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
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ESR2 METHYLATION IN ENDOMETRIAL AND ENDOMETRIOTIC CELLS