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 &amp Metabolism (Impact Factor: 6.21). 09/2007; 92(8):3261-7. DOI: 10.1210/jc.2007-0494
Source: PubMed


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.

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Available from: Magdy Milad, Oct 14, 2015
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    • "Accumulating data suggests that aberrant DNA methylation status may be associated with the molecular features of endometriosis. Previous reports have shown that abnormal promoter methylation occurs in certain genes, such as progesterone receptor B [5], E-cadherin [7], HOXA10 [8], estrogen receptor β [4], steroidgenic factor 1 [2], [3] and aromatase [9], [10]. These results suggest that epigenetic abnormalities are important in the pathogenesis and development of endometriosis; however, these results do not elucidate to what extent DNA methylation status contributes to endometriosis at the genome-wide level. "
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    ABSTRACT: The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa) and without endometriosis (euESCb) and ovarian endometrial cysts (choESC). Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2), which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.
    PLoS ONE 01/2014; 9(1):e83612. DOI:10.1371/journal.pone.0083612 · 3.23 Impact Factor
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    • "Recent studies have indicated that an epigenetic disorder may play a role in the pathophysiology of endometriosis [25]. For instance, Xue et al. have reported that DNA hypomethylation of estrogen receptor 2 (ESR2) and steroidogenic factor-1 (SF-1) were responsible for their strikingly elevated level in endometriosis [26,27]. Additionally, DNA hypermethylation and suppressed expression of HOXA10 have been shown in the endometrium of women with endometriosis [28]. "
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    ABSTRACT: Accumulated evidence reveals that cyclooxygenase-2 (COX-2) was overexpressed in eutopic endometrium of endometriosis, which may play a critical role in the pathogenesis of endometriosis. However, few studies have been performed to explore the molecular mechanisms underlying the abnormal high expression of COX-2 in endometriosis. Considering the fact that a number of recent studies have shown DNA methylation affecting some genes in endometriosis, the present study was therefore aimed to determine whether the observed high expression COX-2 in endometriosis is caused by the hypomethylation of CpG island within the promoter of this gene. The endometrial tissues were collected from 60 women with endometriosis (endometriosis group) and 20 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation specific PCR. Quantitative real-time RT-PCR was performed to measure COX-2 mRNA level in endometrial tissues. The frequency of promoter hypermethylation of COX-2 was lower in eutopic endometrium of the endometriosis group (41.7%) than that in the control group (75.0%), P < 0.05. COX-2 mRNA level in the eutopic endometrium of the endometriosis group was 2.61-fold higher than that in the control group (P < 0.01). COX-2 mRNA level in unmethylated endometrium of the endometriosis group or the control group was 2.39-fold and 2.66-fold, respectively, higher than that in the methylated endometrium of the same group (P < 0.01). The hypomethylation within the promoter of COX-2 may be responsible for the elevated gene expression in eutopic endometrium of endometriosis.
    05/2012; 17(1):12. DOI:10.1186/2047-783X-17-12
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    • "RNA extraction and real-time RT-PCR were performed as described previously [21], [22]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were measured as an internal control. "
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    ABSTRACT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Each leiomyoma is thought to be a benign monoclonal tumor arising from a single transformed myometrial smooth muscle cell; however, it is not known what leiomyoma cell type is responsible for tumor growth. Thus, we tested the hypothesis that a distinct stem/reservoir cell-enriched population, designated as the leiomyoma-derived side population (LMSP), is responsible for cell proliferation and tumor growth. LMSP comprised approximately 1% of all leiomyoma and 2% of all myometrium-derived cells. All LMSP and leiomyoma-derived main population (LMMP) but none of the side or main population cells isolated from adjacent myometrium carried a mediator complex subunit 12 mutation, a genetic marker of neoplastic transformation. Messenger RNA levels for estrogen receptor-α, progesterone receptor and smooth muscle cell markers were barely detectable and significantly lower in the LMSP compared with the LMMP. LMSP alone did not attach or survive in monolayer culture in the presence or absence of estradiol and progestin, whereas LMMP readily grew under these conditions. LMSP did attach and survive when directly mixed with unsorted myometrial cells in monolayer culture. After resorting and reculturing, LMSP gained full potential of proliferation. Intriguingly, xenografts comprised of LMSP and unsorted myometrial smooth muscle cells grew into relatively large tumors (3.67 ± 1.07 mm(3)), whereas xenografts comprised of LMMP and unsorted myometrial smooth muscle cells produced smaller tumors (0.54 ± 0.20 mm(3), p<0.05, n = 10 paired patient samples). LMSP xenografts displayed significantly higher proliferative activity compared with LMMP xenografts (p<0.05). Our data suggest that LMSP, which have stem/reservoir cell characteristics, are necessary for in vivo growth of leiomyoma xenograft tumors. Lower estrogen and progesterone receptor levels in LMSP suggests an indirect paracrine effect of steroid hormones on stem cells via the mature neighboring cells.
    PLoS ONE 05/2012; 7(5):e36935. DOI:10.1371/journal.pone.0036935 · 3.23 Impact Factor
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