StAR/β-actin. C, Densitometry results of YY1/β-actin. The relative values of StAR/β-actin and YY1/β-actin are both set to 1 U by normalization of the pretreatment mean value in each subgroup. One-way ANOVA followed by Tukey post hoc test was performed. The P value of vehicle effect on StAR/β-actin is 0.852, of PGF2{alpha} effect on StAR/β-actin is less than 0.001, of vehicle effect on YY1/β-actin is 0.933, and of PGF2{alpha} effect on YY1/β-actin is less than 0.001. *, P < 0.05 for StAR/actin or YY1/actin in PGF2{alpha} treatment group compared with pretreatment.

 
PGF2{alpha} suppresses StAR protein expression at the mRNA transcript level
To determine which gene expression event accounted for the altered StAR protein levels, we looked at changes at the mRNA level in rat luteal cells treated with 1.0 µM PGF2{alpha} over a 24-h time period. Northern blots were performed to detect StAR mRNA transcripts. After hybridizing the blot with a 32P-labeled StAR cDNA probe, we found that StAR mRNA levels started to fall after 1 h of treatment and decreased 5-fold at 24 h in comparison to the pretreatment (Fig. 3Go, upper panel).


Figure 3
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  		FIG. 3. PGF2{alpha} treatment reduced StAR mRNA transcript levels. Rat luteal cells were obtained and cultured. Three days later, they were treated with 1.0 µM PGF2{alpha} then collected at different time intervals. RNA extraction and Northern blotting were performed to measure the mRNA transcript levels. Detection of 28S and 18S rRNA in the gel served as loading control. Results shown are representative of three independent experiments.

 
Detection of 28S and 18S rRNA in the RNA gels demonstrated equal lane loading of total RNA on the blot and the integrity of the RNA molecules (Fig. 3Go, lower panel). These data suggest that PGF2{alpha} regulates StAR protein expression at the transcription step.

YY1 knockdown increases StAR protein levels in luteal cells
RNA interference was used to knockdown YY1 mRNA transcripts to determine whether YY1 negatively regulates StAR protein expression. Figure 4AGo shows that YY1 protein levels declined to a negligible level in the YY1-specific siRNA-treated cells compared with that observed in the control siRNA-treated cells. Conversely, StAR protein expression was enhanced in YY1-specific siRNA-treated cells in either the presence or absence of PGF2{alpha} treatment. β-Actin protein expression served as a lane loading control in the Western blot. Densitometric analysis of three independent experiments showed that PGF2{alpha} treatment enhanced YY1 protein expression in the control siRNA-transfected cells by 4.7 ± 0.8-fold (P = 0.008) but not in the YY1 siRNA-transfected cells. Correspondingly, PGF2{alpha} treatment suppressed StAR protein expression in the control siRNA-transfected cells by 67 ± 2% (P = 0.007), but only slightly affected StAR protein levels, approximately a 10 ± 11% (P = 0.4) decrease was observed, in the YY1 siRNA-transfected cells (Fig. 4BGo). This result indicates that YY1 is likely to be a strong transcriptional repressor of and a mediator of PGF2{alpha} effect on StAR gene expression.


Figure 4
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  		FIG. 4. YY1 knockdown enhanced StAR protein expression. Rat luteal cells were obtained and cultured. Cells were transfected with YY1 siRNA or the control siRNA, and 24 h later, treated with vehicle or 1.0 µM PGF2{alpha}. Forty-eight hours after transfection, cell lysates were prepared for and subjected to SDS-PAGE then transferred to nitrocellulose and immunoblotted. A, Immunoblots. Results shown are representative of three independent experiments. B, Densitometry results. The relative values of YY1/β-actin and StAR/β-actin are both set to 1 U in control siRNA-transfected luteal cells by normalization using the mean value of the control siRNA group. Results represent the average ± SEM of three independent experiments. *, Induction (P < 0.01) of YY1 protein by PGF2{alpha} treatment compared with vehicle treatment (PGF2{alpha} treatment did not significantly induce YY1 protein in YY1 siRNA-transfected cells). **, Reduction (P < 0.005) of YY1 protein by YY1 siRNA compared with the control siRNA (PGF2{alpha} treatment did not significantly reduce StAR protein in YY1 siRNA-transfected cells). #, Reduction (P < 0.01) of StAR protein by PGF2{alpha} treatment compared with the vehicle treatment. ##, Induction (P < 0.05) of StAR protein by YY1 siRNA compared with control siRNA.

 
YY1 suppresses StAR promoter activity
To determine how the YY1 protein regulated StAR gene expression, we examined the effect of YY1 on StAR promoter activity. The rat primary luteal cells were extremely inefficient in taking up exogenous plasmids, thus the HEK 293T cell line was used in the transient transfection studies. Using a luciferase assay, we cotransfected HEK 293T cells with wild-type YY1 and a fully functional StAR promoter, p1862 (Fig. 5AGo), coupled to the luciferase gene. We found that StAR promoter activity was reduced by the wild-type YY1 protein by 62 ± 4% (P = 0.006), but not by the mutant YY1 {Delta}154–199 that has a deletion in a region that is essential for the transcriptional repression effect (43) but not for the transactivation effect (44) (Fig. 5AGo). YY1, but not its deletion mutant, also suppressed SREBP-1a-induced StAR promoter activity (Fig. 5BGo). These results demonstrate a specific inhibitory effect of YY1 protein on the StAR promoter.


Figure 5
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  		FIG. 5. StAR promoter activity was suppressed by the wild-type YY1 but not its deletion mutant. HEK 293T cells were transfected with the indicated plasmids together with equal amount of renilla luciferase plasmid in each well. The luciferase activity was measured 2 d after transfection. The graph represents the relative average luciferase activity ± SEM of three independent experiments after normalization to the renilla luciferase activity. *, P < 0.05, and **, P < 0.01, reduction of StAR promoter activity by YY1 compared with that in presence of empty vector.

 
We next took a closer look at what region(s) of the StAR promoter were affected by YY1 and SREBP-1a. Another set of luciferase assays was conducted using p1862 as well as two StAR partial promoter constructs, p1413 and p998 (Fig. 6AGo). We found that YY1 inhibited the basal transcriptional activities of p1862 and p1413 but not of p998 in HEK 293T cells (Fig. 6BGo). Although SREBP-1a induced the promoter activity of all the constructs, only the activity of the longest two promoter constructs could be repressed by YY1. These data show that YY1 exerts its regulatory effect mainly in the region of 998 to 1413 nucleotides upstream of the StAR transcript, which includes one of three YY1 potential binding sequences found in the StAR promoter (10) (Fig. 6AGo).


Figure 6
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  		FIG. 6. Effects of YY1 and SREBP-1a on StAR promoter activity. A, Schematic of StAR promoter structure. The positions of five potential SREBP-1a binding regions (SRE 1–5) and three potential YY1 binding regions (YY1 BS 1–3) are indicated. Three luciferase reporter constructs used in this study are shown. B, Effects of YY1 and SREBP-1a on luciferase expression under control of the StAR promoter. HEK 293T cells were transfected with the p1862, p1413, or p998 rat StAR promoter construct either in the presence or absence of pCMV5-expression vectors for SREBP-1a and/or YY1 together with equal amount of renilla luciferase plasmid in each well. The luciferase activity was measured 2 d after transfection. The graph represents the relative average luciferase activity ± SEM of three independent experiments after normalization to the renilla luciferase activity. *, Induction (P < 0.001) of StAR promoter activity by SREBP-1a compared with that in presence of empty vector. #, Reduction (P < 0.01) of StAR promoter activity by YY1 compared with that in presence of empty vector (YY1 did not significantly suppress p998 luciferase activity either in the presence or absence of SREBP-1a).

 
The binding of YY1 and SREBP-1a to the StAR promoter
ChIP assays were performed to determine whether YY1 and SREBP-1a were bound directly to the StAR promoter in vivo. There are five potential SREBP-1a binding regions, SRE1–5, and three potential YY1 binding regions, YY1 BS1–3, in the StAR promoter (10) (Fig. 6AGo). Five pairs of PCR primers were synthesized to cover all these binding regions. Among the three potential YY1 binding sequences, only the most proximal one, YY1 BS1 in region 4, was bound by the YY1 protein (Fig. 7BGo). This binding pattern corresponded to the luciferase assay data, implicating the essentiality of YY1 binding to BS1 for its repressive effect on the StAR promoter.


Figure 7
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  		FIG. 7. The binding patterns of YY1 and SREBP-1a on the StAR promoter region in vivo. Rat luteal cells were cultured and ChIP assays were performed. A, Sequences of five PCR primer pairs to cover the five regions in StAR promoter as shown in Fig. 6AGo. B, Anti-YY1 antibody was used to pull down YY1 protein and its associated DNA fragments. C, Anti-SREBP-1a antibody was used to pull down SREBP-1a and its associated DNA fragments. D, Input DNA was PCR amplified using the five pairs of primers. Results in B, C, and D are representative of three independent experiments.

 
In contrast, among the five potential sterol regulator elements, only the two distal regions were associated with SREBP-1a binding. One of them is 30 nucleotides away from the YY1 BS1 (Fig. 7CGo). Figure 7DGo shows the PCR products generated using the five primer pairs on the input DNA, indicating that the absence of YY1 and SREBP-1a binding to the other potential binding regions shown in Fig. 7Go, B and C, was not due to PCR failure.

To explore whether YY1 binding to the YY1 BS1 was the regulatory event in mediating PGF2{alpha} regulation of StAR protein expression, we examined the time-dependent pattern of YY1 binding. Figure 8Go shows that PGF2{alpha} promoted YY1 binding to StAR promoter region 4 from 1–24 h after treatment, with a pattern inversely correlated to the change in StAR protein levels (Fig. 2Go).


Figure 8
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  		FIG. 8. The effect of PGF2{alpha} treatment on YY1 binding affinity in StAR promoter region 4. Rat luteal cells were cultured and treated with 1.0 µM PGF2{alpha}, and ChIP assays were performed. The pair of PCR primers for region 4 was used to measure the effect of PGF2{alpha} treatment on YY1 binding affinity to StAR promoter. A, Detection of PCR-amplified DNA fragments in ChIP and input. Results are representative of three independent experiments. B, Densitometry results of ChIP/input. The value was set to 1 U for cells before PGF2{alpha} treatment by normalization of the mean density of the pretreatment group. The results are the average ± SEM of three independent experiments. One-way ANOVA followed by Tukey post hoc test shows P = 0.038 for the time-dependent effect of PGF2{alpha} on YY1 binding in the promoter region. *, P < 0.05; **, P < 0.005, induction of YY1 binding to StAR promoter by PGF2{alpha} treatment compared with pretreatment.

 
YY1 increases HDAC activity on the StAR promoter
The HDAC inhibitor TSA was used in a luciferase assay to examine whether the effect of YY1 on StAR protein expression was mediated by altered acetylation levels in the StAR promoter. Figure 9AGo shows that TSA treatment in HEK 293T cells dramatically enhanced p1982 promoter activity. YY1 overexpression in the cells inhibited basal promoter activity by 63 ± 3% (P < 0.001) but only slightly decreased the TSA-induced StAR promoter activity, indicating that regulation of deacetylation activity was a major downstream event of the YY1 effect. Strikingly, TSA treatment dramatically enhanced StAR protein levels in the primary luteal cells (Fig. 9BGo).


Figure 9
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  		FIG. 9. Effect of HDAC inhibitor TSA on StAR promoter activity and protein expression. A, Luciferase assay results. HEK 293T cells were transfected with the p1862 StAR promoter construct with the YY1-pCMV5 or the empty pCMV5 expression vector together with equal amount of renilla luciferase plasmid in each well, and treated with 100 nM TSA or vehicle for 24 h. Luciferase assays were performed. The graph represents the average luciferase activity ± SEM of three independent experiments after normalization to the renilla luciferase activity. *, Reduction (P < 0.001) of StAR promoter activity by YY1 compared with the empty pCMV5 vector. #, Induction (P < 0.001) of StAR promoter activity by TSA compared with vehicle. B, Immunoblot. Rat luteal cells were obtained and cultured. Three days later, cells were treated with TSA at various concentrations. Twenty-four hours later, cell lysates were prepared for and subjected to SDS-PAGE then transferred to nitrocellulose and immunoblotted with StAR and β-actin antibodies.

 
It has been previously reported that YY1 binds HDACs and regulates their activity (11, 45). Therefore, we attempted to detect whether YY1 altered HDAC binding to the StAR promoter. ChIP assays detected the association of HDAC1 protein to StAR promoter region 4 (Fig. 10Go). When YY1 was knocked down by RNA interference, we observed a decrease to 37 ± 3% (P < 0.005) in HDAC1 binding compared with control siRNA-treated cells (Fig. 10Go, A and B).


Figure 10
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  		FIG. 10. Effects of YY1 knockdown and PGF2{alpha} on HDAC1 binding affinity to StAR promoter region 4. A, YY1 siRNA knockdown abrogated HDAC1 binding to StAR promoter region 4. YY1 protein expression was knocked down in rat luteal cells by siRNA transfection. ChIP assays were performed 48 h later to determine HDAC1 binding to StAR promoter using the region 4 PCR primer pair for PCR amplification. Result shown was the representative of three independent experiments. B, Densitometry results for A. C, PGF2{alpha} treatment enhanced HDAC1 binding to StAR promoter region 4. Rat luteal cells were treated with 1 µM PGF2{alpha} for 12 h. ChIP assays were performed to detect HDAC1 binding to StAR promoter region 4. Result shown was representative of three independent experiments. D, Densitometry results for C. The relative density of ChIP/input was set to 1 U for both the control siRNA-transfected luteal cells in B and the vehicle-treated cells in D by normalization of the mean values of the respective control groups. The graphs represent the average ± SEM. *, Change (P < 0.005) in HDAC1 binding to StAR promoter by YY1 knockdown (B) or by PGF2{alpha} treatment (D) compared with their respective control group.

 
Conversely, PGF2{alpha} treatment, which increased YY1 protein expression (Figs. 1Go and 2Go), enhanced HDAC1 binding to StAR promoter region 4 by 3.1 ± 0.2-fold (P < 0.005) (Fig. 10Go, C and D). These results indicated that the binding affinity of HDAC1 to its cis-element in this promoter region is proportional to YY1 protein expression.

ChIP assays using an antibody against acetyl histone H3 were performed to test whether HDAC1 recruitment regulated histone deacetylation in StAR promoter region 4. Figure 11Go shows that the knockdown of YY1 enhances basal histone H3 acetylation levels by 2.7 ± 0.1-fold (P < 0.001) and PGF2{alpha} treatment decreases acetyl histone H3 levels by 78 ± 2% (P = 0.002) in control siRNA-treated compared with a decrease of only 11 ± 4% (P = 0.13) observed in YY1 siRNA-treated luteal cells.


Figure 11
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  		FIG. 11. Effects of YY1 knockdown and PGF2{alpha} on histone H3 acetylation in StAR promoter region 4. Rat luteal cells were subjected to control or YY1 siRNA transfection for 48 h and treated with vehicle or PGF2{alpha} for 24 h before cell lysis. ChIP assays were performed to determine the nucleosome acetyl histone H3 in StAR promoter region 4 using the corresponding PCR primer pair for PCR amplification. A, Detection of PCR-amplified DNA fragments in ChIP and input. The result shown is the representative of three independent experiments. B, Densitometry results. The relative value of ChIP/input is set to 1 U in control siRNA-transfected luteal cells treated with vehicle by normalization of the mean value of this control group. The graph represents the average ± SEM of three independent experiments. *, Reduction (P < 0.005) of acetylated histone H3 level by PGF2{alpha} treatment compared with vehicle treatment. #, Induction (P < 0.001) of acetyl histone H3 level by YY1-siRNA compared with control siRNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides evidence that PGF2{alpha} treatment induces YY1 and suppresses StAR protein expression via the recruitment of HDAC1 by YY1 to the StAR promoter in rat luteal cells. YY1 binds to one region in the StAR promoter, which is essential for the suppressive effect of YY1 on StAR promoter activity. HDAC1 binds to the same region and YY1 is essential for this binding. The prevention of deacetylation activity by a HDAC inhibitor abrogates the effect of YY1 on StAR gene expression. These results suggest a regulatory pathway for how PGF2{alpha} represses StAR protein expression under physiological conditions.

Although the effects of PGF2{alpha} on YY1 and StAR protein gene expression have been previously observed in separate reports (10, 39), the present study is the first to elucidate the time-dependent patterns of the two distinct events and to examine their relationship. In a rat luteal cell culture model, we observe that the increase in YY1 protein levels in PGF2{alpha}-treated cells is followed by a reduction in StAR protein expression. Considering that YY1 is a transcriptional repressor for many genes, the sequential effects of PGF2{alpha} on YY1 induction and StAR repression suggests that the first event is responsible for the second one. The decrease in StAR mRNA transcript levels by PGF2{alpha} treatment also indicates that StAR gene expression is regulated at the transcription stage. Wild-type YY1, but not its mutant {Delta}154–199, suppresses StAR gene promoter activity in the luciferase assay, implying that the transcriptional repression domain in YY1 is essential for the effect. The fact that knockdown of YY1 using the RNA interference technique in luteal cells leads to an increase in StAR protein levels and negates the PGF2{alpha} effect further supports the causal role of YY1 on StAR gene expression.

We also show that the binding of YY1 to the StAR gene promoter is essential for the effect of YY1 as a transcriptional repressor. In the luciferase assay, YY1 suppresses the StAR gene promoter constructs p1862 and p1413, but has no effect on the short form, p998. ChIP assays demonstrate that YY1 binds directly to BS1 in StAR gene promoter region 4 (–1193 to –1382 nucleotides), which is included in p1862 and p1413 but not in p998. Knockdown of YY1 gene transcripts reduces the binding of HDAC1 to the StAR promoter in the same region, suggesting that the binding of YY1 to the promoter might recruit the HDAC1 protein to, and enhance deacetylation activity in the same region of the promoter. Because HDAC1 is a protein that generally suppresses the transcriptional activity of many genes (46), these data demonstrate that YY1 invokes the negative regulation of StAR gene expression through binding to region 4 of the StAR promoter.

The importance of HDAC1 recruitment by YY1 to the StAR promoter is further supported by the promoter activity assay. The fact that the HDAC inhibitor TSA increases p1862 luciferase activity indicates that StAR gene promoter activity is negatively regulated by deacetylation. The observation that YY1 exerts a negligible effect on TSA-induced StAR promoter activity implies that deacetylation is a downstream event of the regulation by YY1 of StAR gene expression. The demonstration that HDAC1 binding to the StAR promoter in the same region of the YY1 BS1 is enhanced by PGF2{alpha} treatment further confirms the involvement of HDAC1 on both PGF2{alpha}- and YY1-regulated StAR gene expression. The results of the promoter activity assay are in agreement with what is observed in the primary luteal cells. TSA treatment significantly enhances StAR protein expression in luteal cells, providing a rationale for using the luciferase assay system to assess transcriptional activity. The effects of PGF2{alpha} and YY1 on regulating deacetylation activity are further confirmed by the observation that nucleosome acetyl histone H3 levels display a reverse pattern compared with the binding affinity of HDAC1 within the same region. These experiments demonstrate that the effects of PGF2{alpha} and YY1 on StAR protein expression are mediated by regulating deacetylation activity in the promoter region.

The unresolved question in this study is whether SREBP-1a is involved in the regulation of the effect of YY1 on StAR gene expression. Although YY1 suppresses SREBP-1a-induced StAR promoter activity, we could not determine whether YY1 directly interacts with SREBP-1a or exerts its general inhibition on StAR promoter function independent of the SREBP-1a transactivation effect. In humans, one of the SREBP-1a response elements is overlaid with one of the YY1 binding sequences in the StAR promoter proximal region (9), which is analogous to the StAR promoter region 1 in the rat. Mutation of this YY1-binding site increases the responsiveness of the StAR promoter to exogenous SREBP-1a. However, the rat StAR promoter sequence has several nucleotide changes in this area (10). These changes were found to have a major impact on the binding profile such that this site of the rat StAR promoter is not associated with either SREBP-1a or YY1 binding in the cells used for this study and in the EMSA in a previous report (10). There are two sterol regulatory elements in regions 4 and 5 that are identified to bind SERBP-1a in this study, both of which are upstream of the YY1 binding sequence, and one is very close to YY1 BS1. Because SREBP-1a activity is regulated by acetylation/deacetylation (47), it is possible that YY1-recruited HDAC1 might deacetylate SREBP-1a, hence decreasing its transactivation activity. In contrast, it has been noticed that SREBP-1a is an inefficient transcriptional regulatory factor by itself, and requires a coregulatory factor to efficiently activate the SREBP target genes (48, 49). Because there are two NF-Y binding sites close to these two SRE sequences, it has been suggested that mature SREBP enters the nucleus and activates transcription of the StAR gene by a process that is dependent upon binding of SREBP and NF-Y to the promoter (10). Therefore, we cannot rule out the possibility that YY1 might disrupt the formation of a SREBP-1a/NF-Y complex and/or obstruct the binding of the complex to the StAR promoter, thereby achieving transcriptional repression of StAR gene expression.

We have previously reported that PGF2{alpha} treatment increases DAX-1 protein levels and DAX-1 suppresses StAR gene expression using overexpression studies and reporter gene assays in a heterologous system (39). The effect of DAX-1 on StAR gene expression remains to be confirmed in the physiological state. Although DAX-1 binds to the StAR promoter proximal region hairpin (nucleotide positions –65 to –31) (50) and YY1 binds to the distal promoter region in rat, a more precise study is required to explore the relationship of these two molecular changes.

PGF2{alpha} is believed to be the physiological agent responsible for corpus luteum regression at the end of a nonfertile cycle (51, 52). The luteolytic effect of PGF2{alpha} is likely to be mediated by the decrease in progesterone in luteal cells (41, 51, 52, 53, 54, 55, 56). PGF2{alpha} stimulates the metabolism of progesterone by inducing the expression of 20{alpha}-hydroxysteroid dehydrogenase (57), which converts progesterone into a biologically inactive steroid. PGF2{alpha} also reduces progesterone anabolism by reducing cholesterol transport to the inner mitochondrial membrane through the suppression of StAR gene expression. Therefore, PGF2{alpha} exerts its function by reducing progesterone availability through two mechanisms: decreasing synthesis and increasing consumption. In this study, we demonstrate that YY1 induction and increased deacetylation mediate the effect of PGF2{alpha} on StAR gene expression. Therefore, the physiological effect of PGF2{alpha} on the corpus luteum might be modulated by these two downstream events.


   Acknowledgments
 
We thank Dr. Tom Shenk (Howard Hughes Medical Institute, Princeton University, Princeton, NJ) for the gift of full-length pCMV-YY1 plasmid, Dr. Bernhard Luscher (Institute for Molecular Biology, Hannover Medical School, Hannover, Germany) for the gift of the YY1 deletion mutant plasmid, and Dr. Tao Wang (Department of Epidemiology and Biostatistics, University of South Florida, Tampa, FL) for statistical advice.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD35163 to M.P.M.

The authors have nothing to disclose.

First Published Online August 16, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; HDAC, histone deacetylase; NF-Y, nuclear factor Y; PGF2{alpha}, prostaglandin F2{alpha}; siRNA, small interfering RNA; SREBP-1a, sterol regulatory element binding protein-1a; StAR, steroidogenic acute regulatory; TSA, trichostatin A; YY1, Yin Yang 1.

Received March 8, 2007.

Accepted for publication August 7, 2007.


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Endocrinology, May 1, 2008; 149(5): 2090 - 2097.
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