BIOLOGY OF REPRODUCTION 77, 681–687 (2007)
Published online before print 11 July 2007.
Promoter Methylation Regulates Estrogen Receptor 2 in Human Endometrium and
Qing Xue,3,6Zhihong Lin,3You-Hong Cheng,3Chiang-Ching Huang,5Erica Marsh,3,4Ping Yin,3
Magdy P. Milad,6Edmond Confino,6Scott Reierstad,3Joy Innes,3and Serdar E. Bulun2,3,6
Division of Reproductive Biology Research,3and Division of Reproductive Endocrinology and infertility,4Department of
Obsterics and Gynecology, and Department of Preventative Medicine,5Feinberg School of Medicine, Northwestern
University, Chicago, Illinois 60611
Department of Obstetrics and Gynecology,6First Hospital of Peking University, Beijing, China
Steroid receptors in the stromal cells of endometrium and
its disease counterpart tissue endometriosis play critical
physiologic roles. We found that mRNA and protein levels of
estrogen receptor 2 (ESR2) were strikingly higher, whereas levels
of estrogen receptor 1 (ESR1), total progesterone receptor
(PGR), and progesterone receptor B (PGR B) were significantly
lower in endometriotic versus endometrial stromal cells.
Because ESR2 displayed the most striking levels of differential
expression between endometriotic and endometrial cells, and
the mechanisms for this difference are unknown, we tested the
hypothesis that alteration in DNA methylation is a mechanism
responsible for severely increased ESR2 mRNA levels in
endometriotic cells. We identified a CpG island occupying the
promoter region (?197/þ359) of the ESR2 gene. Bisulfite
sequencing of this region showed significantly higher methyla-
tion in primary endometrial cells (n ¼ 8 subjects) versus
endometriotic cells (n ¼ 8 subjects). The demethylating agent
5-aza-20-deoxycytidine significantly increased ESR2 mRNA
levels in endometrial cells. Mechanistically, we employed serial
deletion mutants of the ESR2 promoter fused to the luciferase
reporter gene and transiently transfected into both endometri-
otic and endometrial cells. We demonstrated that the critical
region (?197/þ372) that confers promoter activity also bears
the CpG island, and the activity of the ESR2 promoter was
strongly inactivated by in vitro methylation. Taken together,
methylation of a CpG island at the ESR2 promoter region is a
primary mechanism responsible for differential expression of
ESR2 in endometriosis and endometrium. These findings may be
applied to a number of areas ranging from diagnosis to the
treatment of endometriosis.
CpG island, DNA methylation, endometriosis, endometrium,
ESR2, estradiol receptor, ovary, uterus
Endometriosis is defined as the presence of endometrium-
like tissue outside of the uterine cavity. It is a common
gynecologic condition, affecting 1 in 10 women in the
reproductive age group . Endometriosis is associated with
severely painful menstruation, chronic pelvic pain, and
infertility [1, 2]. Although the etiology and exact mechanism
for the development of endometriosis is unclear, there is a large
body of laboratory and circumstantial evidence that suggests a
crucial role for estrogen in the establishment and maintenance
of this disease [3–5].
Despite its sensitivity to estrogen, endometriosis appears to
contain a unique complement of steroid hormone receptors
compared with that of its normal tissue counterpart, the eutopic
endometrium. For example, a number of investigators reported
markedly higher levels of estrogen receptor 2 (ESR2) and lower
levels of estrogen receptor 1 (ESR1) in human endometriotic
tissues and primary stromal cells compared with eutopic
endometrial tissues and cells [6, 7]. Moreover, the levels of
both isoforms of progesterone receptor (PGR), particularly
progesterone receptor B (PGR B), are significantly lower in
endometriosis compared with eutopic endometrium [8, 9]. The
classical human ESR1 was cloned in 1986, and a second
estrogen receptor, ESR2, was cloned from rat prostate and
human testis in 1996 [10–12]. Both ESRs act as transcription
factors and are believed to play a key role in endometrial and
endometriosis growth regulation.
Hypermethylation of a CpG island has been associated with
the transcriptional inactivation of genes. Recently, key nuclear
receptor genes, such as ESR1, ESR2, and PGR, were shown to
be regulated by methylation of their promoter regions in breast,
prostate, and endometrial cancer tissues [13–15]. To assess the
relative expression levels of these nuclear receptors and the
DNA methylation mechanism in endometrium and endometri-
osis, an in vitro model of primary stromal cells from these two
tissue sources was developed.
Currently, the biologic roles of ESR2 in endometrium and
endometriosis are not well understood. We chose to investigate
the molecular mechanism responsible for differential expres-
sion of ESR2 for two reasons. First, the most striking difference
between endometriosis and endometrium was observed with
respect to ESR2 levels compared with other steroid receptors,
and ESR2 mRNA levels were very low or nearly undetectable
in the endometrial stromal cells. Second, an ESR2-selective
compound was shown to be therapeutic in a rodent
endometriosis model [16, 17]. At present, no evidence has
been provided to indicate whether DNA methylation is causally
linked to differential ESR2 expression in endometriotic stromal
cells and endometrial stromal cells. Direct evidence in support
of the cytosine methylation of specific 50CpGs that leads to
transcriptional inactivation has not been reported.
1Supported by National Institutes of Health/National Institute of Child
Health & Human Development grant U54-HD40093.
2Correspondence: Serdar E. Bulun, Division of Reproductive Biology
Research, Department of Obstetrics and Gynecology, Northwestern
University, 303 East Superior Street, Suite 4–250, Chicago, IL 60611.
FAX: 312 503 0095; e-mail: firstname.lastname@example.org
Received: 26 March 2007.
First decision: 9 April 2007.
Accepted: 9 July 2007.
? 2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
MATERIALS AND METHODS
Subjects and Primary Cell Culture
Eutopic endometrium from disease-free subjects (n ¼ 8) and ectopic
endometrium from the cyst walls of ovarian endometriomas (defined as a cystic
ovarian lesion composed of endometrium-like tissue in its cyst wall and bloody
fluid filled in this cyst; n ¼ 8) were obtained immediately after surgery. The
mean ages of subjects in each group were 42 6 3 to 39 6 3 years, respectively,
and there were no significant differences between the two groups with respect
to age or cycle phase. Moreover, we obtained three paired samples of ovarian
endometriomas and eutopic endometrium from the same subjects. 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 histologic examination. Written informed consent
was obtained before surgical procedures, including a consent form and protocol
approved by the Institutional Review Board of Northwestern University.
Stromal cells were isolated from these two types of tissues using a protocol
previously reported by Ryan et al. with minor modifications [18, 19]. Briefly,
tissues were rinsed with sterile PBS, minced finely, and digested with
collagenase (Sigma, St. Louis, MO) and DNase (Sigma) at 378C for 60 min.
Stromal cells were separated from epithelial cells by filtration through a 70- and
a 20-lm sieve, then they were suspended in Dulbecco modified Eagle medium
(DMEM)/F12 1:1 (GIBCO/BRL, Grand Island, NY) containing 10% FBS and
in a humidified atmosphere with 5% CO2at 378C.
RNA Extraction and Quantitative Analysis by Real-Time RT-
Total RNA was isolated from stromal cells using TRIzol reagent (Sigma)
and following the manufacturer’s protocol. Total RNA was first treated with
DNaseI (Ambion, Austin, TX) to remove contaminating genomic DNA from
the RNA samples, then 1 lg RNA was used to generate cDNA with the
Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Real-
time quantitative PCR was performed with the ABI 7900 Sequence Detection
System and the ABI Taqman Gene Expression system (purchased from Applied
Biosystems, Foster City, CA) for ESR1, ESR2, and eukaryotic 18S rRNA (18S).
The SYBR Green assay was used for total PGR, PGR B, and 18S. 18S values
were used for normalization. Primers used for SYBR Green assay were: total
PGR, forward: 50-GTCCTTACCTGTGGGAGCTG-30; reverse: 50-CAACAG
CATCCAGTGCTCTC-30. PGR B, forward: 50-GTACGGAGCCAGCA
GAAGTC-30; reverse, 50-TCTCTGGCATCAAACTCGTG-30. 18S, forward:
50-AGGAATTCCCAGTAAGTGCG-30; reverse: 50-GCCTCACTAAAC
CATCCAA-30. Relative quantification of mRNA species was performed using
the comparative threshold cycles (CT) method. In brief, CTwas used to
determine the mRNA level normalized to the average mRNA level in
endometrial stromal cells. Thus, mRNA levels were expressed as an n-fold
difference. For each sample, the gene CTvalue was normalized using the
formula: DCT¼CTgene? CT18S. To determine relative expression levels, the
following formula was used: DDCT¼DCTsample? DCTcalibrator. This value
was used to plot the gene expression employing the formula 2?D DCT.
Bisulfite Modification and Sequencing Analysis
Genomic DNA was extracted from the primary stromal cells using the
DNeasy Tissue kit (Qiagen, Valencia, CA). DNA (500 ng) was treated with
sodium bisulfite following the manufacturer’s protocol (Zymo Research,
Orange, CA). Purified DNA was dissolved in 10 ll M-Elution Buffer (Zymo
Research). For PCR amplification, 3 ll bisulfite-modified DNA was added to a
final volume of 20 ll. AmpliTaq Gold PCR Master Mix (Applied Biosystems)
was used for all PCR amplifications. PCR amplifications were performed using
the following primers for ESR2: forward: 50-ATTATTTTTGTGGGTGGAT-
TAGGAG-30, and reverse: 50-AACCCCTTCTTCC-TTTTAAAAACC-30. The
thermal cycle conditions were as follows: 958C for 10 min, followed by 40
cycles of denaturation at 958C for 30 sec, annealing at 508C for 2 min, and
elongation at 728C for 2 min, then followed by an incubation at 728C for 7 min.
PCR products (166 bp) were gel purified and cloned into the pGEM-Teasy
vector (Promega, Madison, WI). Following transformation, six to eight clones
with the correct insert were randomly picked for each PCR and were sequenced
using an Applied Biosystems 377 instrument.
5-Aza-20-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 20 lM DNA
methyltransferase inhibitor, 5-aza-dC (Sigma) for 5 days, and medium was
(D), and PGR B (E) mRNA levels were quantified using real-time PCR. The values were first normalized to 18S and are expressed as fold-difference of the
levels determined byWestern blot of ESR1 and ESR2 in endometrial and endometriotic stromal cells (eight subjectsin eachgroup),Student’s t test,P , 0.05.
Expression levels of ESR1, ESR2, total PGR, and PGR B in endometrial (n¼8) and endometriotic (n¼8) stromal cells. ESR1 (A), ESR2 (B), total PGR
XUE ET AL.
changed each day. Total RNA was isolated from the treated cells using TRIzol
reagent. All experiments were conducted in triplicate and repeated three times
in primary cultured cells from at least three different subjects.
Reporter plasmid vectors containing the ESR2 promoter sequences were
constructed by PCR cloning. Genomic DNA from endometriotic stromal cells
was used as the template for amplification. The primers were: reverse primer 50-
GATATCTTAGCACAATCAACCCAGAG-C-30(position þ564 relative to
the transcription start site); forward primers 50-GGTACCTTCCC-AGT
(?197), and 50-GGT-ACCTGTTTGAAATCCTGCGGTGAG-30(þ372). Re-
striction sites (Kpn2site for forward primers and EcoRV site for reverse
primers) were added to the 50-end of primers, and promoter sequences were
amplified using TAKARA LA Taq with GC buffer (TaKaRa, Otsu, Japan).
PCR products were cloned into a modified 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 final plasmids containing ESR2 promoter sequences were ?525/þ564,
?197/þ564, and þ372/þ564.
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 ll FuGENE 6 was mixed with 240 ng reporter plasmid and 60 or 80 ng
pSV-b-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).
Beta-galactosidase activity was used to normalize transfection efficiency. All of
the experiments were repeated three times in triplicate.
In Vitro Methylation of Reporter Plasmids
In vitro methylation assays were carried out according to the methods
described by Robertson and Ambinder  and Singal et al. . Briefly,
region-specific methylation was carried out on the ESR2 promoter fragments of
?525/þ564 and ?197/þ564 after excision and isolation. DNA was incubated
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 re-ligated into their respectively unmethylated vectors. All
constructs were sequenced to confirm the correct region of the ESR2 gene, and
the efficiency of the methylation was determined through methylation-sensitive
and methylation-insensitive restriction enzyme digestion with HpaII and MspI.
Western Blot Analysis
Cells were washed with ice-cold PBS and suspended in the protein
extraction reagent (Pierce, Rockford, IL). Lysates were cleared by centrifuga-
tion at 15 7003g for 10 min. Equal amounts of protein (15 lg) were resolved
on 4%–15% Tris-HCL gels, transferred onto nitrocellulose membranes, and
incubated with anti-human ESR1 or ESR2 antibodies diluted 1:100 or 1:2000
(purchased from Calbiochem, Darmstadt, Germany and Upstate, Chicago, IL).
Anti-ACTB antibody was used as a loading control. Detection was performed
using a supersignal west femto maximum sensitivity substrate system (Pierce).
Band intensity of protein expression was quantified using the Quantity One
Analysis Software (Bio-Rad Laboratories, Los Angeles, CA).
For mRNA levels and luciferase assays, the values are expressed as means
6 SEM of measurements for primary cells cultured in triplicate. The results
were representative of at least three independent experiments. Percent
methylation of each clone obtained from each of the eight patients in each
group was treated as a single value for the statistical analysis of bisulfite
sequencing. The data were analyzed using Student’s t-test with statistical
significance at the level of P , 0.05. Spearman’s rank correlation coefficient
was calculated for the correlation between ESR2 mRNA levels and percent
methylation, and a permutation test was used to assess its statistical significance.
ESR1, ESR2, Total PGR, and PGR B mRNA Levels in
Endometrial and Endometriotic Stromal Cells
Real-time RT-PCR was used to quantify the mRNA levels
of nuclear receptors in endometrial (n ¼ 8 subjects) and
endometriotic (n ¼ 8 subjects) stromal cells. ESR1 mRNA
levels were somewhat lower (7-fold; P ¼ 0.037) in endome-
triotic stromal cells compared with endometrial stromal cells.
ESR2 mRNA was strikingly higher (approximately 34-fold; P
¼ 0.015) in endometriotic stromal cells, whereas it was much
lower or nearly absent in endometrial stromal cells. Thus, the
ratios of ESR1 to ESR2 were, on average, 841 and 21 in
endometrial and endometriotic stromal cells, respectively (P ,
ESR2 in three paired endometrial and
endometriotic stromal cells. ESR1 (A) and
ESR2 (B) mRNA levels were quantified using
real-time PCR. The values are expressed as
fold-difference of the average value found in
endometrial stromal cells. C) Protein levels
determined by Western blot of ESR1 and
ESR2 in endometrial and endometriotic
stromal cells (three subjects in each group),
Student’s t test, P , 0.05.
Expression levels of ESR1 and
ESR2 METHYLATION IN ENDOMETRIAL AND ENDOMETRIOTIC CELLS
0.001). Total PGR and PGR B mRNA levels in endometriotic
stromal cells were also significantly lower than those in
endometrial stromal cells (P ¼ 0.027 and P ¼ 0.029; Fig. 1).
Western blot showed that ESR2 protein levels in endometriotic
cells (n ¼ 8 subjects) were significantly higher compared with
endometrial cells (n¼8 subjects), whereas ESR1 protein levels
in endometriotic cells were significantly lower compared with
endometrial cells (P , 0.05; Fig. 1F). We also compared ESR1
and ESR2 expression in matched endometrial versus endome-
triotic stromal cells obtained simultaneously from separate
groups of three subjects (P , 0.05; Fig. 2). Both mRNA and
protein levels of ESR1 and ESR2 were significantly different in
these two groups similar to the findings illustrated in Figure 1.
DNA Methylation Profile of the ESR2 Promoter Region
Among the four steroid receptors that we examined, ESR2
mRNA levels displayed the highest and most strikingly
differential expression between the two homologous cell types.
Since this observation made promoter methylation a likely
mechanism for the regulation of ESR2 in endometriosis versus
endometrium, we pursued this line of investigation. We
identified an approximately 550-bp classic CpG island (?197/
þ359) within the promoter and its downstream untranslated
exon 0N region of the ESR2 gene. Methylation status of ESR2
promoter region was determined by bisulfite genomic sequenc-
ing. The detailed CpG methylation status of endometrial and
endometriotic stromal cells was shown in Figure 3. There was a
statistically significant difference in the methylation status
within this region (?189/?24; P , 0.0001). It was heavily
methylated in the majority of endometrial stromal cells (n¼8)
that expressed lower levels of ESR2 and largely unmethylated
in endometriotic stromal cells (n ¼ 8) that expressed higher
levels of ESR2 mRNA. Also, significant negative correlation
was found between percentage of methylation of ESR2
promoter region and ESR2 mRNA expression (in logarithmc
promoter region in endometrial and endo-
metriotic stromal cells. A) Schematic dia-
gram indicating the classic CpG island on
ESR2 50-flanking region. The transcription
start site (TSS) is indicated as þ1. Upper
black bar, predicted CpG island; lower
black bar, bisulfite sequencing fragment
containing the promoter region. B) methyl-
ation status of 13 CpG sites in ESR2
promoter region obtained from bisulfite
sequencing in endometrial and endometri-
otic stromal cells. Open and filled circles
represent unmethylated and methylated
cytosines, respectively. The numbers indi-
cate the positions of cytosine residues of
CpGs relative to the transcription start site
(þ1), and the numbers 1 to 8 on each side
represent subjects from whom primary
stromal cells were obtained. Cells were
obtained from a total of 16 subjects. C)
Percent methylation of ESR2 promoter
region in endometrial and endometriotic
cells, *P , 0.0001. D) Significant negative
correlation (Spearman’s rank correlation
coefficient: ?0.89, P , 0.001) between
percentage of methylation of ESR2 promoter
region and ESR2 mRNA expression (in
logarithmc scale) among eight endometrial
stromal cells and eight endometriotic stro-
DNA methylation status of ESR2
XUE ET AL.
scale) among eight endometrial stromal cells and eight
endometriotic stromal cells (Fig. 3D, Spearman’s rank
correlation coefficient ?0.89, permutation test; P , 0.001).
Induction of ESR2 mRNA Expression by 5-aza-dC
To determine the correlation between DNA methylation and
downregulation of the ESR2 gene or its near-silencing, the
endometrial stromal cells (with hypermethylation of ESR2
promoter) were treated with demethylating agent 5-aza-dC.
The level of ESR2 mRNA was measured using real-time RT-
PCR. As shown in Figure 4, the treatment in endometrial
stromal cells with 5-aza-dC significantly increased ESR2
mRNA levels (P ¼ 0.025).
Regulation of ESR2 Promoter Activity by Methylation of
To elucidate the critical region in the ESR2 gene 50-flanking
sequence, which regulates promoter activity, we transfected
serial deletion mutants (?525/þ564,?197/þ564, þ372/þ564)
of the ESR2 promoter region fused to the luciferase reporter
gene into endometriotic and endometrial stromal cells. The
relative luciferase activities of the reporter gene constructs were
determined in triplicate. We did not detect a significant
difference in luciferase activity between?525/þ564 and?197/
þ564 constructs, whereas the þ372/þ564 construct exhibited
significant decreases (60.1% and 48.6%) in ESR2 promoter
activity compared with the ?197/þ564 construct in endome-
triotic or endometrial cells (Fig. 5, A and B). This indicated
that the ?197/þ372 bp region containing the CpG island is
critical for baseline promoter activity in both endometriotic and
endometrial cells (P , 0.01).
Next, in vitro methylation analysis was performed to
determine whether ESR2 promoter activity was regulated by
the methylation of the ESR2 CpG island. Figure 5, C and D,
showed that in vitro methylation of the CpG island in the?525/
þ564 or?197/þ564 luciferase constructs significantly reduced
ESR2 promoter activity in both cell types (Student’s t-test, P ,
0.001, and P , 0.01).
Development and progression of endometriosis depends on
the presence of estrogen [22, 23]. However, the biologic
influence of estrogen on target organs is modulated by changes
in tissue hormone levels and the local distribution of its
receptors ESR1 and ESR2. Studies in knockout mice and its
nonidentical tissue distribution compared with ESR1 would
mRNA in endometrial stromal cells. ESR2 mRNA levels following treatment
with vehicle or 5-aza-dC (20 lM) were quantified by real-time PCR and
normalized to their expression in vehicle-treated cells. Experiments were
performed using triplicate dishes of cells. This is a representative of three
independent experiments using cells from different subjects.
Effect of the demethylating agent 5-aza-dC on the levels of ESR2
methylation. A, B) Serial deletion analysis. The promoter constructs were transfected into endometriotic (A) and endometrial (B) stromal cells. C, D) In
vitro mock-methylated and in vitro methylated constructs were transfected into endometriotic (C) and endometrial stromal cells (D). Open and filled
circles represent the unmethylated and methylated regions of DNA. The results are presented as means 6 SEM. *P , 0.01; **P , 0.001.
Identification of the critical ESR2 promoter region using luciferase reporter gene assays and repression of ESR2 promoter activity by DNA
ESR2 METHYLATION IN ENDOMETRIAL AND ENDOMETRIOTIC CELLS
suggest that ESR2 has a biologic function distinct from that of
ESR1 . We demonstrated that ESR1 expression was
downregulated and ESR2 was upregulated in endometriotic
stromal cells compared with endometrial stromal cells, which
confirmed previous reports [7, 25]. This raises the possibility
that at least some of the critical functions of estradiol are
mediated by ESR2. Recently uncovered biologic roles of ESR2
regulating inflammatory processes in autoimmune diseases and
endometriosis lend further credence to these findings [16, 17].
In fact, an ESR2-selective drug has been shown to treat
endometriosis in a rodent model. This highly selective ESR2
agonist, ERB-041, is found to be inactive on classic estrogenic
targets, such as the uterus, mammary gland, and bone.
However, it has potent anti-inflammatory activity in two in
vivo models: the HLA-B27 transgenic rat and Lewis rat
adjuvant-induced arthritis. In a rodent model of endometriosis,
the beneficial actions of this compound were interpreted to be
independent of ESR2 in the lesion . ESR2 has also been
shown to induce cell proliferation and may cause growth of
endometriosis via this mechanism .
ESR2 is regulated by two alternatively used promoters
(exons 0N and 0K) upstream of a common coding region. Both
promoter regions contain the CpG islands. We elected to
evaluate the DNA methylation status of the CpG island, located
at the proximal promoter-exon 0N, because this region was
shown to be differentially methylated previously in normal
versus malignant breast lesions [27, 28].
Differences in the ESR1 to ESR2 ratio between endometri-
otic and endometrial stromal cells could have important
functional implications, since these ESRs have different
ligand-binding characteristics [29, 30]. It also has been
proposed that heterodimers of ESR1 and ESR2 can associate
with estrogen-responsive elements in vitro . Because it was
reported that one possible role of ESR2 is to modulate ESR1
activity, the relative expression levels of two ESR subtypes are
an important determinant of target genes regulated by estrogens
and antiestrogens . Therefore, it is conceivable that the set
of estrogen target genes vary significantly in endometriotic
versus endometrial stromal cells.
We observed a clear inverse relationship between the extent
of methylation in the ESR2 promoter CpG island and its
mRNA levels in endometrial and endometriotic stromal cells.
This was verified mechanistically using treatments with
demethylating agent and isolation of the regulatory region
subject to methylation by assaying promoter activity. This is
consistent with a large body of literature showing that DNA
methylation at the transcription regulatory region is generally
associated with gene silencing or downregulation [33–35]. In
general, DNA methylation-mediated control of gene expression
may be a major mechanism for the regulation of steroid
receptor mRNA levels in various tissues in view of
accumulating published evidence [13–15].
It has been demonstrated that DNA methylation can
interfere with protein-DNA interaction, recruitment of histone
deacetylases, and the induction of chromatin condensation
necessary for gene inactivation [36, 37]. Methylation can
directly interfere with the DNA binding of certain transcrip-
tional factors. Also, some methyl-CpG binding proteins are
shown to bind to methylated DNA and alter its DNA
conformation, thus affecting the binding of various transcrip-
tional regulators [38, 39]. These molecular alterations associ-
ated with the methylation of the ESR2 promoter may be
responsible for its repression in endometrial stromal cells. Also,
the expression of ESR2 in the stromal cells of endometriosis
may be regulated by factors other than methylation. For
example, sequence analysis of the 50-flanking region of the
ESR2 promoter 0N has shown the presence of several
consensus transcriptional factor binding sites and cis-regulatory
This is the first demonstration of a methylation-dependent
mechanism responsible for strikingly elevated levels of ESR2
in endometriosis. This finding may have several clinical
applications. Because the methylation of a specific gene can
be detected in DNA from diagnosis biopsies , ESR2
methylation status could be a potentially helpful adjunct to
morphologic criteria for the diagnosis of endometriosis.
Moreover, testing for ESR2 promoter methylation in endome-
triotic lesions may identify patients who are candidates for
treatment with ESR2-selective compounds. Finally, new drugs
that regulate methylation may be used as potential therapeutics
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ESR2 METHYLATION IN ENDOMETRIAL AND ENDOMETRIOTIC CELLS