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Endocrinology Vol. 143, No. 3 1085-1096
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Repression of the Steroidogenic Acute Regulatory Gene by the Multifunctional Transcription Factor Yin Yang 1

Anna C. Nackley, Wendy Shea-Eaton, Dayami Lopez and Mark P. McLean

Departments of Obstetrics and Gynecology (A.C.N., W.S.-E., D.L., M.P.M.) and Biochemistry and Molecular Biology (M.P.M.), University of South Florida, Tampa, Florida 33606

Address all correspondence and requests for reprints to: Mark P. McLean, Ph.D., Department of Obstetrics and Gynecology, University of South Florida, Harbourside Medical Tower, Suite 529, 4 Columbia Drive, Tampa, Florida 33606. E-mail: . mmclean{at}hsc.usf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transport of cholesterol to the inner mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein is a critical step in steroidogenesis. The current study was designed to examine whether the multifunctional transcription factor Yin Yang 1 (YY1) was able to repress activation of the StAR gene. YY1 bound to three putative YY1-binding sites in the rat StAR promoter. Cotransfection of the StAR promoter linked to a luciferase reporter gene and YY1 in the presence or absence of sterol regulatory element binding protein-1a (SREBP-1a) resulted in transcriptional repression. YY1 was found to colocalize in the nucleus with SREPB-1a. YY1 inhibited SREBP-1a/nuclear factor Y (NF-Y) enhancement of StAR activation and YY1 decreased SREBP-1a binding to a sterol regulatory element in the presence or absence of NF-Y. YY1 also decreased NF-Y binding to a nonconsensus NF-Y-binding motif in the StAR promoter. These studies provide novel information on the mechanisms of YY1-mediated repression of the StAR gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRODUCTION OF STEROID hormones is essential to human survival and reproductive function. Steroid hormone biosynthesis can occur under basal conditions or through acute and chronic regulation by tropic hormones originating from the hypothalamus and anterior pituitary gland (1, 2). The binding of tropic hormone to its receptor activates adenylate cyclase with the subsequent activation of cAMP, which promotes steroidogenesis through induction of steroidogenic genes (1, 2). The rate-limiting step in steroidogenesis is the transfer of cholesterol from the outer to the inner mitochondrial membrane, which is mediated by the steroidogenic acute regulatory (StAR) protein (3, 4). StAR was found to play a vital role in adrenal and gonadal steroidogenesis by providing a continuous supply of cholesterol to the P450 side-chain cleavage enzyme, the first enzymatic step in steroid hormone biosynthesis (5). A rapid increase in steroid hormone production in ovarian tissue in response to tropic hormone stimulation was associated with increased expression of the StAR gene (6). A similar treatment regimen with tropic hormones in ovarian tissue resulted in an increase in the transcription factor, sterol regulatory element binding protein 1a [SREBP-1a (7)]. SREBP-1a is synthesized as a membrane-bound precursor that is attached to the endoplasmic reticulum membrane and nuclear envelope (8, 9, 10, 11, 12). Under conditions of sterol depletion, a two-step proteolytic process releases the NH2-terminal segment of the SREBP-1a, which then enters the nucleus, binds to the sterol regulatory element (SRE), and activates transcription of sterol sensitive genes (8, 9, 10, 11, 12). The rat StAR promoter was found to contain five SRE-binding sites through which SREBP-1a binds and activates transcription (13). In addition, Christenson et al. (14) have identified an additional SRE/YY1 (the multifunctional transcription factor Yin Yang 1)-binding site (-81 to -70 from the start site of transcription) through which the human StAR promoter was found to be conditionally responsive to high levels of SREBP-1a.

SREBP-1a is known to be a weak transcriptional activator and optimal activation of sterol responsive genes involves other cofactors that work synergistically with SREBP-1a such as the promoter-specific transcription factor (Sp1) and nuclear factor Y (NF-Y). Sp1 has been shown to be a coactivating factor with SREBP-1a for the low-density lipoprotein receptor (15) and the high-density lipoprotein receptor (5), and NF-Y has been shown to be a coactivating factor with SREBP-1a for the StAR (13), the farnesyl diphosphate synthase (16), and 3-hydroxy-3-methylglutaryl-coenzyme A synthase genes (2). An additional regulatory mechanism was recently identified that revealed a requirement for both Sp1 and NF-Y in sterol regulation of the human fatty acid synthase promoter (17) and the rat 7-dehydrocholesterol reductase gene (18).

Although activators of the StAR gene have been studied in great detail, transcription factors that repress StAR expression have received less attention. One candidate for negative regulation of the StAR gene is YY1, a unique transcription factor that has the capability of acting as repressor, activator, or initiator of gene transcription (19). Multiple mechanisms for YY1 inhibition of transcription have been proposed. One mechanism involves YY1 binding to an activator to occlude its recognition site (20). As an activator-specific repressor, YY1 has been described as both a type I (interfering directly with an activator) and type II (interfering with targets of an activator) repressor (20). The second mechanism involves direct binding of YY1 to a promoter to bend the DNA and disrupt activator interaction (21). Another proposed mechanism involves YY1-mediated recruitment of histone deacetylases that result in transcriptional silencing (22). YY1 has been shown to repress the low-density lipoprotein receptor (LDL-R) (23, 24) and the high-density lipoprotein receptor (HDL-R) (5) gene transcription in a dose-dependent manner by specifically targeting SREBP-1a interaction with Sp1.

In the current study, the effect of YY1 on StAR gene transcription was examined under basal conditions (promoter in the absence of SREBP-1a) and after SREBP-1a-mediated induction in the presence and absence of the cofactors Sp1 and NF-Y.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Integrated DNA Technologies, Inc. (Coralville, IA) synthesized all oligonucleotides and primers. The 2.0-kb rat StAR promoter was cloned and used in the preparation of the StAR promoter-luciferase gene constructs as previously described (13). The pGL3-basic luciferase vector, renilla luciferase vector, and dual luciferase reporter assay system were obtained from Promega Corp. (Madison, WI). The QuickChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). The human bladder HTB-9, COS-7, and mouse Y1 adrenal cells were obtained from American Type Culture Collection (Manassas, VA). The GST-YY1 and pCMV-YY1 expression plasmids were a gift from Thomas Shenk (Department of Molecular Biology, Princeton University, Princeton, NJ). The GG-CL cells were a generous gift from Geula Gibori (Department of Physiology and Biophysics, University of Illinois at Chicago). NF-Y A, B, and C in pCITE were obtained from Dr. Sankar Maity (M.D. Anderson Cancer Center, Houston, TX). After introduction of an EcoRI site into NF-Y B and C, all three cDNAs were transferred into pcDNA 3.1 using EcoRI and XhoI. The NH2-terminal segment (active fragment) of SREBP-1a under the control of the cytomegalovirus (CMV) promoter (SREBP-1a-pCMV5) and SREBP-1a-polyhistidine-tagged in the pRSET B vector was kindly provided by Dr. Tim Osborne (Department of Molecular Biology and Biochemistry, University of California, Irvine). The pEYFP-C1 and pECYFP-C1 vectors were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). All Promega Corp. restriction enzymes, Biomax-MR film, FBS, and chemicals were obtained from Fisher Scientific (Norcross, GA).

Animal model
Twenty-eight-day-old Sprague Dawley rats were purchased from Harlan Industries (Madison, WI). All procedures for PG treatment and the methods for tissue sampling were approved by the University of South Florida Animal Care Committee. 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 rats (25) by injection of 8 IU PMSG (sc). Ten days following ovulation, rats were given a single injection (im) of PGF2{alpha} (250 µg). Ovaries were removed before PGF2{alpha} injection (control) and at 2 and 4 h post PGF2{alpha} treatment.

SDS-PAGE and Western blots
Ovarian tissue (100–150 mg) obtained from control or PGF2{alpha}-treated rats was homogenized and 50 µg extracted protein was loaded on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and Western blotting was performed as previously described (7). The blots were probed with a rabbit polyclonal antibody against YY1 (Geneka Biotechnologies, Montréal, Québec, Canada). Immunoreactive proteins were visualized using horseradish peroxidase-conjugated goat antirabbit antisera (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the SuperSignal ULTRA Chemiluminescent Substrate method (Pierce Chemical Co., Rockford, IL).

Cell transfection and luciferase assays
HTB-9 or COS-7 cells were transfected with the specified StAR promoter-luciferase reporter gene construct either in the presence or absence of SREBP-1a-pCMV5 and/or YY1-pCMV5. Cell culture conditions have been described in detail elsewhere (5). Purified plasmids were transfected using Fugene 6 (Roche, Indianapolis, IN), treated with a passive lysis buffer, and luciferase assays were performed using the dual luciferase reporter assay system (Promega Corp.). Cotransfection of a plasmid containing the renilla luciferase gene in the pGL3 (basic) vector was used as a control to correct for differences in transfection efficiencies.

Site-directed mutagenesis
Site-directed mutants were obtained using the QuickChange site-directed mutagenesis kit (Stratagene) as described previously (13). The mutations were confirmed by sequencing using the T7 Sequenase DNA sequencing kit and [35S]-dATP. An oligonucleotide (with mutations underlined) used to mutate the (-80 to -76) NF-Y binding site was (5'-CAGTGACTTTTGAATTTTTTAT-3'). The primer (5'-CAGTTACTGGGTACCTAAGTGAATG-3') and its complement were used to introduce a KpnI restriction site in the StAR promoter in the preparation of the p-291 construct. The primers used to mutate the YY1-binding sites (YY1 BS) were YY1 BS1 (5'-GGCTTGGCTATTTATGTGGTTCCA-3'), YY1 BS2 (5'GTTGTGCGTCGTTTTTTGGTTGCT-3'), and YY1 BS3 (5'CCAGGTTAGATTATCTTACTTTTTTAG-3').

EMSA
YY1 oligonucleotide probes are as follows: YY1 BS1 (5'-GGCTGTCCATGTGGTTCCACAAAT-3'), YY1 BS2 (5'-GCGTCGTCATGTGGTTCCTGGGAA-3'), YY1BS3 (5'-GAGGTTAGACATTTTTACTTTTTT-3'), SRE/YY1 (5'-GGGTGCACAGTGACTGTTGGCTT-3'), and SRE 3 (5'-TGGATGTGTCATCTCATATCCAGA-3'). The probes were labeled using T4 polynucleotide kinase (Promega Corp.) and [{gamma}32P] ATP (NEN Life Science Products, Boston, MA). An oligonucleotide containing the YY1-binding site from the intracisternal A-particle (5'-AAGACGTCGCCATCTTGTCTTACGTCA-3') was used as a positive control (5). Unlabeled wild-type oligonucleotides (50–100x excess) were used as specific competitors (cold) in some experiments and antibodies against YY1 (Geneka Biotechnologies Inc.), NF-Y (Chemicon, Temecula, CA), or SREBP-1a (Santa Cruz Biotechnology, Inc.) were used for supershift analysis. A mutant oligonucleotide (5'-GGGGATCAGGGTCTTTGTTTTGAAGCGGGATCTCCC-3'; YY1 gel shift kit, Geneka Biotechnology Inc.) was used as a nonspecific competitor in the YY1 mobility shift assays. GST-YY1 fusion protein and histidine-tagged SREBP-1a fusion protein were prepared as described previously (26). For preparation of nuclear extracts, HTB-9 cells were transfected with NF-Y (A, B, and C) in pcDNA 3.1, nuclear proteins prepared, and EMSA was performed as previously described (13).

Data analysis
Luciferase data were expressed as the fold difference from activity seen with the StAR promoter alone ± SEM. The luciferase activity of promoter alone was set to 1. Each transfection experiment was done three times and performed in triplicate. Quantitation of DNA-protein complex signals in the mobility shift assay autoradiographs was performed using an image acquisition system (UVP, Upland, CA). Data from the individual parameters were compared by ANOVA followed by Student-Newman-Keuls multiple comparison test when applicable. All analysis was completed using the Statview program [+graphics (Abacus Concepts, Berkeley, CA)]. Values of P < 0.05 were considered significant for all tests.

Fluorescence resonance energy transfer
Y1 adrenal cells were directly cultured onto Lab-Tek chamber glass slides and cotransfected with cyan fluorescent protein SREBP-1a-pECFP-C1 and yellow fluorescent protein YY1-pEYFP-C1 plasmids using standard transfection methods as previously described (5). The fluorescence resonance energy transfer (FRET) imaging microscope used for these experiments was equipped for epifluorescence and transmitted illumination (BX60, Olympus Corp. America, Inc., Melville, NY). Fluorescence images were acquired 24 h after transfections. The excitation light source was a 100-watt mercury arc lamp (Hamamatsu Corp., Middlesex, NY) coupled to excitation and neutral density filters, Chroma cyan/Topaz FRET filter set (535/30 nm). After fixing the slides, the images were captured using an Optronics DEI-750 3-chip cooled video camera and analyzed using ImagePro Plus and FluoroPro data analysis software (Media Cybernetics, Silver Spring, MD). The slides were viewed using specific FRET filter sets, and the values obtained for the background fluorescence (mock transfection with one empty vector and YY1 or SREBP-1a in the second vector) were subtracted from the values obtained for each transfected sample. The corrected sample fluorescence was measured with each of the three separate filter sets and relative differences in the presence of a single or both proteins were compared and calculated mathematically as described elsewhere (27, 28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of YY1 protein levels in rat ovaries
Previous studies have demonstrated that StAR gene expression in the rat ovary is regulated by gonadotropins (6) and PGF2{alpha} (25). To examine whether expression of YY1 was regulated in a manner consistent with regulation of the StAR gene, YY1 protein levels were measured up to 4 h after treatment with PGF2{alpha} (Fig. 1Go, A and B) or human CG [hCG (Fig. 1BGo)]. By 4 h of PGF2{alpha} treatment, YY1 protein levels detected on a Western blot were almost 3-fold higher than the time zero controls (Fig. 1AGo). Figure 1BGo depicts a graphical representation of three Western blots after PGF2{alpha} treatment, including the Western blot shown in Fig. 1AGo, plotted simultaneously with two other Western blots run on hCG-treated samples (data not shown). The levels of YY1 protein increased under conditions (treatment with PGF2{alpha}) previously shown to decrease StAR protein (25); similarly, YY1 protein levels were low under conditions (treatment with hCG) previously shown to increase StAR gene expression (6), suggesting a possible scenario of repression of StAR gene activity by YY1.



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  		Figure 1. Regulation of YY1 protein levels in rat ovaries. Twenty-eight-day-old Sprague Dawley rats were injected with 8 IU PMSG and 10 d after ovulation were injected with 250 µg PGF2{alpha} (A and B) or 25 U hCG (B), and ovarian tissue was harvested at the indicated times. A, Western blots of two ovarian protein extracts probed with YY1 and ß-actin antibodies. This experiment was repeated three times in duplicate and one time in triplicate. B, Graphical representation of three separate YY1 Western blots for PGF2{alpha} and two Western blots for hCG. Quantitation of the immunoreactive YY1 and actin protein signals were performed using the UVP chemiluminescence system and were normalized to the time (0) control. *, P < 0.005, compared with the time (0) control.

 
YY1 binding to the StAR promoter
A schematic showing the location of the YY1-, SRE-, Sp1-, and NF-Y-binding sites, including the SRE/YY1 site previously reported for the human StAR promoter (29) and an additional putative NF-Y-binding site that overlaps the SRE/YY1 site is shown in Fig. 2AGo. The nucleotide sequences for the three putative YY1 BS in addition to the SRE/YY1-binding site are shown in Fig. 2BGo. It should be noted that the rat SRE/YY1 sequence has two nucleotides that are different from the human StAR SRE/YY1 binding motif and that one of these changes disrupts the inverted CCAT motif necessary for YY1 binding. EMSA was performed (Fig. 2BGo) using oligonucleotides containing one of the three putative YY1 sites, the rat SRE/YY1 site, or a consensus YY1-binding site previously described (5). All three YY1-binding sites in the rat StAR promoter and the control consensus sequence showed high binding affinity for recombinant YY1 protein (rYY1), but the SRE/YY1 site did not bind rYY1 under similar conditions. The binding of the YY1 protein to the three YY1-binding sites in the StAR promoter was specific because 50-fold molar excess of unlabeled oligonucleotide (cold) was able to prevent YY1 binding to the DNA (Fig. 2BGo), whereas the same amount of a mutated oligonucleotide, provided as a YY1 noncompleting negative control in the gel shift kit (see Materials and Methods), had no effect on YY1 binding (data not shown).



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  		Figure 2. Recombinant YY1 bound to the YY1 BS in the StAR promoter. A, Schematic drawing of the portion of the rat StAR promoter containing the putative SREBP-1a-, Sp1-, NF-Y-, and SREBP/YY1-binding sites. B, Electrophoretic mobility shift assays using 32P-labeled double-stranded oligonucleotide probes shown above (50,000 cpm/lane) and 500 ng of recombinant YY1 were performed in the presence or absence of 50x-fold molar excess unlabeled oligonucleotide (cold) as described in Materials and Methods. This experiment was repeated four times, and a representative mobility shift assay autoradiograph is shown. The consensus YY1 BS was used as a positive control.

 
YY1 repression of basal and SREBP-1a-induced StAR promoter activation
The p-1862 construct of the rat StAR gene, which contains the three YY1-binding sites and the SRE/YY1-binding site as well as all five SREBP-1a binding sites (SRE), was cotransfected into HTB-9 cells in the presence or absence of SREBP-1a and YY1 and luciferase assays were performed. As shown in Fig. 3AGo using a standard ratio of StAR promoter DNA:YY1 DNA of 2:1, YY1 had little effect on basal (promoter activity in the absence of SREBP-1a) activity but caused a significant reduction in SREBP-1a induction of StAR promoter. However, when the concentration of YY1 to p-1862 promoter is increased up to 2 µg (1:1 ratio) or higher, there was a significant reduction in basal StAR promoter activity in HTB-9 cells (Fig. 3BGo). These dose response studies were repeated with the rat ovarian GG-CL cell line with similar results (data not shown). This inhibitory effect was specifically because of YY1 and not because of a nonspecific quenching effect seen when high amounts of DNA are transfected as the amount of DNA was kept constant for all groups by the transfection of vector alone where necessary.



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  		Figure 3. Effects of YY1 on luciferase expression under the control of the StAR promoter. A, HTB-9 cells were cotransfected with the p-1862 rat StAR promoter construct (2 µg) either in the presence or absence of pCMV5-expression vectors for SREBP-1a and YY1 (1 µg each), and luciferase assays were performed as described in Materials and Methods. This graph represents results from three separate experiments and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. *, P < 0.001, compared with the p-1862 promoter alone; **, a reduction (P < 0.005) by YY1, compared with the promoter in the presence of SREBP-1a. B, HTB-9 cells were transfected with the p-1862 construct (2 µg) in the presence or absence of expression vectors for YY1 as indicated. This graph represents results from three separate experiments, and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. **, A reduction (P < 0.05 or less) by YY1, compared with the promoter alone.

 
Mutation of YY1-binding sites did not prevent YY1-mediated repression of SREBP-1a-induced StAR activation
To determine whether YY1 BS 1, 2, and 3 were required for YY1-mediated repression of the rat StAR promoter, site-directed mutagenesis was performed on the p-1862 promoter construct. The wild-type p-1862 and the mutated promoter (mBS1/2/3/p-1862) were transfected into HTB-9 cells and luciferase assays were performed (Fig. 4Go). There was no specific binding of rYY1 to any of the mutated YY1 BS as assessed by EMSA (data not shown). Mutation of all three YY1 BS did not affect basal promoter activation or YY1 repression of SREBP-1a induced promoter luciferase activation (Fig. 4Go) suggesting that YY1 binding to these sites was not necessary for the repression that occurred.



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  		Figure 4. Effect of site-specific mutation of the three-major YY1 BS on YY1-mediated repression. A, Site-directed mutagenesis was used to remove YY1 BS 1, 2, and 3 from the p-1862 StAR promoter construct (mBS1/2/3/p-1862) as listed in Materials and Methods, and HTB-9 cells were transfected with 2 µg each of the wild-type p-1862 construct or mBS1/2/3/p-1862 in the presence or absence of pCMV5-expression vectors for SREBP-1a (1 µg) and/or YY1 (1 µg), and luciferase assays were performed. This graph represents results from three separate experiments, and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. *, P < 0.0005, compared with the p-1862 promoter alone; **, a reduction (P < 0.001) by YY1, compared with the promoter in the presence of SREBP-1a.

 
YY1 interaction with SREBP-1a
Because an intact YY1 BS was not required for inhibition of SREBP-1a mediated activation of the rat StAR promoter, it was probable that YY1 was repressing StAR activity via interaction with SREBP-1a protein. Previous two-hybrid studies by this laboratory have demonstrated direct protein-protein interaction between YY1 and SREBP-1a with the subsequent disruption of SREBP-1a-mediated activation of the rat high-density lipoprotein receptor (5). To confirm these results and determine whether YY1 and SREBP-1a colocalize in the nucleus, FRET analysis was performed in Y1 cells. Y1 represents a widely characterized steroidogenic cell line, and we have found that the Y1 cell growth pattern is well suited for FRET analysis. A control luciferase experiment was initially performed to demonstrate that the presence of a fluorescent protein tag did not interfere with SREBP-1a and YY1 protein functions (Fig. 5AGo). Fluorescent-tagged SREBP-1a protein was still able to induce the rat StAR promoter and fluorescent-tagged YY1-repressed SREBP-1a induced activation. The relative activation with SREBP-1a using the FRET vectors is lower (Fig. 5AGo). This is because of the presence of the fluorescent tag on the expression vectors. The fluorescence emitted from the FRET vectors enhances the overall activity obtained from both the luciferase and renilla (control to correct for differences in transfection efficiencies) vectors causing an overcorrection in the transfection efficiency, and consequently, the fold-induction appears lower. The 2-fold induction in the luciferase activity following SREBP-1a expression and the repression by YY1 remain significant (P < 0.001 and P < 0.005, respectively). Expression of mature SREBP-1a protein with a cyan-tagged fluorescent protein was located mainly in the nucleus of Y1 cells (Fig. 5BGo, panel 1). When the YY1 protein labeled with a yellow fluorescent tag was coexpressed with the cyan-tagged SREBP-1a protein and viewed under a specialized FRET filter, both proteins were found to colocalize in the nucleus in Y1 cells (Fig. 5BGo, panel 2). Measurement of the average nuclear fluorescence in Y1 cells demonstrated a loss in cyan emission as fluorescent energy was transferred to the YY1-pECFP acceptor protein (Fig. 5CGo, 1 vs. 2) and an increase in fluorescence when both SREBP-1a and YY1 proteins are in close proximity and detected under the FRET filter (Fig. 5CGo, 5 vs. 6).



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  		Figure 5. FRET analysis. A, Effect of the fluorescent-protein-tagged SREBP-1a and YY1 on luciferase expression under the control of the StAR promoter. HTB-9 cells were transfected with the p-1862 rat StAR promoter construct either in the presence or absence of expression vectors for SREBP-1a and YY1, which link these proteins to a fluorescent protein (pECFP-C1 vector for cyan or pEYFP-C1 for yellow) and luciferase assays were performed as described in Materials and Methods. This graph represents results from three separate experiments, and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. *, P < 0.001, compared with the p-1862 promoter alone; **, a reduction (P < 0.005) by YY1, compared with the promoter in the presence of SREBP-1a. B, A total of 2 x 105 Y1 adrenal cells were seeded on sterile glass slide cassettes and fed with DME: F12 media containing 10% FBS for 48 h. The cells were transfected with 1 µg each of two of the following vectors: SREBP-1a in pECFP-C1 or YY1 in pEYFP-C1. After 24 h, the slides were fixed in 4% freshly prepared paraformaldehyde, and three successive digital images (40x or 60x) were acquired using three separate filter settings (cyan, yellow, or FRET) and processed (after background subtraction) using FluoroPro Plus software. The cyan or blue donor CFP images (B, panel 1, 40x) were acquired by exciting the CFP donor at 433 ± 10 nm and detecting CFP donor emission at 480 ± 15 nm. The FRET images (B, panel 2, 40x) were acquired from the YFP acceptor (emission 535 ± 15 nm) while exciting the CFP donor at 433 nm. Under these conditions, excitation of the cyan donor will transfer energy to the yellow acceptor and the selected FRET filter set will pick up the yellow acceptor emissions. The loss of cyan emission because of transfer of cyan emission energy to the yellow acceptor in FRET was 8–15% over two separate experiments, and the actual FRET emission (after subtraction of the mock "empty" vector in one FRET vector, protein in second vector transfection and single protein controls in both FRET vectors) was routinely between 8 and 10%. C, Table depicting results of a typical series of FRET experiments in Y1 cells. The amount of nuclear fluorescence units was detected using the cyan or the yellow filters in the presence of one or both proteins (YY1/SREBP-1a) and compared with the fluorescence detected using the FRET filter to give an estimate of the efficiency of FRET. The data represent three separate experiments performed in duplicate. The average nuclear fluorescence in each experiment represents n = 50 or more cells.

 
Cofactors partially protect against YY1 repression of SREBP-1a binding and activation of rat StAR promoter
To determine whether the presence of a cofactor could prevent YY1 repression of SREBP-1a induction of StAR activity, the p-1862 rat StAR promoter was cotransfected into COS-7 cells with expression vectors for YY1, SREBP-1a, and/or NF-Y. COS-7 cells were chosen because they respond well to enhancement by NF-Y and SREBP-1a. NF-Y could not prevent YY1 repression of SREBP-1a induction of the p-1862 rat StAR promoter (Fig. 6AGo). EMSA studies were undertaken using SRE 3 from the StAR promoter. SRE 3 was chosen because it exhibited the highest affinity for SREBP-1a (13); however, a similar binding-inhibition pattern in the presence of YY1 was seen with the other four SREs (data not shown). The presence of rYY1 was able to decrease the major SREBP-1a/SRE binding complex in the absence of NF-Y in a dose-dependent manner and resulted in the appearance of several smaller sized bands (small arrows) when analyzed by EMSA (Fig. 6BGo, lanes 4–6). Subsequent analysis with specific YY1 antibody resulted in a diffuse smear located between the two smaller bands (data not shown), suggesting that YY1 was a component in this complex. The presence of NF-Y also enhanced SREBP-1a binding to SRE 3 (Fig. 6CGo, lane 2) and partially protected against YY1 inhibition of SREBP-1a binding even in the presence of twice the amount of rYY1 (Fig. 6CGo, lane 3 vs. 6B, lane 6). Higher doses of rYY1 were able to almost completely prevent SREBP-1a binding to SRE 3 in the presence of NF-Y (Fig. 6CGo, lanes 4 and 5). The SREBP-1a enhancement experiments were repeated with another transcription factor, Sp1, to determine whether the ability of YY1 to repress SREBP-1a/coactivator induction was cofactor specific (Fig. 7Go). The p-1862 rat StAR promoter was cotransfected into COS-7 cells in the presence or absence of Sp1, SREBP-1a, or YY1 (Fig. 7AGo). This graph is a composite of three separate experiments presented as fold difference relative to promoter activity alone to increase sample number. There was no synergistic activation of the rat p-1862 StAR promoter by SREBP/Sp1; in addition, YY1 did not inhibit SREBP-1a/Sp1 induction of StAR promoter activity (Fig. 7AGo). Site mutation of the single Sp1 site in the rat StAR promoter had previously shown a minor decrease SREBP-1a induction of luciferase activity in a StAR promoter construct lacking SREs 3–5 (13), and using this same mutated construct, there was no change in StAR promoter activity after the addition of YY1 (data not shown). The addition of recombinant Sp1 (rSp1) enhanced SREBP-1a binding to SRE 3 (Fig. 7BGo, lane 2) and attenuated YY1-inhibition of SREBP-1a binding even with higher concentrations of YY1 (Fig. 7BGo, lanes 3–5 vs. Fig. 6BGo, lanes 4–6). The data in Fig. 7Go suggest that Sp1 enhancement of SREBP-1a binding does not necessarily result in Sp1/SREBP-1a-enhanced activation potential.



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  		Figure 6. Effects of YY1 on NF-Y/SREBP-1a enhancement of StAR. A, NF-Y enhanced SREBP-1a-induced activation of the rat StAR promoter but did not protect against YY1 repression. COS-7 cells were transfected with the p-1862 construct (2 µg) of the StAR gene in the presence of SREBP-1a (1 µg) and increasing amounts of NF-Y and YY1 (1x = 1 µg) and luciferase assays were performed as described in Materials and Methods. This graph represents results from three separate experiments, and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. *, P < 0.001. compared with the p-1862 promoter alone; **, a reduction (P < 0.025) by YY1, compared with the promoter in the presence of SREBP-1a. B, YY1 decreased formation of the major SREBP-1a/SRE binding complex. EMSA using a 32P-labeled double-stranded oligonucleotide probe containing SRE 3 (50,000 cpm/lane) and 250 ng of rSREBP-1a was performed in the presence or absence of 50x-fold molar excess unlabeled oligonucleotide (cold) or increasing amounts of rYY1 as described in Materials and Methods. This experiment was repeated twice and a representative mobility shift assay autoradiograph is shown. C, YY1 decreased NF-Y enhancement of SREBP-1a binding in a dose-dependent manner. EMSA using a 32P-labeled double-stranded oligonucleotide probe containing SRE 3 (50,000 cpm/lane) and 250 ng of rSREBP-1a was performed in the presence or absence of NF-Y nuclear extract (2 µg) and increasing amounts of rYY1. The percentage of SREBP-1a binding in the presence of both SREBP-1a and NF-Y is set at 100%, and the intensity of the bands in lanes 3–5 are compared with lane 2. This experiment was repeated twice, and a representative mobility shift assay autoradiograph is shown.

 


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  		Figure 7. Effects of YY1 on Sp1/SREBP-1a enhancement of StAR. A, Sp1 protected against YY1 repression of SREBP-1a-induced activation of the rat StAR promoter. COS-7 cells were transfected with the p-1862 construct of the StAR gene (2 µg) in the presence of SREBP-1a, Sp1, and YY1 (1 µg each), and luciferase assays were performed as described in Materials and Methods. This graph represents results from three separate experiments, and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. *, P < 0.001, compared with the p-1862 promoter alone; **, a reduction (P < 0.01) by YY1, compared with the promoter in the presence of SREBP-1a. B, Sp1 stabilized SREBP-1a binding to an SRE from the rat StAR promoter and attenuated YY1-induced inhibition of SREBP-1a binding. EMSA using a 32P-labeled double-stranded oligonucleotide probe containing SRE 3 (50,000 cpm/lane) and 500 ng of rSREBP-1a was performed in the presence or absence of rSp1 or rYY1 as listed on the figure. The percent of SREBP-1a binding in the presence of both SREBP-1a and Sp1 is set at 100%, and the intensity of the bands in lanes 3–6 are compared with lane 2. This experiment was repeated twice, and a representative mobility shift assay autoradiograph is shown.

 
The rat StAR promoter was then cotransfected into COS-7 cells in the presence or absence of SREBP-1a, Sp1, NF-Y, and YY1. Sp1 and NF-Y alone had little effect on basal promoter activity (Fig. 8AGo), but NF-Y significantly enhanced SREBP-1a-mediated activation of the StAR promoter. In the presence of Sp1, there was enhancement of SREBP-1a/NF-induced activation of the rat StAR promoter although the activity was not statistically different from the promoter activity seen with SREBP-1a/NF-Y (Fig. 8AGo). However, the combined action of Sp1 and NF-Y did not prevent YY1-mediated repression of SREBP-1a-induced activation of the p-1862 rat StAR promoter in COS-7 cells (Fig. 8AGo). EMSA performed in the presence of rSREBP-1a, rSp1, and NF-Y nuclear extract demonstrated an increase in SREBP-1a binding, compared with binding in the presence of SREBP-1a alone (Fig. 8BGo, lane 2 vs. lane 1). Higher amounts of rYY1 were able to decrease SREBP-1a binding to SRE 3 by 66–72% (Fig. 8BGo, lanes 3 and 4 vs. lane 2).



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  		Figure 8. Effects of YY1 on SREBP-1a enhancement of StAR in the presence of both Sp1 and NF-Y. A, Sp1 and NF-Y enhanced SREBP-1a-induced activation of the rat StAR promoter, but their combined action did not protect against YY1 repression. COS-7 cells were transfected with the p-1862 construct (2 µg) in the presence of SREBP-1a and Sp1-pCMV5, NF-Y (A, B, and C) in pcDNA 3.1 (1 µg each) or increasing amounts of YY1 (1x = 1 µg), and luciferase assays were performed as described in Materials and Methods. *, P < 0.05 or less, compared with the p-1862 promoter alone; **, a reduction (P < 0.005) by YY1, compared with the promoter in the presence of SREBP-1a; ***, enhancement over activity of the StAR promoter in the presence of SREBP-1a. B, YY1 decreased Sp1/NF-Y enhancement of SREBP-1a. EMSA using a 32P-labeled double-stranded oligonucleotide probe containing SRE 3 (50,000 cpm/lane) and 250 ng of rSREBP-1a was performed in the presence or absence of rSp1 (500 ng), nuclear extract from HTB-9 cells overexpressing NF-Y (2 µg), and increasing amounts of rYY1. The percentage of SREBP-1a binding in the presence of SREBP-1a, Sp1, and NF-Y is set at 100%, and the intensity of the bands in lanes 3 and 4 are compared with lane 2. This experiment was repeated twice, and a representative mobility shift assay autoradiograph is shown.

 
YY1 regulation of rat StAR activity through NF-Y
To better study the role of YY1 in SREBP-1a/NF-Y enhancement of the rat StAR promoter, the characterization of NF-Y binding sites was examined by EMSA. The NF-Y binding site (-80 to -76) for the rat StAR promoter was found serendipitously when nuclear extract prepared from cells overexpressing NF-Y was found to bind to an oligonucleotide containing the SRE/YY1-binding site and the identity of the specific DNA sequence necessary for NF-Y binding to this site was confirmed after site-directed mutation (data not shown). Recombinant SREBP-1a (unpublished observation) and YY1 (Fig. 2BGo) did not bind to this site, but NF-Y bound specifically to this site and a supershift was evident after addition of NF-Y antibody (Fig. 9Go, lane 4). The addition of SREBP-1a slightly enhanced NF-Y binding in the presence or absence of Sp1 (Fig. 9Go, lanes 5–7), but all binding to this NF-Y-binding site was lost in the presence of rYY1 (Fig. 9Go, lane 8). NF-Y was found to bind with very low affinity to the four classical NF-Y-binding sites in the rat StAR promoter in the absence of additional cofactors (data not shown).



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  		Figure 9. YY1 inhibited NF-Y binding to a putative NF-Y binding site in the rat StAR promoter. EMSA using a 32P- labeled double-stranded oligonucleotide probe containing an NF-Y-binding site (50,000 cpm/lane) and 2 µg of nuclear extract from HTB-9 cells overexpressing NF-Y was performed in the presence or absence of 100x-fold molar excess unlabeled oligonucleotide (cold), 1 µg of NF-Y antibody (AB), rSREBP-1a, rYY1, or rSp1 (500 ng each). This experiment was repeated three times, and a representative mobility shift assay autoradiograph is shown.

 
The contribution of the -80 to -76 NF-Y site for YY1 repression of SREBP-1a induction of StAR promoter activity was examined using site-directed mutation and several different rat StAR promoter constructs (Fig. 10Go). The p-1862 rat StAR promoter contains four classical NF-Y sites in addition to the nonconsensus NF-Y site. NF-Y was able to synergistically activate the p-1862 StAR promoter in the presence of SREBP-1a, and YY1 was able to reduce the synergistic activation (Fig. 10AGo). Site-specific mutation of the NF-Y-binding site at -80 to -76 (mp-1862) slightly decreased the synergistic activation of the promoter by SREBP-1a/NF-Y, and YY1 was able to reduce this activation although the difference was not statistically significant (Fig. 10AGo). Deletion of the four classical NF-Y binding sites in the p-291 StAR promoter construct (Fig. 10BGo) and the p-150 promoter construct (Fig. 10CGo) did not affect SREBP-1a/NF-Y enhancement and YY1-mediated repression of the rat StAR promoter luciferase activity. However, site-specific mutation of the -80 to -76 NF-Y-binding site in smaller rat StAR promoters [mp-291 (Fig. 10BGo) and mp-150 (Fig. 10CGo)], which lack the four classical NF-Y-binding sites, abolished SREBP-1a induction of the rat StAR promoter in both the presence and absence of NF-Y and prevented YY1 repression.



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  		Figure 10. Mutation of the putative NF-Y-binding site attenuated YY1 repression of the rat StAR promoter. HTB-9 cells were transfected with the wild-type [p-1862 (A), p-291 (B), or p-150 (C)] or NF-Y-binding site mutated [mp-1862 (A), mp-291 (B), or mp-150 (C)] rat StAR promoter constructs (2 µg) either in the presence or absence of expression vectors for SREBP-1a, NF-Y, and YY1 (1 µg each), and luciferase assays were performed as described in Materials and Methods. This graph represents results from three separate experiments, and the data are presented as fold difference, compared with the StAR promoter alone, in which the promoter is given a value of 1. *, A significant (P < 0.05 or less) enhancement over activity seen with promoter alone; **, a significant (P < 0.01 or less) repression by YY1, compared with the activity seen with the appropriate promoter in the presence of SREBP-1a and NF-Y.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although there have been several earlier studies establishing YY1 as a negative regulatory factor for other sterol responsive genes, this is the first study that establishes YY1 as a negative regulatory factor for the rat StAR gene and defines multiple mechanisms for YY1’s repressive effects through displacement of NF-Y binding and disruption of SREBP-1a binding in the presence or absence of positive regulatory factors. This study is also the first to demonstrate that SREBP-1a and YY1 proteins colocalize in the nucleus at the site necessary for transcriptional regulation. The YY1 protein expression data exhibited a pattern that was inversely proportional to the pattern previously demonstrated for rat StAR protein levels after PGF2{alpha} or hCG administration (30). In earlier studies, StAR was shown to be a mitochondrial protein synthesized in response to gonadotropins and cAMP and was found to be essential for steroid hormone production (31). PGF2{alpha} administration was shown to reduce steroidogenesis by causing a decrease in the expression of basal and gonadotropin-induced intracellular cholesterol transport protein expression (32, 33, 34, 35). This lab has previously shown that PGF2{alpha} resulted in a decrease in StAR mRNA levels (25). This is in contrast to a study by Fiedler et al. (36), which found that the rapid decrease in StAR protein expression after PGF2{alpha} was not accompanied with a decline in mRNA expression. However, the study by Sandhoff and McLean (25) examined rat ovaries isolated from d-10-postovulatory rats when luteal tissue is extremely sensitive to PGF2{alpha} luteolytic action rather than tissue isolated earlier (d 7 postovulatory rats) when rats are somewhat less sensitive to PGF2{alpha} luteolytic effect (36), which may explain the discrepancy. The exact mechanism for negative regulation of the StAR gene by PGF2{alpha} has not been determined, but studies have shown that activation of PKC (34), the NUR77 transcription factor (37), dosage-sensitive sex adrenal hypoplasia congenital critical region on the X-chromosome, gene-1 (38), c-fos (39), and YY1 (present study) occur concurrently with a reduction in StAR gene expression, and it is probable that multiple mechanisms are involved in reducing steroidogenesis.

The transcription factor YY1 has been shown to act as a negative regulator of transcription for numerous genes (19). For the 3-hydroxy-3-methyl-glutaryl coenzyme A synthase, farnesyl diphosphate synthase, and LDL-R promoter (24) and basal activity of the HDL-R promoter (5), YY1 achieved repression through direct binding to the DNA resulting in displacement of NF-Y and/or Sp1, the hypothesis being that YY1 maintains sterol-sensitive genes in a transcriptionally repressed state in the absence of nuclear SREBP-1a. When cholesterol levels are depleted and SREBP-1a levels are increased, YY1 repression is overcome through synergistic interactions with SREBP-1a, NF-Y, and/or Sp1 proteins and DNA-binding sites. However, NF-Y enhancement of most SREBP-activated genes requires NF-Y binding to one or more adjacent CCAAT sites that are usually found within 21 bp of an SRE (40). The classical NF-Y-binding sites in the rat StAR promoter are not near any of the five SREs (the distance being greater than 110 bp). However, Christenson et al. (29) identified a nonconsensus SRE/YY1 site in the human StAR promoter, which displayed binding to both SREBP-1a and YY1. Although YY1 was able to bind directly to three sites in the rat StAR promoter with high affinity, elimination of these sites by site-directed mutation did not prevent YY1-mediated repression, and recombinant YY1 did not bind to the SRE/YY1 site in the rat StAR promoter. However, the rat StAR promoter sequence was shown to have several nucleotide changes in this area, compared with the human StAR promoter SRE/YY1 sequence AGTGA(G to C)TG(A to T)TGG. These changes were found to have a major impact on the EMSA-binding profile in that SREBP-1a (unpublished observation) and YY1 (current study) were unable to bind to this site in the rat StAR promoter. In contrast, NF-Y displayed specific binding but with low affinity to this site, which lacks the final T of the inverted CCAAT box/NF-Y-binding motif. These EMSA results are in contrast to those of Keri et al. (41), which identified the T of the CCAAT motif as being critical for NF-Y binding in the LH ß-subunit gene. The discrepancy between the current results and those of Keri et al. most likely reflect differences in nucleotides adjacent to the actual NF-Y binding motif, which may contribute to NF-Y binding. In addition, YY1 was able to prevent NF-Y from binding to the -80 to -76 nonconsensus NF-Y-binding site. However, only higher doses of YY1 were able to decrease basal luciferase activity of the rat StAR promoter suggesting that maximal repression by YY1 could involve interaction with additional transcriptional regulatory factors. Studies have shown that YY1 was able to repress both the LDL-R promoter (23) and the HDL-R promoter (5) by disrupting interactions between positive transcription factors like SREBP-1a and Sp1. In the present study, YY1 was not able to repress the activation of the StAR promoter in the presence of SREBP-1a and Sp1 and was able to decrease SREBP-1a binding only in the presence of Sp1 at a very high dose, suggesting Sp1/SREBP-1a interactions enhance binding but not activation. However, the presence of Sp1 somehow prevented YY1-mediated repression of SREBP-1a induction of StAR promoter activity.

The dual actions of SREBP-1a and the heterotrimer NF-Y have been shown to enhance DNA binding and cause synergistic activation of the rat farnesyl diphosphate synthase gene (16) and rat StAR gene (13). NF-Y was also shown in this study to enhance SREBP-1a binding to an SRE and enhance SREBP-1a induced activation of the rat StAR promoter. The combined action of both Sp1 and NF-Y with SREBP-1a resulted in maximal stimulation of the rat StAR promoter but YY1 was still able to easily repress StAR promoter activity suggesting that YY1 was acting mainly through disruption of SREBP-1a/NF-Y protein interactions. Although technical variations in the amount of SREBP-1a enhancement in binding and activation of the StAR promoter were noted, Sp1 has consistently been found to enhance SREBP-1a binding and act as a deterrent for the YY1-mediated disruption of SREBP-1a binding or activation. In addition, NF-Y has consistently been found to enhance SREBP-1a binding and to partially deter YY1 disruption of SREBP-1a binding. NF-Y was never able to prevent YY1-mediated inhibition of SREBP-1a induction of StAR promoter activity.

The observation that YY1 was also able to directly inhibit NF-Y binding to a site on the rat StAR promoter demonstrates a novel inhibitory mechanism whereby YY1 is able to amplify its repressive activity toward the StAR gene. We hypothesize that when there is adequate steroidogenesis occurring in cells, YY1 is present ubiquitously in amounts that prevent the low levels of endogenous SREBP-1a proteins from being enhanced by endogenous Sp1 and NF-Y proteins, and this interaction with YY1 prevents constitutive activation of the StAR gene. We believe YY1 accomplishes this repressive state by binding to SREBP-1a and preventing SREBP-1a interaction with its DNA-binding motif or any positive regulatory factors that could encourage SREBP-1a binding. In addition, YY1 has been shown to be associated with histone deacetylases (22) that are known to maintain chromatin in a conformation that discourages transcription. Disruption of NF-Y binding would be an additional mechanism to prevent NF-Y protein from remaining in close association with the DNA and therefore NF-Y would not be available as an SREBP-1a binding enhancement factor in the nucleus. Our results suggest that YY1-repression involves complex protein/DNA interactions, and analysis of additional regulatory factors that interact with YY1 and SREBP-1a to control StAR transcription will be required to fully understand YY1’s molecular action on the StAR gene.


   Footnotes
 
This work was supported by grants from the NIH RO1 HD-35163 (to M.P.M.) and an American Heart Association Florida Affiliate PostDoctoral Fellowship 9703004 (to D.L.).

Abbreviations: CMV, Cytomegalovirus; FRET, fluorescence resonance energy transfer; hCG, human CG; HDL-R, high-density lipoprotein receptor; LDL-R, low-density lipoprotein receptor; NF-Y, nuclear factor Y; rSp1, recombinant Sp1; rYY1, recombinant YY1 protein; Sp1, promoter-specific transcription factor; SRE, sterol regulatory element; SREBP-1a, sterol regulatory element binding protein-1a; StAR, steroidogenic acute regulatory; YY1, transcription factor Yin Yang 1; YY1 BS, YY1-binding site.

Received July 10, 2001.

Accepted for publication November 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Menon KMJ, Gunaga KP 1974 Role of cyclic AMP in reproductive processes. Fertil Steril 25:732–750[Medline]
  2. Waterman MR, Keeney DS 1996 Signal transduction pathway combining peptide hormones and steroidogenesis. Vit Horm 52:129–148[Medline]
  3. Sugawara T, Holt JA, Driscoll D, Strauss III JF, Lin D, Miller WL, Patterson D, Clancy KP, Hart IM, Clark BJ, Stocco DM 1995 Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13. Proc Natl Acad Sci USA 92:4778–4782[Abstract/Free Full Text]
  4. Stocco DM, Clark BJ 1997 The role of the steroidogenic acute regulatory protein in steroidogenesis. Steroids 62:29–36[CrossRef][Medline]
  5. Shea-Eaton W, Lopez D, McLean MP 2001 Yin Yang 1 protein negatively regulates high-density lipoprotein receptor gene transcription by disrupting binding of sterol regulatory element binding protein to the sterol regulatory element. Endocrinology 142:49–58[Abstract/Free Full Text]
  6. Sandhoff TW, McLean MP 1996 Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary. Endocrine 4:259–267
  7. Lopez D, McLean MP 1999 Sterol regulatory element-binding protein-1a binds to cis elements in the promoter of the rat high density lipoprotein receptor SR-BI gene. Endocrinology 140:5669–5681[Abstract/Free Full Text]
  8. Dooley KA, Millinder S, Osborne TF 1998 Sterol regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene through a direct interaction between sterol regulatory element binding protein and the trimeric CCAAT-binding factor/nuclear factor Y. J Biol Chem 273:1349–1356[Abstract/Free Full Text]
  9. Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, Wang X 1993 SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci USA 90:11603–11607[Abstract/Free Full Text]
  10. Wang X, Sato R, Brown MS, Hua X, Goldstein JL 1994 SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77:53–62[CrossRef][Medline]
  11. Horton JD, Shimomura I, Brown MS, Hammer RE, Goldstein JL, Shimano H 1998 Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 101:2331–2339[Medline]
  12. Sato R, Inoue J, Kawabe Y, Kodama T, Takano T, Maeda M 1996 Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2. J Biol Chem 271:26461–26464[Abstract/Free Full Text]
  13. Shea-Eaton WK, Trinidad MJ, Lopez D, Nackley A, McLean MP 2001 Sterol regulatory element binding protein-1a regulation of the steroidogenic acute regulatory protein gene. Endocrinology 142:1525–1533[Abstract/Free Full Text]
  14. Christenson LK, Osborne TF, McAllister JM, Strauss III JF 2001 Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a. Endocrinology 142:28–36[Abstract/Free Full Text]
  15. Sanchez HB, Yieh L, Osborne TF 1995 Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene. J Biol Chem 270:1161–1169[Abstract/Free Full Text]
  16. Jackson SM, Ericsson J, Mantovani R, Edwards PA 1998 Synergistic activation of transcription by nuclear factor Y and sterol regulatory element binding protein. J Lipid Res 39:767–776[Abstract/Free Full Text]
  17. Xiong S, Chirala SS, Wakil SJ 2000 Sterol regulation of human fatty acid synthase promoter requires nuclear factor-Y-and Sp-1-binding sites. Proc Natl Acad Sci USA 97:3948–3953[Abstract/Free Full Text]
  18. Kim JH, Lee JN, Paik YK 2001 Cholesterol biosynthesis from lanosterol: a concerted role for Sp1 and NF-Y binding sites for sterol-mediated regulation of rat 7-dehydrocholesterol reductase gene expression. J Biol Chem 276:18153–18160[Abstract/Free Full Text]
  19. Shi Y, Lee JS, Galvin KM 1997 Everything you wanted to know about Yin Yang 1. Biochim Biophys Acta 1332:F49–F66
  20. Galvin KM, Shi Y 1997 Multiple mechanisms of transcriptional repression by YY1. Mol Cell Biol 17:3723–3732[Abstract]
  21. Natesan S, Gilman MZ 1993 DNA bending and orientation-dependent function of YY1 in the c-fos promoter. Genes Dev 7:2497–2509[Abstract/Free Full Text]
  22. Yang WM, Yao YL, Sun JM, Davie JR, Seto E 1997 Isolation and characterization of cDNAs corresponding to an additional member of the human histone deacetylase gene family. J Biol Chem 272:28001–28007[Abstract/Free Full Text]
  23. Bennett MK, Ngo TT, Athanikar JN, Rosenfeld JM, Osborne TF 1999 Co-stimulation of promoter for low density lipoprotein receptor gene by sterol regulatory element-binding protein and Sp1 is specifically disrupted by the Yin Yang 1 protein. J Biol Chem 274:13025–13032[Abstract/Free Full Text]
  24. Ericsson J, Usheva A, Edwards P 1999 YY1 is a negative regulator of transcription of three sterol regulatory element-binding protein-responsive genes. J Biol Chem 274:14508–14513[Abstract/Free Full Text]
  25. Sandhoff TW, McLean MP 1996 Prostaglandin F2{alpha} reduces steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary. Endocrine 5:183–190
  26. Lopez D, Sandhoff TW, McLean MP 1999 Steroidogenic factor-1 mediates 3',5'-cyclic adenosine monophosphate regulation of the high density lipoprotein receptor. Endocrinology 140:3034–3044[Abstract/Free Full Text]
  27. Mahajan NP, Linder K, Berry G, Gordon GW, Heim R, Herman B 1998 Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol 16:545–552
  28. Llopis J, Westin S, Ricote M, Wang J, Cho CY, Kurokawa R, Mullen TM, Rose DW, Rosenfeld MG, Tsien RY, Glass CK 2000 Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are requires for transcription. Proc Natl Acad Sci USA 97:4363–4369[Abstract/Free Full Text]
  29. Christenson LK, Johnson PF, McAllister JM, Strauss III JF 1999 CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene. J Biol Chem 274:26591–26598[Abstract/Free Full Text]
  30. Chung PH, Sandhoff TW, McLean MP 1998 Hormone and prostaglandin F2{alpha} regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in human corpora lutea. Endocrine 8:153–160[CrossRef][Medline]
  31. Stocco DM, Kilgore MW 1988 Induction of mitochondrial proteins in MA-10 leydig tumor cells with human chorionic gonadotropin. Biochem J 249:95–103[Medline]
  32. Stocco DM, Chen W 1991 Presence of identical mitochondrial proteins in unstimulated constitutive steroid producing R2C rat leydig tumor and stimulated nonconstitutive steroid-producing MA-10 mouse leydig tumor cells. Endocrinology 128:1918–1926[Abstract]
  33. Morohashi K, Honda S, Inomata Y, Handa H, Omura T 1992 A common trans-acting factor, Ad4-binding protein, binds to the promoter of steroidogenic P450s. J Biol Chem 267:17913–17919[Abstract/Free Full Text]
  34. Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes an essential regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract]
  35. Honda SI, Morohash KI, Nomura M, Takeya H, Kitajima M, Omura T 1993 AD4BP regulating steroidogenic P450 gene is a member of steroid hormone receptor superfamily. J Biol Chem 268:7494–7502[Abstract/Free Full Text]
  36. Fiedler EP, Plouffe Jr L, Hales DB, Hales KH, Khan I 1999 Prostaglandin F2{alpha} induces a rapid decline in progesterone production and steroidogenic acute regulatory protein expression in isolated rat corpus luteum without altering messenger ribonucleic acid expression. Biol Reprod 61:643–650[Abstract/Free Full Text]
  37. Stocco CO, Zhong L, Sugimoto Y, Ichikawa A, Lau LF, Gibori G 2000 Prostaglandin F2{alpha}-induced expression of 20{alpha}-hydroxysteroid dehydrogenase involves the transcription factor NUR77. J Biol Chem 275:37202–37211[Abstract/Free Full Text]
  38. Sandhoff TW, McLean MP 1999 Repression of the rat steroidogenic acute regulatory (StAR) protein gene by PGF2{alpha} is modulated by the negative transcription factor DAX-1. Endocrine 10:83–91[CrossRef][Medline]
  39. Shea-Eaton W, Sandhoff TW, Lopez D, Hales DB, McLean MP, Transcriptional repression of the rat steroidogenic acute regulatory (StAR) protein gene by the AP-1 family member c-Fos. Mol Cell Endocrinol, in press
  40. Edwards PA, Ericsson J 1999 Sterols and isoprenoids: signaling molecules derived from the cholesterol biosynthetic pathway. Annu Rev Biochem 68:157–185[CrossRef][Medline]
  41. Keri RA, Bachmann DJ, Behrooa A, Herr BD, Ameduri RK, Quirk CC, Nilson JH 2000 An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone stimulated expression of the luteinizing hormone ß subunit gene. J Biol Chem 275:13082–13088[Abstract/Free Full Text]



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M. D. Pisarska, J. Bae, C. Klein, and A. J. W. Hsueh
Forkhead L2 Is Expressed in the Ovary and Represses the Promoter Activity of the Steroidogenic Acute Regulatory Gene
Endocrinology, July 1, 2004; 145(7): 3424 - 3433.
[Abstract] [Full Text] [PDF]