Article

Transcriptional Activation of Steroidogenic Factor-1 by Hypomethylation of the 5′ CpG Island in Endometriosis

Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois 60611, USA.
Journal of Clinical Endocrinology & Metabolism (Impact Factor: 6.21). 09/2007; 92(8):3261-7. DOI: 10.1210/jc.2007-0494
Source: PubMed
ABSTRACT
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.
The objective of the study was to unravel the mechanism for differential SF-1 expression in endometrial and endometriotic stromal cells.
We identified a CpG island flanking the SF-1 promoter and exon I region and determined its methylation patterns in endometrial and endometriotic cells.
The study was conducted at Northwestern University.
Eutopic endometrium from disease-free subjects (n = 8) and the walls of cystic endometriosis lesions of the ovaries (n = 8) were investigated.
Stromal cells were isolated from these two types of tissues.
Measures are mentioned in 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.
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.

Full-text

Available from: Magdy Milad
Transcriptional Activation of Steroidogenic Factor-1 by
Hypomethylation of the 5 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. Steroido-
genic factor-1 (SF-1), a transcriptional factor essential for activation
of multiple steroidogenic genes for estrogen biosynthesis, is unde-
tectable in normal endometrial stromal cells and aberrantly ex-
pressed 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 dis-
ease-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.
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 in-
creased 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 pro-
moter activity in both cell types (P 0.01). Chromatin immuno-
precipitation 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-de-
pendent regulation of SF-1 in any mammalian tissue. These find-
ings point to a new mechanism for targeting local estrogen bio-
synthesis in endometriosis. (J Clin Endocrinol Metab 92:
3261–3267, 2007)
E
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 endometri-
um-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 in-
cluding steroidogenic acute regulatory protein (StAR) and aro-
matase, giving rise to local estrogen production in endometri-
otic 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 C
19
steroids to estrogens (10). Both StAR and aromatase are ex-
pressed 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 steroido-
genic 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 stud-
ies 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-pi-
tuitary-adrenal or hypothalamic-pituitary-gonadal endocrine
axes (15–17). We found that SF-1, which is expressed in endo-
metriosis but not in its normal counterpart tissue, eutopic en-
dometrium, is the key activator of the aromatase gene promoter
in endometriotic stromal cells (5).
Methylation of DNA at the CpG dinucleotides is a pos-
treplication event catalyzed by the DNA (cytosine-5)-meth-
yltransferase (18), which establishes normal methylation pat-
terns during embryogenesis and reproduces these patterns
during replication of adult cells (19, 20). DNA methylation,
an important mechanism of epigenetic gene regulation, is
First Published Online May 22, 2007
Abbreviations: 5-aza-dC, 5-Aza-2-deoxycytidine; ChIP, chromatin
immunoprecipitation; CT, comparative threshold cycle; MeCP2, methyl-
CpG-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 en-
docrine community.
0021-972X/07/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 92(8):3261–3267
Printed in U.S.A. Copyright © 2007 by The Endocrine Society
doi: 10.1210/jc.2007-0494
3261
Page 1
involved in genomic imprinting, X chromosome inactivation,
aging, mutagenesis, and regulation of tissue-specific gene
expression during development and adult life (21–23). Ab-
errant 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 endometri-
otic 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 exem-
plified 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 men-
strual 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 en-
dometrial samples were obtained from women undergoing hysterec-
tomy 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-CTG-
GAGCCGGATGAGGAC-3, reverse: 5-ACCTGGCGGTAGATGTGGT-
3. 18S primers were forward: 5-AGGAATTCCCAGTAAGTGCG-3,
reverse: 5-GCCTCACTAAACCATCCAA-3.
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 bisulfite-
modified 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-AAC-
RAACAAAACCAACCTACTATCC-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-GATATCA-
GAGAGAGCCACAGAGACAAC-3 (position 524 relative to the tran-
scription start site), forward primers 5-GGTACCACTGGCCTGTCCT-
GACTCT-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-GATATCTAAGTGGAGCAGGCAGTGG-
3(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 (Pro-
mega). The construct was then named as pGL4-SV40) to construct plas-
mids 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 Ap-
plied Science, Indianapolis, IN) according to the manufacturer’s proto-
col. 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 mea-
sured using a luciferase assay system (Promega).
-Galactosidase ac-
tivity was used to normalize transfection efficiency. All of the experi-
ments 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, region-
specific methylation was carried out on the SF-1 promoter fragment of
85/239 after excision and isolation of the fragment. DNA was in-
3262 J Clin Endocrinol Metab, August 2007, 92(8):3261–3267 Xue et al. Methylation of SF-1 and Endometriosis
Page 2
cubated with SssI CpG methylase (New England Biolabs, Ipswich, MA)
in the presence (methylated) or absence (mock-methylated) of S-adeno-
sylmethionine, as recommended by the manufacturer for 2 h. Methyl-
ated and mock-methylated fragments were ligated again into their re-
spectively 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 meth-
ylation-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 man-
ufacturer’s protocol (Upstate Biotechnology, Inc., Lake Placid, NY). Immu-
noprecipitation 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-GGTACCGTGGGGGC-
AGAGACCAAT-3, reverse primer 5-GATATCTAAGTGGAGCAGGC-
AGTGG-3. The PCR products (324 bp) were analyzed on 1% agarose gel.
Three independent experiments were preformed.
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-
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 mem-
branes, and incubated with antihuman SF-1 or MeCP2 antibodies di-
luted 1:1000 purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
and Abcam. Anti-
-actin antibody was used as a loading control. De-
tection was performed using an enhanced chemiluminescence system
(Amersham Pharmacia Biotech, Piscataway, NJ). Band intensity of pro-
tein 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 as-
says were used to quantify mRNA levels of SF-1 gene in
endometrial and endometriotic stromal cells. Each assay in-
dicated 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
FIG. 1. mRNA levels of SF-1 in endometrial and endometriotic stro-
mal 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 elec-
trophoresis 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 endo-
metrial and endometriotic stromal cells. Top, A schematic diagram
indicating the CpG island on SF-1 5-flanking region. 1 is the tran-
scription start site. Black bar, Bisulfite sequencing fragment con-
taining 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 cy-
tosines, 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.
Xue et al. Methylation of SF-1 and Endometriosis J Clin Endocrinol Metab, August 2007, 92(8):3261–3267 3263
Page 3
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 se-
quencing. 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 ex-
press 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 modifi-
cation 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-
dent increase in SF-1 mRNA in originally SF-1-negative en-
dometrial stromal cells. To confirm the demethylating effect
of 5-aza-dC, bisulfite sequencing was performed on un-
treated 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 respon-
sible for SF-1 gene silencing.
Methylation of SF-1 promoter inhibits its activity
To elucidate the critical region of the SF-1 promoter re-
sponsible 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 endo-
metrial and endometriotic cells under the same serum-
starved 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-
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 nor-
malized 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 ob-
tained from bisulfite sequencing in en-
dometrial stromal cells before and after
10
M 5-aza-dC treatment for5d(P
0.05; Student’s t test). Open and filled
circles represent unmethylated and
methylated cytosines, respectively.
3264 J Clin Endocrinol Metab, August 2007, 92(8):3261–3267 Xue et al. Methylation of SF-1 and Endometriosis
Page 4
ylation of the CpG island in the 85/239 region. We gen-
erated naturally methylated and unmethylated forms of the
85/239 region fused to the luciferase vector using endo-
metrial or endometriotic cells. Additional luciferase con-
structs were generated from treating the unmethylated con-
struct from endometriotic cells with a methylase in the
presence or absence of S-adenosylmethionine (in vitro meth-
ylated 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 chro-
matin-associated protein to the SF-1 promoter as a function
of the methylation status of its promoter region. We deter-
mined 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 pro-
moter (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, suggest-
ing 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 C
19
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 control-
ling the expression of the SF-1 gene may provide new op-
portunities for targeted therapies of endometriosis (5, 8).
In the present study, we chose to investigate the methyl-
ation status of the CpG island in the SF-1 promoter and exon
FIG. 4. Identification of the critical SF-1 promoter region using lu-
ciferase 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).
FIG. 5. A, Recruitment of MeCP2 to the methylated SF-1 promoter
CpG island. ChIP assay using antibody against MeCP2 were per-
formed 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).
Xue et al. Methylation of SF-1 and Endometriosis J Clin Endocrinol Metab, August 2007, 92(8):3261–3267 3265
Page 5
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 show-
ing 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 hypermethyl-
ation may inhibit transcription by interfering with the re-
cruitment and function of basal transcription factors or tran-
scriptional coactivators. Also, hypermethylation of CpG
dinucleotides near the transcriptional regulatory region may
initiate the recruitment of the methylation-dependent DNA-
binding proteins that mediate silencing of genes via facili-
tation 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 methyl-
ation 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 ex-
pression of the SF-1 gene. Sry-type high-mobility-group box
transcription factor-9 up-regulates SF-1 expression via bind-
ing its regulatory motif in its promoter. Regulation by Sry-
type 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 ste-
roidogenic 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 ob-
servations indicate that differentially up-regulated upstream
stimulatory factor-2 in endometriosis may contribute to SF-1
expression.
In conclusion, our data suggest that differential methyl-
ation 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 en-
dometrial 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 mam-
malian tissue. Therefore, these findings significantly increase
our understanding of mammalian physiology and clinically
point to a new target for developing novel therapeutic strat-
egies 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/Na-
tional 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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    • "NR5A1 binds to its consensus DNA sequence and activates the transcription of target genes [20, 21] such as aromatizing enzyme, luteinizing hormone , follicle-stimulating hormone, prolactin, gonadotropin releasing hormone receptor, corticotropin releasing hormone, and others. Zeitoun and Xue found that the mRNA and protein levels of NR5A1 in endometriotic stromal cells were significantly higher than those in normal endometrial stromal cells [22, 23]. The aberrant expression of NR5A1 in endometrial stromal Dysfunction of Liver Receptor Homolog-1 in Decidua Abbreviations: PE, preeclampsia; SPE, severe preeclampsia; NP, normal pregnancy; IGFBP1, insulin-like growth factor binding protein 1; PRL, prolactin; EVT, extravillous trophoblastic; SF-1, NR5A1, steroidogenic factor-1; LRH-1, NR5A2, liver receptor homolog-1; LH, luteinizing hormone; FSH, follicle-stimulating hormone; BMP2, morphogenetic protein; hESC, human endometrial stromal cell; MPA, medroxyprogesterone-17-acetate; cAMP, N6, 2 0 -O- dibutyryladenosine. "
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    • "Among these nuclear receptors, ER β expression is maybe 100 times higher in endometriotic tissue than in endometrium. ER β suppresses oestrogen receptor-α (ER α ) expression and results in strikingly high ER β -to-ER α ratios in endometriotic cells (Falconer et al. 2007; Xue et al. 2007a). Th e methylation of a CpG island at the ER β promoter region is a primary mechanism responsible for diff erential expression of ER β in endometriosis and endometrium . "
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    • "Trichostatin A (HDAC pan inhibitor) inhibits NFκB signaling, COX-2 expression, and cell proliferation ; by contrast, increases expression of PR-B and E-cadherin in endometriotic cells in vitro ( Guo, 2007, 2008; ). The DNA methylation inhibitor 5-Aza differentially regulates expression of ERβ and SF-1 in endometrial and endometriotic cells (Izawa et al., 2008; Xue et al., 2007a Xue et al., , 2007b). Although PGE2 plays an important role in the pathogenesis of endometriosis (Banu et al., 2008; Chuang et al., 2010; Cobellis et al., 2004; Laschke et al., 2007; Matsuzaki et al., 2004; Olivares et al., 2008; Wu et al., , 2010), the underlying epigenetic mechanisms of PGE2 action are largely unknown. "
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