Transcriptional Activation of Steroidogenic Factor-1 by ...

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The Journal of Clinical Endocrinology & Metabolism 92(8):3261–3267 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2007-0494

Transcriptional Activation of Steroidogenic Factor-1 by Hypomethylation of the 5ⴕ CpG Island in Endometriosis Qing Xue, Zhihong Lin, Ping Yin, Magdy P. Milad, You-Hong Cheng, Edmond Confino, Scott Reierstad, and Serdar E. Bulun Divisions of Reproductive Biology Research (Q.X., Z.L., P.Y., Y.-H.C., S.R., S.E.B.) and Reproductive Endocrinology and Infertility (M.P.M., E.C., S.E.B.), Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611; and Department of Obstetrics and Gynecology (Q.X.), First Hospital of Peking University, Beijing, People’s Republic of China Context: Endometriosis is an estrogen-dependent disease. Steroidogenic factor-1 (SF-1), a transcriptional factor essential for activation of multiple steroidogenic genes for estrogen biosynthesis, is undetectable in normal endometrial stromal cells and aberrantly expressed in endometriotic stromal cells. Objective: The objective of the study was to unravel the mechanism for differential SF-1 expression in endometrial and endometriotic stromal cells. Design: We identified a CpG island flanking the SF-1 promoter and exon I region and determined its methylation patterns in endometrial and endometriotic cells. Setting: The study was conducted at Northwestern University. Patients or Other Participants: Eutopic endometrium from disease-free subjects (n ⫽ 8) and the walls of cystic endometriosis lesions of the ovaries (n ⫽ 8) were investigated. Intervention(s): Stromal cells were isolated from these two types of tissues.

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NDOMETRIOSIS IS AN estrogen-dependent disease that affects 6 –10% of women of reproductive age and is the most common disorder associated with chronic pelvic pain. Endometriosis is characterized by the presence of endometrium-like tissue outside the uterine cavity, primarily on ovaries and pelvic peritoneum (1). Endometriotic lesions grow in an estrogenic environment and tend to regress when local and systemic estrogen concentrations are low (2, 3). We and others demonstrated abundant expressions of steroidogenic genes including steroidogenic acute regulatory protein (StAR) and aromatase, giving rise to local estrogen production in endometriotic tissue (4 –7). StAR and aromatase catalyze the key steps of steroidogenesis (8). StAR facilitates the entry of cholesterol into the mitochondria (9). Aromatase, on the other hand, catalyzes the final step of estrogen production via conversion of C19 First Published Online May 22, 2007 Abbreviations: 5-aza-dC, 5-Aza-2⬘-deoxycytidine; ChIP, chromatin immunoprecipitation; CT, comparative threshold cycle; MeCP2, methylCpG-binding domain protein 2; 18S, 18S rRNA; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Main Outcome Measure(s): Measures are mentioned in Results. Results: SF-1 mRNA and protein levels in endometriotic stromal cells were significantly higher than those in endometrial stromal cells (P ⬍ 0.001). Bisulfite sequencing showed strikingly increased methylation in endometrial cells, compared with endometriotic cells (P ⬍ 0.001). Demethylation by 5-aza-2⬘-deoxycytidine increased SF-1 mRNA levels by up to 55.48-fold in endometrial cell (P ⬍ 0.05). Luciferase assays showed that the ⫺85/⫹239 region bearing the CpG island regulated its activity (P ⬍ 0.01). Natural or in vitro methylation of this region strikingly reduced SF-1 promoter activity in both cell types (P ⬍ 0.01). Chromatin immunoprecipitation assay showed that methyl-CpG-binding domain protein 2 binds to the SF-1 promoter in endometrial but not endometriotic cells. Conclusions: This is the first demonstration of methylation-dependent regulation of SF-1 in any mammalian tissue. These findings point to a new mechanism for targeting local estrogen biosynthesis in endometriosis. (J Clin Endocrinol Metab 92: 3261–3267, 2007)

steroids to estrogens (10). Both StAR and aromatase are expressed in the stromal cell compartment of endometriosis, whereas they are undetectable in eutopic endometrial stromal cells from disease-free women (8, 11). StAR, aromatase, and some of the other key steroidogenic enzymes are regulated by a nuclear receptor termed steroidogenic factor-1 (SF-1), which is also known as adrenal 4-binding protein, and encoded by the NR5A1 gene in humans (12, 13). SF-1 was originally cloned from adrenal cells (14). Further studies have established that SF-1 is responsible for the development of the posterior hypothalamus, adrenals, and gonads. In the adults, SF-1 regulates multiple genes in the hypothalamic-pituitary-adrenal or hypothalamic-pituitary-gonadal endocrine axes (15–17). We found that SF-1, which is expressed in endometriosis but not in its normal counterpart tissue, eutopic endometrium, is the key activator of the aromatase gene promoter in endometriotic stromal cells (5). Methylation of DNA at the CpG dinucleotides is a postreplication event catalyzed by the DNA (cytosine-5)-methyltransferase (18), which establishes normal methylation patterns during embryogenesis and reproduces these patterns during replication of adult cells (19, 20). DNA methylation, an important mechanism of epigenetic gene regulation, is

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involved in genomic imprinting, X chromosome inactivation, aging, mutagenesis, and regulation of tissue-specific gene expression during development and adult life (21–23). Aberrant methylation of CpG islands, located in the 5⬘-promoter region of genes, is commonly associated with transcriptional inactivation. Such inactivation is observed in various human cancers, especially in tumor suppressor genes (24). Here we identified a classical CpG island at the promoter region of the SF-1 gene and present evidence that promoter methylation is a major mechanism of SF-1 silencing in normal endometrial cells and its aberrant expression in endometriotic cells. This is the first demonstration that the SF-1 gene is regulated by DNA methylation in any mammalian tissue. Because SF-1 expression in endometriosis leads to aromatase expression and local estrogen production, our findings are clinically significant. The clinical relevance is further exemplified by the therapeutic role of aromatase inhibitors in endometriosis (3, 8). Subjects and Methods Subjects and primary cell culture Eutopic endometrium from disease-free subjects (n ⫽ 8) and the walls of cystic endometriosis lesions of the ovaries (n ⫽ 8) were obtained immediately after surgery. The age ranges of subjects were 40.75 ⫾ 3.37 (endometrium) to 38.88 ⫾ 2.95 yr (endometriosis), and there were no differences between the two groups with respect to age or cycle phase. None of the patients had received any preoperative hormonal therapy. All samples were histologically confirmed, and the phase of the menstrual cycle was determined by preoperative history and histological examination. Half of the tissue samples were in the proliferative phase and the other half in the secretory phase in both groups. Eutopic endometrial samples were obtained from women undergoing hysterectomy for cervical dysplasia or uterine leiomyoma. Written informed consent was obtained before surgical procedures, including a consent form and protocol approved by the Institutional Review Boards at Northwestern University. Stromal cells were isolated from these two types of tissues using a protocol previously reported by Ryan et al. (25) with minor modification and then were suspended in DMEM/F12 1:1 (GIBCO/BRL, Grand Island, NY) containing 10% fetal bovine serum. All cells were passed once.

RNA extraction and quantitative analysis by real-time RT PCR Total RNA were isolated with TRIzol (Sigma, St. Louis, MO) from stromal cells according to the manufacturer’s protocol. One microgram of total RNA was used to generate cDNA with the Superscript III first-strand synthesis system (Invitrogen, Carlsbad, CA). Real-time quantitative PCR was performed using the ABI 7900 sequence detection system and the ABI Taqman gene expression system (purchased from Applied Biosystems, Foster City, CA) for SF-1 and eukaryotic 18S rRNA (18S). 18S values were used for normalization. Relative quantification of SF-1 gene was analyzed by comparative threshold cycles (CT) method. In brief, CT was used to determine the expression level normalized to the expression in endometrial stromal cells. For each sample, the SF-1 CT value was normalized using the formula: ⌬ CT ⫽ CT SF-1 ⫺ CT 18S. To determine relative expression levels, the following formula was used: ⌬ ⌬CT ⫽ ⌬CT sample ⫺ ⌬CT calibrator. The value was used to plot the SF-1 expression using the expression 2-⌬ ⌬CT. Thus, expression levels were expressed as n-fold difference relative to the calibrator. Also, we performed semiquantitative RT-PCR, and the products were analyzed on 1% agarose gel. The primers for the SF-1 were: forward: 5⬘-CTGGAGCCGGATGAGGAC-3⬘, reverse: 5⬘-ACCTGGCGGTAGATGTGGT3⬘. 18S primers were forward: 5⬘-AGGAATTCCCAGTAAGTGCG-3⬘, reverse: 5⬘-GCCTCACTAAACCATCCAA-3⬘.

Xue et al. • Methylation of SF-1 and Endometriosis

Bisulfite modification and sequencing analysis Genomic DNA was extracted from the cells by using DNeasy tissue kit (QIAGEN, Valencia, CA). Five hundred nanograms of DNA was treated with sodium bisulfite following the manufacturer’s protocol (Zymo Research, Orange, CA). For PCR amplification, 3 ␮l of bisulfitemodified DNA were added to a final volume of 20 ␮l. AmpliTaq Gold PCR master mix (Applied Biosystems) was used for all the PCR. PCR amplifications were performed using the following primers: forward: 5⬘-TGTAGAAGGAGGTTGGTTATTAGAG-3⬘, and reverse: 5⬘-AACRAACAAAACCAACCTACTATCC-3⬘. The thermal cycle conditions were as follows: 95 C for 10 min followed by 40 cycles of denaturation at 95 C 30 sec, annealing at 50 C for 2 min, and elongation at 72 C for 2 min, followed by an extension at 72 C for 7 min. PCR products (213 bp) were gel purified and cloned into the pGEM-Teasy vector (Promega, Madison, WI). After transformation, six to eight clones with the right insert were randomly picked from each PCR and sequenced on an Applied Biosystems 377 instrument.

5-Aza-2⬘-deoxycytidine (5-aza-dC) treatments At approximately 40% confluence, endometrial stromal cells were placed in serum-free DMEM/F12 for 24 h and then treated with various concentrations (0, 1, 5, and 10 ␮m) of DNA methyltransferase inhibitor, 5-aza-dC (Sigma) for 5 d. The medium was changed daily. Total RNA and genomic DNA were isolated from the treated cells using TRIzol reagent and DNeasy tissue kit. All of the experiments were repeated three times in different primary cultured cells in triplicate.

Plasmid construction Reporter plasmid vectors containing the SF-1 promoter sequences were constructed by PCR cloning. Genomic DNA (unmethylated at SF-1 promoter) from endometriotic stromal cells was used as the template for amplification, and the primers were: reverse primer 5⬘-GATATCAGAGAGAGCCACAGAGACAAC-3⬘ (position ⫹524 relative to the transcription start site), forward primers 5⬘-GGTACCACTGGCCTGTCCTGACTCT-3⬘ (⫺465), 5⬘-GGTACCGTGGGGGCAGAGACCAAT-3⬘ (⫺85), 5⬘-GGTACCGTGACCGGTGCCCCCTGCT-3⬘ (⫹239). Genomic DNA from endometrial and endometriotic stromal cells was used as the template for the amplification of the ⫺85/⫹239 construct. In this case, reverse primer was 5⬘-GATATCTAAGTGGAGCAGGCAGTGG3⬘(⫹239), and the forward primer was the same (⫺85). Restriction sites (KpnI site for forward primers and EcoRV site for reverse primers) were added to the 5⬘-end of primers, and promoter sequences were amplified using TAKARA LA Taq with GC buffer (TaKaRa, Otsu, Japan). PCR products were cloned into the pGL4 vector-SV40 (the SV40 minimal promoter was digested with BglII and HindIII from pGL2-promoter vector and cloned into BglII and HindIII digested pGL4.10 vector (Promega). The construct was then named as pGL4-SV40) to construct plasmids SF-1 (⫺465/⫹524) Luc, SF-1(⫺85/⫹524) Luc, SF-1(⫹239/⫹524) Luc, and SF-1(⫺85/⫹239) Luc, respectively.

Transfection and luciferase reporter gene assay Transfection experiments of endometrial and endometriotic stromal cells were performed using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol. Briefly, the cells were grown in 24-well tissue culture plates so that the cell layer was 50 – 60% confluent on the day of transfection. For each well, OPTI-MEM I containing 1.5 ␮l of FuGENE 6 was mixed with 240 ng of reporter plasmid and 60 or 80 ng of pSV-␤-galactosidase vector (Promega) for endometrial or endometriotic stromal cells. The cells were harvested 48 h after transfection, and the luciferase activity was measured using a luciferase assay system (Promega). ␤-Galactosidase activity was used to normalize transfection efficiency. All of the experiments were repeated three times in triplicate.

In vitro methylation assays In vitro methylation assays were carried out according to the methods by Robertson and Ambinder (26) and Singal et al. (27). Briefly, regionspecific methylation was carried out on the SF-1 promoter fragment of ⫺85/⫹239 after excision and isolation of the fragment. DNA was in-


cubated with SssI CpG methylase (New England Biolabs, Ipswich, MA) in the presence (methylated) or absence (mock-methylated) of S-adenosylmethionine, as recommended by the manufacturer for 2 h. Methylated and mock-methylated fragments were ligated again into their respectively unmethylated vector. All constructs were sequenced to confirm the correct region of the SF-1 gene, and the efficiency of the methylation was determined through methylation-sensitive and methylation-insensitive restriction enzyme digestion with HpaII and MspI and also using bisulfite sequence to confirm that all of the CpG sites were indeed methylated or unmethylated.

Chromatin immunoprecipitation (ChIP) assay ChIP assay was carried out using ChIP assay kit according to the manufacturer’s protocol (Upstate Biotechnology, Inc., Lake Placid, NY). Immunoprecipitation was carried out with 3 ␮g of either methyl-CpG-binding domain protein 2 (MeCP2) antibody (Abcam, Cambridge, MA) or rabbit IgG at 4 C overnight with rotation. The recovered DNA was subjected to 33 cycles of PCR using the following primers: 5⬘-GGTACCGTGGGGGCAGAGACCAAT-3⬘, reverse primer 5⬘-GATATCTAAGTGGAGCAGGCAGTGG-3⬘. The PCR products (324 bp) were analyzed on 1% agarose gel. Three independent experiments were preformed.


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trifuge at 13,000 rpm for 10 min. Equal amounts of protein (15 ␮g) were resolved on 4 –15% Tris-HCL gels, transferred onto nitrocellulose membranes, and incubated with antihuman SF-1 or MeCP2 antibodies diluted 1:1000 purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Abcam. Anti-␤-actin antibody was used as a loading control. Detection was performed using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Band intensity of protein expression was quantified using the Quantity One analysis Software (Bio-Rad Laboratories, Los Angeles, CA).

Results SF-1 expression in endometrial and endometriotic stromal cells

Taqman-based real-time and semiquantitative RT-PCR assays were used to quantify mRNA levels of SF-1 gene in endometrial and endometriotic stromal cells. Each assay indicated that SF-1 mRNA levels in primary endometriotic stromal cells (n ⫽ 8 subjects) were dramatically higher than those in primary endometrial stromal cells (n ⫽ 8 disease-free subjects, P ⬍ 0.001, Fig. 1, A and B). Western blot showed that

Western blot analysis Cell were washed with ice-cold PBS and suspended in the protein extraction reagent (Pierce, Rockford, IL). Lysates were cleared by cen-

FIG. 1. mRNA levels of SF-1 in endometrial and endometriotic stromal cells. A, SF-1 mRNA levels in endometriotic stromal cells, which were quantified by real-time PCR, first normalized to 18S, and further calibrated to values in endometrial stromal cells. B, Agarose gel electrophoresis of RT-PCR products verified these results. *, P ⬍ 0.001 (Student’s t test). C, Protein levels determined by Western blot of SF-1 in endometrial and endometriotic stromal cells (eight subjects in each group). P ⬍ 0.01 (Student’s t test).

FIG. 2. DNA methylation status of SF-1 5⬘-flanking region in endometrial and endometriotic stromal cells. Top, A schematic diagram indicating the CpG island on SF-1 5⬘-flanking region. ⫹1 is the transcription start site. Black bar, Bisulfite sequencing fragment containing the promoter region and the first exon. Bottom, Methylation status of 13 CpG sites of SF-1 promoter region obtained from bisulfite sequencing in endometrial and endometriotic stromal cell (P ⬍ 0.001, Student’s t test). Six to eight clones were examined for each subject. Open and filled circles represent unmethylated and methylated cytosines, respectively. The numbers on the top indicate the positions of cytosine residues of CpGs relative to the transcription start site (⫹1), and the numbers 1– 8 on the left represent primary cultured stromal cells from different subjects in the two groups.

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SF-1 protein levels in endometriotic cells (n ⫽ 8 subjects) were significantly higher, compared with endometrial cells (n ⫽ 8 subjects, P ⬍ 0.01, Fig. 1C). DNA methylation profile of the CpG island at the SF-1 promoter region

To determine whether loss of SF-1 expression resulted from promoter hypermethylation, methylation status of a total of 13 CpGs across a 213-bp region in the approximately 252-bp CpG island (⫺84/⫹168) at the SF-1 promoter and exon I region was characterized by bisulfite genomic sequencing. Six to eight clones were checked for each subject. The detailed CpG methylation status of primary endometrial (n ⫽ 8) and endometriotic (n ⫽ 8) stromal cells was shown in Fig. 2. The SF-1-negative endometrial stromal cells, showed a dense methylation pattern at the SF-1 promoter and exon I region. In contrast, the majority of the CpG sites were not methylated in endometriotic stromal cells that express high levels of SF-1. There was a significant difference (P ⬍ 0.001, Student’s t test) in methylation status between the two groups of cells. Induction of SF-1 mRNA expression by 5-aza-dC

Next, we considered the possibility of epigenetic modification of the SF-1 promoter as a mechanism of silencing in endometrial cells. Endometrial stromal cells were treated with the demethylating agent 5-aza-dC to investigate the involvement of DNA methylation in SF-1 gene silencing. SF-1 mRNA levels were then quantified by real-time PCR. As shown in Fig. 3A, 5-aza-dC treatment led to a dose-depen-

FIG. 3. Effect of the demethylating agent 5-aza-dC on expression of SF-1 gene in endometrial stromal cells. A, top, Relative mRNA levels of SF-1 gene after treatment with 5-aza-dC were quantified by real-time PCR and normalized to its expression in nontreated cells. Bottom, RT-PCR result of SF-1 expression after the same treatment. (*, P ⬍ 0.01; #, P ⬍ 0.001; Student’s t test). B, DNA methylation status of SF-1 5⬘-flanking region on the effect of 5-aza-dC treatment in endometrial stromal cells. Methylation status of 13 CpG sites of SF-1 promoter region obtained from bisulfite sequencing in endometrial stromal cells before and after 10 ␮M 5-aza-dC treatment for 5 d (P ⬍ 0.05; Student’s t test). Open and filled circles represent unmethylated and methylated cytosines, respectively.


dent increase in SF-1 mRNA in originally SF-1-negative endometrial stromal cells. To confirm the demethylating effect of 5-aza-dC, bisulfite sequencing was performed on untreated and treated cells. 5-aza-dC significantly decreased methylation status of the SF-1 promoter region (P ⬍ 0.05, Student’s t test) in endometrial stromal cells (Fig. 3B). These data strongly suggested that hypermethylation was responsible for SF-1 gene silencing. Methylation of SF-1 promoter inhibits its activity

To elucidate the critical region of the SF-1 promoter responsible for the constitutively active SF-1 promoter activity, we introduced a series of SF-1 promoter deletion constructs (⫺465/⫹524, ⫺85/⫹524, and ⫹239/⫹524) into the endometrial and endometriotic cells under the same serumstarved conditions, and the relative activities of the fused reporter gene luciferase were determined. We did not detect a significant difference in luciferase activity between ⫺465/ ⫹524 and ⫺85/⫹524 constructs, both of which contain the full-length CpG island, whereas the luciferase activity of the ⫹239/⫹524 construct was significantly (60.0 –74.2%) lower than the ⫺85/⫹524 constructs in both endometriotic and endometrial stromal cells (P ⬍ 0.01, Student’s t test). These results suggested that the constitutive SF-1 promoter activity was conferred by the region between ⫺85 and ⫹239, which bears the CpG island. We did not observe any differences between endometriotic or endometrial cell types with respect to promoter activity of these constructs (Fig. 4, A and B). Next, methylation analysis was performed to examine whether SF-1 promoter activity was regulated by the meth-



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structs were generated from treating the unmethylated construct from endometriotic cells with a methylase in the presence or absence of S-adenosylmethionine (in vitro methylated or mock-methylated constructs). Natural or in vitro methylation significantly (⬃70%) decreased the promoter activities of the luciferase constructs in both cell types (P ⬍ 0.01, Fig. 4, C and D). Recruitment of MeCP2 to the SF-1 promoter

We investigated the differential recruitment of a key chromatin-associated protein to the SF-1 promoter as a function of the methylation status of its promoter region. We determined by ChIP the binding activity of MeCP2, which silences genes via prevention of binding of transcriptional factors or directly acts as a transcriptional repressor, at the SF-1 promoter (28, 29). We found that MeCP2 bound to the SF-1 promoter CpG island region (⫺85/⫹239) in endometrial stromal cells but not in endometriotic stromal cells, suggesting that loss of the methylation status in SF-1 promoter in endometriotic cells is linked to loss of the association of MeCP2 (Fig. 5A). We determined comparable protein levels of MeCP2 in both cell types by Western analysis, indicating that variations in its levels did not account for our findings (Fig. 5B). The proposed mechanism for the regulation of SF-1 by DNA methylation is shown in Fig. 6. Discussion

Estradiol biosynthesis is dependent on the facilitation of the entry of cholesterol into mitochondria followed by six enzymatic steps, in which aromatase is the key enzyme and catalyzes the final and key step, the conversion of C19 steroids to estrogens. Because the presence of SF-1 in endometriotic stromal cells and its absence in endometrial stromal cells is the key event for the differential expression of aromatase, a better understanding of the molecular mechanisms controlling the expression of the SF-1 gene may provide new opportunities for targeted therapies of endometriosis (5, 8). In the present study, we chose to investigate the methylation status of the CpG island in the SF-1 promoter and exon

FIG. 4. Identification of the critical SF-1 promoter region using luciferase activity and repression of SF-1 promoter activity by DNA methylation. A and B, Serial deletion analysis. The constructs were transfected into endometriotic cells (SF-1-expressing cells, A) and endometrial cells (SF-1-negative cells, B). C and D, Four luciferase reporter plasmids, naturally methylated, naturally unmethylated, in vitro mock methylated, and in vitro methylated, were transfected into endometriotic cells (C) and endometrial cells (D). Open and filled circles represent the unmethylated and methylated regions of DNA. *, P ⬍ 0.01 (Student’s t test).

ylation of the CpG island in the ⫺85/⫹239 region. We generated naturally methylated and unmethylated forms of the ⫺85/⫹239 region fused to the luciferase vector using endometrial or endometriotic cells. Additional luciferase con-

FIG. 5. A, Recruitment of MeCP2 to the methylated SF-1 promoter CpG island. ChIP assay using antibody against MeCP2 were performed in endometrial cells (SF-1-negative cells) and endometriotic cells (SF-1-expressing cells). B, Western blot confirming comparable levels of MeCP2 in endometrial and endometriotic stromal cells (three subjects in each group).

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FIG. 6. Proposed mechanism for the regulation of SF-1 expression by DNA methylation of 5⬘-flanking CpG island in eutopic endometrium and endometriosis. DNA methylation associated with the recruitment of MeCP2 to the 5⬘-flanking CpG island exert their inhibitory effects on SF-1 transcription in the endometrium (B), whereas hypomethylation permits SF-1 transcription in endometriosis (A).

I region. Using bisulfite sequencing, transient transfections, treatments with a demethylating compound and ChIP, we demonstrated that methylation of this critical CpG island at the promoter and exon I region regulates SF-1 transcriptional activity in endometriosis or endometrium-derived stromal cells. This is consistent with a large body of literature showing that DNA methylation at the transcriptional regulatory region is generally associated with gene silencing (30 –36). It was previously reported that the methylation status in the promoter and exon I region of a number of genes determines transcriptional activity and levels of mRNA and/or protein encoded by these genes (31–36). CpG island hypermethylation may inhibit transcription by interfering with the recruitment and function of basal transcription factors or transcriptional coactivators. Also, hypermethylation of CpG dinucleotides near the transcriptional regulatory region may initiate the recruitment of the methylation-dependent DNAbinding proteins that mediate silencing of genes via facilitation of a repressive chromatin environment (37, 38) (Fig. 6). The discovery of the function of these proteins, in particular MeCP2, suggests that transcriptional repression by methylation may, in part, be due to the binding of these methyl CpG-binding proteins that prevent the functional binding of transcription factors or may act as transcriptional repressors themselves (28, 29). Although SF-1 mRNA was induced approximately 55-fold from its basal level by 5-aza-dC in endometrial stromal cells, the final amounts of expression were still far below the levels expressed in endometriotic stromal cells. Therefore, other regulatory factors may be required to provide maximal expression of the SF-1 gene. Sry-type high-mobility-group box transcription factor-9 up-regulates SF-1 expression via binding its regulatory motif in its promoter. Regulation by Srytype high-mobility-group box transcription factor-9 accounts partially for the sexually dimorphic expression pattern of SF-1 observed during male gonadal differentiation (39). An E-box and a CCAAT box in the SF-1 promoter region were reported to be required for the expression of SF-1 gene in adrenal and gonadal development and function. Upstream stimulatory factor-1 and -2 that bind to the E-box are likely key regulators of SF-1 in the pituitary gonadotrope and steroidogenic cells (39 – 42). Thus, it is warranted to investigate which factors regulate the expression of SF-1 via binding to

its promoter in endometriosis. In fact, our unpublished observations indicate that differentially up-regulated upstream stimulatory factor-2 in endometriosis may contribute to SF-1 expression. In conclusion, our data suggest that differential methylation of the CpG island at the promoter and exon I of the SF-1 gene may be a key mechanism for SF-1 mRNA expression in endometriotic stromal cells and its silencing in eutopic endometrial stromal cells. This mechanism may be, in part, the basis for activation of StAR, aromatase, and other genes critical for estrogen biosynthesis in endometriosis. This is the first demonstration of a methylation-dependent mechanism responsible for regulation of SF-1 expression in any mammalian tissue. Therefore, these findings significantly increase our understanding of mammalian physiology and clinically point to a new target for developing novel therapeutic strategies in endometriosis. Acknowledgments Received March 5, 2007. Accepted May 15, 2007. Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, 303 East Superior Street, Suite 4-123, Chicago, Illinois 60611. E-mail: s-bulun@northwestern. edu. This work was supported by the National Institutes of Health/National Institute of Child Health and Human Development Grant R01 HD38691 and partly by the World Health Organization Fellowship Program. Disclosure Statement: Q.X., Z.L., P.Y., M.P.M., Y.-H.C., E.C., S.R., and S.E.B. have nothing to declare.

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