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Endocrinology, doi:10.1210/en.2008-0104
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Endocrinology Vol. 149, No. 11 5599-5609
Copyright © 2008 by The Endocrine Society

Deoxyribonucleic Acid Methylation Controls Cell Type-Specific Expression of Steroidogenic Factor 1

Erling A. Hoivik, Linda Aumo, Reidun Aesoy, Haldis Lillefosse, Aurélia E. Lewis, Rebecca M. Perrett, Nancy R. Stallings, Neil A. Hanley and Marit Bakke

Department of Biomedicine (E.A.H., L.A., R.A., H.L., A.E.L., M.B.), University of Bergen, 5009 Bergen, Norway; Stem Cells and Regeneration/Human Genetics Division (R.M.P.), Centre for Human Development, University of Southampton, Southampton SO16 6YD, United Kingdom; Endocrine Sciences Research Group (N.A.H.), University of Manchester & Manchester Biomedical Research Centre, Central Manchester & Manchester Children’s University Hospitals NHS Trust, Manchester M13 9PT, United Kingdom; and Departments of Internal Medicine and Pharmacology (N.R.S.), University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Marit Bakke, Department of Biomedicine, University of Bergen, Jonas Lies vei 9, 5009 Bergen, Norway. E-mail: marit.bakke{at}biomed.uib.no.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DNA METHYLATION IS a major epigenetic mechanism that control developmental gene expression. Coordinated waves of demethylation and de novo methylation establish the genome-wide methylation pattern during embryogenesis, and function to maintain cellular phenotypes through clonal inheritance of spatiotemporal expression of key developmental genes (1). Mammalian cytosine DNA methyl transferases (Dnmts) are divided into two groups based on their preferred DNA substrate. Dnmt1 copies the methylation pattern during DNA replication. In concordance with this, Dnmt1 is expressed constitutively in proliferating cells and is located at the replicating foci during S-phase (2). De novo methylation is carried out by the DNA methyl transferases, Dnmt3a and Dnmt3b. These factors are highly expressed in the developing mouse embryo in which they carry out de novo methylation after implantation (3). Genetic manipulation of the Dnmt genes has demonstrated that appropriate DNA methylation is required for normal mammalian development (4).

Steroidogenic factor 1 (SF1; also called adrenal-4 binding protein and officially designated NR5A1) is a nuclear receptor encoded by the Fushi tarazu factor-1 gene (Ftz-F1; for simplicity we refer to the gene as SF1/Sf1). SF1 plays fundamental roles in the development and function of steroidogenic organs, and targeted deletion of the Sf1 gene causes adrenal and gonadal agenesis and nearly immediate postnatal death due to respiratory distress caused by glucocorticoid deficiency (5). During mouse embryogenesis, SF1 is expressed from embryonic day (E) 9.0 in the adrenogonadal primordium, a population of cells that arises from the coelemic epithelium of the urogenital ridge (6). The adrenal and gonadal anlagen progressively individualize and are separate structures at E12.5. The adrenal cells form the outer cortex with invasion by migratory neural crest cells that form the inner adrenal medulla by E16–E16.5. SF1 expression is confined to the cortical region of the adrenal gland (7). In this location, it is maintained postnatally in which it transactivates the genes encoding cytochrome P450 steroid hydroxylases, the melanocortin type 2 receptor, which binds ACTH, cholesterol transporters, and other genes responsible for the steroidogenic phenotype (8). SF1 is expressed in the bipotential gonad. In the developing testis, it is maintained in the Leydig cells, in which it is essential for male sexual differentiation due to its role, as in the adrenal cortex, in regulating expression of the cytochrome P450 steroid hydroxylases among other genes responsible for testosterone synthesis. In Sertoli cells, SF1 regulates expression of the Müllerian inhibiting substance (MIS) and insulin-like factor 3 (Insl-3) genes. After sex determination, SF1 is gradually down-regulated in the ovary from E12.5 and reappears postnatally in theca and granulosa cells and in the corpus luteum once folliculogenesis commences (9). SF1 is also expressed in the gonadotrophs of the pituitary, ventromedial hypothalamic nucleus (VMH), and spleen (8). A corresponding expression pattern of SF1 is observed in human fetal and adult tissues (10, 11).

Several transcription factors have been implicated in the regulation of the SF1/Sf1 gene: Sp1 (stimulatory protein-1) (12, 13), GATA4 (14), Sox9 (SRY-type high-mobility-group-box protein 9) (15), WT-1 (Wilms tumor suppressor) (16), Lhx-9 (lim homeobox gene-9) (17) and Cited2 (CBP/p300 interacting transactivator with ED-rich tail-2) (18). In transient transfection experiments, expression also depends on a conserved E-box motif in the proximal promoter (13, 19, 20), bound with different consequences by two basic helix-loop-helix factors. Upstream stimulatory factor (USF) family members activate transcription (21, 22, 23), whereas Pod-1 seemingly represses SF1 expression (24). In Pod-1 knockout mice, SF1 is expressed ectopically in cells that would normally have expressed Pod-1, supporting a repressive role of this factor (25). In the last few years, two studies using transgenic mouse models have identified enhancer elements regulating SF1 expression in a tissue-specific fashion. One of these studies identified a fetal adrenal-specific enhancer in intron 4 (26). Transcription from this intronic enhancer is initiated by the binding of heterodimeric complexes of Pbx1/Prep1 and Pbx1/Hox. Subsequently when the expression of SF1 is established, SF1 acts back on fetal adrenal-specific enhancer in a positive autoregulatory manner (26). Furthermore, the same group has also detected a VMH-specific enhancer in intron 6 (27). Despite these advances, we are only starting to reveal the mechanisms underlying tissue-specific expression of SF1, and little attention has focused on potential epigenetic regulation of this gene.

In this study we demonstrate that the proximal promoter of the SF1/Sf1 gene is subject to DNA methylation. It is hypomethylated in cells and tissues that express SF1 but hypermethylated in nonexpressing cells and tissues, indicating that epigenetic mechanisms contribute to the restricted expression of this factor. Analyses of fetal tissues indicate that the DNA methylation pattern is established at an early embryonic stage in structures that are determined to express SF1 and faithfully conserved during development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vitro methylation of reporter plasmids
A fragment spanning –185/+141 bp (ApaI/EcoRI) of the mouse Sf1 gene was inserted upstream of luciferase (lacking a minimal heterologous promoter) to create the reporter plasmid Sf1 (–185/+141)/luciferase (Luc). Sf1 (–185/+141)/Luc was methylated in vitro by using the SssI CpG methylase (New England Biolabs Inc., Beverly, MA).

Cell culture
Mouse adrenocortical tumor cells (Y1), human cervix epitheloid carcinoma cells (HeLa), mouse Sertoli cells (MSC-1), and mouse hepatoma liver cells (Hepa-c1c6) were cultured in DMEM (high glucose) supplemented with 10% fetal calf serum. Human ovarian carcinoma cells (A2780) were maintained in DMEM (low glucose) supplemented with 10% fetal calf serum, and L-glutamine. Human adrenocortical carcinoma cells (H295R) were cultured in a 1:1 mixture of DMEM (high glucose):HAM F12 supplemented with 2% ITS+ (Collaborative Research, Bedford, MA) and 2% Nu-Serum (Collaborative Research). Mouse prepubertal Sertoli cells (SMAT-1) were grown in DMEM (high glucose), 10% fetal calf serum, and amino acids. Human embryonic kidney cells (HEK-293 EBNA) were cultured in DMEM (high glucose), L-glutamine, 10% fetal calf serum, and 25 mM HEPES. Mouse embryonic fibroblast cells (NIH-3T3) were maintained in DMEM (high glucose), 10% fetal calf serum, and 25 mM HEPES. Mouse Leydig tumor cells (MA-10) were cultured in a 1:1 mixture of DMEM (high glucose):HAM F12, 15% horse serum, 20 mm HEPES, and 40 µg/ml gentamicin. All cells were maintained in 5% CO2 humidified atmosphere at 37 C, and growth medium also contained penicillin (100 U/ml) and streptomycin (100 µg/ml) (except for MA-10 cells).

Transfection of cells and luciferase assay
Cells were plated in 12-well plates and transiently transfected the following day using Superfect (QIAGEN, Valencia, Sweden). Cells were transfected with reporter plasmid (Sf1(–185/+141)/Luc, 300 ng/well) and a plasmid encoding β-galactosidase (pCMV5/LacZ, 100 ng/well) to control for transfection efficiency. Twenty-four hours later, the cells were lysed in luciferase buffer [10 mM Tris-HCl (pH 8), 4 mM EDTA, 150 mM NaCl, 0.65% Nonidet P-40], and total cell extracts were analyzed for luciferase and β-gal activity on a LUCY-3 luminometer (Anthos, Salzburg, Austria) using the luciferase assay kit from BIO Thema AB (Dalarö, Sweden).

Immunoblotting
Total cell extracts were separated by SDS-PAGE and blotted to nitrocellulose membranes. The membranes were incubated with 6% milk/primary antibodies/secondary antibodies, each incubation step performed for 1 h at room temperature. Antibody dilutions were 1:1000 for the anti-SF1 antibody [06-431; Upstate Biotechnology, Lake Placid, NY; 28740 (H-60) from Santa Cruz Biotechnology (Santa Cruz, CA) for verification of SF1 in HeLa cells], whereas anti-β-actin (ab-6276; Abcam, Cambridge, UK) and secondary horseradish peroxidase (HRP)-conjugated antibodies were used at 1:10000 [antimouse conjugated to HRP was from Santa Cruz Biotechnology; antirabbit HRP conjugated antibody was from Pierce (Rockford, IL)]. Chemiluminescence signals were developed by using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology).

Genomic DNA preparation
Genomic DNA from cell lines, paraffin sections, and human or mouse tissue was isolated using the QIAGEN DNeasy kit or with a standard protocol using proteinase K. Mouse embryonic tissues were pooled before DNA preparation. Mice used in this study were of C57/BL6 background. Ethical approval, collection, and staging of human fetal material were carried out as described previously, using the Carnegie classification and fetal foot length to provide a direct assessment of gestational age as weeks after conception (28).

DNA methylation analysis by HpaII/MspI digestion
Genomic DNA (250 ng) from human and murine cell lines were digested with HpaII (methylation sensitive restriction enzyme, recognizing 5'-CCGG-3') or its isoschizomer MspI (methylation insensitive). All samples were codigested with NcoI (digests outside SF1 and control PCR targets) to aid digestion and PCR. Digested samples were subjected to PCR amplification using appropriate primers (see Table 1Go) and resolved on a 2 or 3% agarose gel. Selected regions with no HpaII/MspI restriction sites of human IGF-II exon 9 or mouse β-globin genes were used as internal controls in the PCRs.


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  		TABLE 1. Overview of the PCR primers used in this study

 
Bisulfite sequencing
Genomic DNA (100–500 ng) was subjected to sodium bisulfite modification with the EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer‘s instructions. PCR amplification was carried out using primers specific for bisulfite-converted DNA sequence of the selected gene area to target either mouse or human origin (see Table 1Go). The amplified PCR product was subcloned into the pGEM-T-easy vector (Promega, Madison, WI) and subsequently sequenced and analyzed. Only PCR clones with at least 95% C-to-T conversion outside CpG sites were analyzed further.

Laser dissection from paraffin embedded tissues
Laser dissections were performed with a system comprised by an inverted microscope (Axiovert 200; Zeiss, New York, NY) and an air-cooled nitrogen laser (model VSL-337 ND-S) from P.A.L.M. Microlaser Technologies GmbH (Munich, Germany). Microdissection was performed on 7-µm-thick paraffin sections. Paraffin embedding and sectioning were performed according to standard protocols.

Chromatin immunoprecipitation (ChIP) assay
Cells were grown to 95% confluence on a 500-cm2 plate. The cells were rinsed with PBS and fixed in 1% formaldehyde at room temperature for 10 min, rinsed again in PBS, and collected into 100 mM Tris-HCl (pH 8.7), 10 mM dithiothreitol by scraping. Cells were collected by centrifugation and sequentially washed in ice-cold PBS, buffer I [0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5)] and buffer II [200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5)]. Nuclei were resuspended in lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 1x protease inhibitor cocktail (Roche, Indianapolis, IN)] and sonicated to an average length of 400–800 bp. The lysates were centrifuged at 14,000 rpm for 10 min and the supernatant diluted 1:10 in dilution buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1), 1x protease inhibitor cocktail (Roche)]. One milliliter of the chromatin solution was precleared with the addition of recombinant protein G agarose (Invitrogen, Carlsbad, CA), sheared herring sperm DNA (2 µg), and normal rabbit IgG (sc-2027; Santa Cruz). Precleared chromatin was then incubated with 5 µg of specific antibody or normal rabbit IgG and rotated at 4 C overnight. Polyclonal antibodies for the ChIP experiment were against USF2 (N-18; Santa Cruz), methyl-CpG-binding domain protein 2 (MeCP2; ab2828; Abcam), Dnmt3a (H-295; Santa Cruz), and RNA polymerase II (ab5131, Abcam). The beads were harvested by centrifugation at 3000 rpm for 15 sec and washed sequentially for 20 min with the following buffers: low-salt buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1)], high-salt buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl (pH 8.1)] and buffer III [1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 0.25 M LiCl, 10 mM Tris-HCl (pH 8.1)] and four times with Tris/EDTA buffer. Immunoprecipitates were eluted three times with 100 µl 1% SDS and 0.1 M NaHCO3, and the eluates were pooled. After dilution, 100 µl of the supernatant were saved as total input of chromatin and was processed with the eluted immunoprecipitates beginning at the cross-link reversal step. Reverse cross-linking of samples was performed by incubation at 65 C overnight. After 1 h proteinase K digestion, samples were purified using QIAquick PCR purification kit (QIAGEN), resuspended in 40 µl of elution buffer [10 mM Tris-Cl (pH 8.5)]. Subsequently quantitative PCRs were performed with 1 µl of immunoprecipitate and serial dilution of input material by using the iQ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) with primers spanning the SF1 proximal promoter area (–128/+70) or an upstream region (–8500/–8305; see Table 1Go for primers). The quantitative PCR was performed on an iQ5 real-time PCR detection system (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The proximal promoter contains CpG sites
The sequences analyzed in this study correspond to the regions spanning –129/+122 of the human SF1 gene and –123/+125 of the mouse Sf1 gene containing the proximal promoter region and part of exon 1 (Fig. 1Go, A and B; for simplicity, these regions will hereafter be referred to as the proximal promoter). The proximal promoter is conserved among species, contains binding sites for several transcription factors, and can promote reporter gene activity in vitro (12, 13). Sequence analyses of the selected region revealed 14 putative CpG sites in the human sequence and 10 in the corresponding mouse sequence. Many of the sites were conserved between the two species including one that coincided with the E-box motif (Fig. 1Go, A and B). Notably, according to the cpgplot program (www.ebi.ac.uk/emboss/cpgplot), the mouse and human proximal promoter does not reside within CpG islands. [A CpG island is frequently defined as a region of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 (29)].


Figure 1
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  		FIG. 1. Overview of the CpG sites in the proximal promoter. A, Overview of CpG sites (indicated as lollipops) in the proximal promoters of the human (top) and mouse (bottom) SF1/Sf1 genes. The regions shown correspond to the region spanning –129/+122 in the human gene and the region spanning –123/+125 in the mouse gene. The positions of DNA regulatory elements present in the proximal promoters are indicated (12 13 14 15 16 19 20 ). A putative INR element (46 ) is present at the start site of transcription (+1) (47 48 ). B, Alignment of the human and mouse SF1/Sf1 genes over the region selected for bisulfite sequencing. The CpG sites are indicated as circles, and the numbering corresponds to that in A. As also shown in A, the positions of functional DNA-regulatory elements are indicated.

 
The proximal promoter is inactive when methylated
To investigate whether the activity of the proximal promoter is dependent on methylation status, we constructed a reporter gene plasmid in which the fragment spanning –185/+141 of the mouse Sf1 gene was inserted upstream of luciferase [Sf1(–185/+141)/Luc]. The plasmid was propagated in the Escherichia coli strain DH5{alpha} (which does not contain CpG methylases) and subjected to in vitro methylation using the SssI CpG methylase. To verify that the original reporter plasmid was indeed unmethylated and that in vitro methylation had occurred, the plasmids were digested with HpaII, which is sensitive to methylation, and with its isoschizomer MspI, which is methylation insensitive (see Fig. 2AGo for HpaII/MspI sites). As demonstrated in Fig. 2BGo, HpaII readily digested unmethylated Sf1 (–185/+141)/Luc (Fig. 2BGo, left panel), whereas it did not digest the methylated plasmid (Fig. 2BGo, right panel). As expected, MspI acted similarly on both methylated and unmethylated plasmid (Fig. 2BGo). To correlate methylation status with reporter gene activity, unmethylated and in vitro methylated Sf1 (–185/+141)/Luc were transfected into the steroidogenic cell lines H295R (human adrenocortical tumor cell line) and Y1 (mouse adrenocortical tumor cell line). As expected, based on the fact that these cell lines express high levels of SF1, Sf1 (–185/+141)/Luc gave rise to relatively high luciferase activity when untreated with SssI CpG methylase (Fig. 2CGo). In vitro methylation resulted in a 95.4 and 99.5% repression of luciferase activity in H295R and Y1 cells, respectively (Fig. 2CGo). These experiments thus suggest that the proximal promoter of the Sf1 gene is inactive when methylated.


Figure 2
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  		FIG. 2. In vitro methylation inhibits transcription from the proximal promoter. A and C, Overview of the positions of the primers used for the HpaII/MspI digestion-based methylation analyses (arrows) and the bisulfite sequencing (arrowheads). The HpaII sites are indicated as diamonds (six sites in the human promoter, four sites in the mouse promoter). Open symbols (top of gene diagram) indicate positions of primers and HpaII sites in the human gene, and closed symbols (below gene diagram) denote HpaII sites and primers for the mouse gene. The transcriptional start site is indicated as +1 (bent arrow). B, Sf1 (–185/+141)/Luc was left untreated or methylated in vitro by the methyltransferase SssI and the methylation status confirmed by HpaII/MspI digestion. IVM, In vitro methylated; L, 1-kb DNA ladder; U, undigested Sf1 (–185/+141)/Luc; MspI, MspI-digested Sf1 (–185/+141)/Luc; HpaII, HpaII-digested Sf1 (–185/+141)/Luc). C, H295R and Y1 cells were transfected with Sf1 (–185/+141)/Luc (300 ng) or the same plasmid that had been methylated in vitro (IVM) (300 ng). Luciferase activity was determined 24 h after transfection and normalized against β-gal activity from LacZ/pCMV (100 ng) that was included in all transfection experiments (n = 6). The experiment was repeated with different batches of in vitro methylated Sf1 (–185/+141)/Luc, all giving the same result.

 
The proximal promoter is hypomethylated in cells and tissues that express SF1
To determine whether the methylation status of the proximal promoter corresponded to the expression profiles of SF1 in various cell lines of mouse and human origin, genomic DNA was isolated and digested with HpaII/MseI followed by PCR (see Fig. 2AGo for HpaII/MspI sites and positions of PCR primers). The presence of a band on gel electrophoresis after PCR indicates that HpaII is unable to digest the template DNA and hence that it is methylated. As demonstrated in Fig. 3AGo, the proximal promoter was unmethylated (i.e. no PCR product was generated after HpaII digestion) in human (Fig. 3AGo, left panel) and murine (Fig. 3AGo, right panel) cells of steroidogenic origin (i.e. adrenocortical H295R and Y1 cells and the mouse Leydig cell line, MA10). Furthermore, it was unmethylated in the murine Sertoli-derived cell lines SMAT-1 and MSC-1 (Fig. 3AGo, right panel). In contrast, PCR on HpaII-treated DNA isolated from HEK-293 cells, HeLa cells (human cervix epithelial carcinoma), A2780 cells (human epithelial ovarian carcinoma), NIH-3T3 cells (mouse embryonic fibroblasts), and Hepa cells (mouse hepatoma cells) generated PCR products indistinguishable from undigested DNA. Thus, the proximal promoter was methylated in these cell lines (Fig. 3AGo, left and right panels). By immunoblotting, SF1 was detected in cells with hypomethylated proximal promoter (Fig. 3BGo). In contrast, a hypermethylated proximal promoter correlated with lack of SF1 expression with one exception: HeLa cells expressed low levels of SF1 (Fig. 3BGo, left panel). The presence of SF1 in HeLa cells was confirmed by an antibody raised against a different epitope (data not shown). The band corresponding to SF1 migrated at a slightly higher molecular weight than observed for H295R cells. The reason for this is unknown. Searching the literature, we have not found other reports describing the expression of SF1 in HeLa cells. However, SF1 is expressed in the uterus (30) that, like the cervix, originates from the Müllerian ducts.


Figure 3
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  		FIG. 3. DNA methylation analyses of the proximal promoter by HpaII digestion. A, Genomic DNA from cell lines of human (left panel) and murine (right panel) origin was submitted to HpaII/MspI digestion followed by PCR. (Please see Materials and Methods for origin of cell lines.) Selected regions of the human IGF-II and mouse β-globin genes (containing no HpaII sites) were used as controls. U, Undigested DNA; MspI, MspI digested DNA; HpaII, HpaII digested DNA; –, no DNA. Representative data from two or three independent sample preparations for each cell line are shown. PCR analyses were performed three times for each sample, and full reproducibility was observed. B, Total cell lysates was prepared from the human (left panel) and mouse (right panel) cell lines and subjected to Western blot analyses using an anti-SF1 antibody. Equal protein loading was verified using an anti-β-actin antibody. The experiments were performed three times with similar results and representative blots are shown. *, Unspecific band.

 
To investigate the precise methylation status of each individual putative CpG site, we performed bisulfite sequencing on genomic DNA isolated from the different cell lines. In concordance with the results in Fig. 3Go, practically all potential sites in the human (Fig. 4AGo) and mouse (Fig. 4BGo) proximal promoter were unmethylated in cell lines that were sensitive to HpaII digestion and that expressed SF1 (i.e. H295R, Y1, MA-10, SMAT-1 and MSC-1 cells). Similarly, the proximal promoter was hypermethylated in cells that did not express SF1 (i.e. HEK-293, A2870, NIH-3T3, and HeLa cells). Despite the apparent presence of SF1 protein in Hela cells, the proximal promoter was heavily methylated (Fig. 4AGo), in agreement with the HpaII digestion analysis in Fig. 3AGo. However, interestingly, all clones examined from this cell line were demethylated at the CpG site at position 2 (Fig. 4AGo), located within the E-box of the human proximal promoter (Fig. 1BGo).


Figure 4
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  		FIG. 4. Site-specific CpG methylation analyses of the proximal promoter in cell lines. Bisulfite sequencing was performed on DNA isolated from the human (A) and murine (B) cell lines used in Fig. 3Go. The analysis covered 14 CpG sites (in the region spanning –129/+122) in the human gene and 10 CpG sites (in the region spanning –123/+125) in the mouse gene. Filled circles indicate methylated CpG sites, and open circles represent unmethylated CpG-sites. Horizontal lines represent individual analyzed clones. The CpG sites are numbered corresponding to the overview in Fig. 1Go.

 
Cell lines, especially those of tumorigenic origin, can exhibit aberrant methylation patterns that are unrepresentative of the normal tissues from which they originate. Therefore, we examined the methylation status of the proximal promoter in adult mouse tissues. The adrenal gland consists of a medulla and cortex, with only the cortical cells expressing SF1. Thus, genomic DNA isolated from the whole gland yielded clones that were either completely unmethylated or completely methylated (Fig. 5AGo). Laser capture microdissection separated the cortex and medulla and established that genomic DNA isolated from the cortex was responsible for the unmethylated clones, whereas DNA from the medulla was completely methylated (Fig. 5AGo). The capsule and subcapsular areas were avoided in the laser capture microdissection. Thus, capsular cells that are devoid of SF1 (31) and cells in the subcapsular area that have been suggested to lack SF1 protein (7) were not included in our analyses. In the adult mouse ovary, SF1 is expressed in both granulosa and theca cells as well as the corpus luteum (32). In agreement with this expression pattern, we found 11 of 13 clones generated from the ovary to be almost completely demethylated across the proximal promoter (Fig. 5BGo, left panel). In the mouse adult testis, SF1 expression is confined to Leydig and Sertoli cells and is not expressed in germ cells or peritubular myoid cells (31). In the adult rodent testis, Leydig and Sertoli cells constitute approximately 10 and 8% of the total cell number, respectively, whereas spermatogonia, spermatocytes, and spermatids account for approximately 70% (33). When bisulfite sequencing was performed on adult mouse testis, it became evident that approximately 24% of the CpG sites were methylated (Fig. 5BGo, middle panel), which would suggest that the proximal promoter is demethylated in a greater number of cells than could be accounted for by the Leydig and Sertoli cell lineages. (In total 56 PCR clones, generated from four testes, were analyzed; mean 24 ± 8%; one representative experiment is shown in Fig. 5BGo, middle panel.) Probably, demethylated promoter is present in cells of the germ cell lineage because primordial germ cells become demethylated around E10.5 and E13.5 and is not completely remethylated until the pachytene stage (34). As expected, nearly complete methylation was apparent in mature sperm (Fig. 5BGo, right panel). The proximal promoter was hypermethylated in genomic clones derived from the tail and liver (Fig. 5CGo). Taken together, these results indicate that the methylation status of the proximal promoter strongly correlates with the expression of the protein in both adult mouse tissues and cell lines.


Figure 5
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  		FIG. 5. Site-specific CpG methylation analyses of the proximal promoter in adult mouse tissue. A, B, and C, The analysis covered 10 CpG sites (in the region spanning –123/+125) in the mouse Sf1 gene. CpG sites are numbered corresponding to overview in Fig. 1Go. Filled circles indicate methylated CpG-sites, and open circles represent unmethylated CpG sites. A, Paraffin sections of mouse adrenal glands were subjected to laser capture microdissection to isolate cortical and medullary cells. Bisulfite sequencing was performed on whole sections (total adrenal; left panel) or isolated cortical cells (cortex; middle panel) and medullary cells (medulla; right panel). B, Bisulfite sequencing on DNA isolated from mouse ovary (left panel), testis (middle panel), and mature sperm (right panel). C, Bisulfite sequencing on DNA isolated from tail (left panel) and liver (right panel). Horizontal lines represent individual analyzed clones. CpG sites are numbered corresponding to the overview in Fig. 1Go.

 
Methylation of the proximal promoter during development
As sexual differentiation occurs, SF1 expression is maintained in the developing testis but declines to undetectable levels in the ovary (9). To determine whether this differential expression is reflected in the methylation status, we performed bisulfite sequencing on DNA isolated from murine gonads at E13.5 and 16.5 (Fig. 6AGo). The proximal promoter was virtually completely demethylated in both sexes at both stages (Fig. 6AGo). The lack of methylated clones in the developing testis is in agreement with the fact that SF1-expressing cells constitute the majority of the testicular cells and that the germ cells contain mainly demethylated DNA at these developmental stages (34, 35). Because SF1 expression is undetectable in the ovary at these time points, our results would indicate that the Sf1 gene is demethylated in the proximal promoter early in development and that it remains in this state in cells programmed to express SF1 at later stages. As expected, the proximal promoter was heavily methylated in the developing liver (Fig. 6BGo). A similar pattern was observed in the kidney at E13.5. However, at E16.5, a number of putative CpG sites were demethylated in the kidney, particularly in the more distal part of the proximal promoter region (Fig. 6BGo). Neither the developing nor adult kidney expresses SF1 (Refs. 30 , 36 and Hanley, N., unpublished data). The observed level of demethylation is therefore unlikely to allow SF1 expression in this tissue.


Figure 6
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  		FIG. 6. Site-specific CpG methylation analyses of the proximal promoter in embryonic tissue. Bisulfite sequencing was performed on DNA isolated from mouse testes, ovaries, kidney, and liver at E13.5 and E16.5 as indicated. The analysis covered 10 CpG sites (in the region spanning –123/+125) of the Sf1 gene. Filled circles indicate methylated CpG sites, and open circles represent unmethylated CpG sites. Horizontal lines represent individual analyzed clones. CpG sites are numbered corresponding to the overview in Fig. 1Go.

 
During human development, as in the mouse, SF1 is expressed in the bipotential gonad and throughout testicular development (10). At 9 wk after conception, when sex cords have formed, the proximal promoter was essentially demethylated in the developing testis (Fig. 7Go). Interestingly, similarly to the mouse, DNA from human kidney also revealed relative low levels of methylation. SF1 is not expressed in the human kidney (Hanley, N., unpublished data), and at present it is not clear why the proximal promoter is partially demethylated in this organ during development.


Figure 7
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  		FIG. 7. Site-specific CpG methylation analyses of the proximal promoter in fetal human tissue. Bisulfite sequencing was performed on DNA isolated from the testis and kidney from a human embryo (8 mm foot length, around 9 wk of development). Filled circles indicate methylated CpG sites, and open circles represent unmethylated CpG sites. Horizontal lines represent individual analyzed clones. CpG sites are numbered corresponding to the overview in Fig. 1Go.

 
The unmethylated proximal promoter recruits transcriptional activators.
ChIP experiments were performed to determine whether the methylation status affected the recruitment of transcription factors to the proximal promoter. Occupation of three factors was analyzed: USF2, which has previously been demonstrated to interact with the unmethylated active proximal promoter (23); MeCP2, which interacts with methylated DNA (37); and RNA polymerase II (polII). As evident from the results presented in Fig. 8Go, the transcriptional activator USF2 was specifically located at the proximal promoter in Y1 cells, whereas no interaction above background levels were detected in Hepa cells. Moreover, antibodies against polII also specifically precipitated the proximal promoter from Y1 cells (Fig. 8Go), further supporting that the unmethylated status correlates with a transcriptionally active promoter. As expected, MeCP2 interacted only with Sf1 in Hepa cells (Fig. 8Go, lower panel). We found that MeCP2 was recruited to a region upstream of the proximal promoter. This is partly in conflict with the study by Xue et al. (38), who suggested that MeCP2 interacts with the proximal promoter in SF1-negative endometrial stroma cells. The recruitment of MeCP2 might differ between nonexpressing cells, but we note that Xue at al. did not use upstream or downstream primer pairs, hindering a direct comparison of the two studies.


Figure 8
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  		FIG. 8. Differential recruitment of transcriptional regulators to the proximal promoter, depending on methylation status. ChIP experiments were performed on Y1 and Hepa cell extracts using antibodies against USF2, MeCP2, and RNA polII as indicated. The quantitative PCR values were related to preimmune IgG control values and are presented as relative fold enrichment in relation to 0.1% input material. Statistical analyses were t tests with pooled variances. a, P = 0.015; b, P = 0.005; c, P = 0.012 (Y1: n = 9; Hepa: n = 6).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies have established that DNA methylation plays regulatory roles in developmental gene expression (39, 40, 41, 42, 43). We demonstrate a striking correlation between SF1 protein expression and methylation status of the proximal promoter, indicating that DNA methylation is a major mechanism to regulate cell and tissue-specific expression of SF1. Whereas our work was ongoing, Xue et al. (38) published a study that links methylation and SF1 protein expression in endometrial and endometriotic cells. In normal endometrial cells that do not express SF1, the proximal promoter was found to be hypermethylated, whereas DNA from endometriotic cells, which do express SF1, was hypomethylated over the same region (38). This study also revealed that MeCP2, which contributes to the repressed state of methylated DNA, is recruited to the proximal promoter in endometrial cells (38). Thus, together, these studies demonstrate that methylation of the proximal promoter directs SF1 expression in both normal tissues and diseased tissue exhibiting aberrant levels of SF1 protein.

We observed low, but consistent, levels of SF1 in HeLa cells (Fig. 3BGo). Interestingly, all PCR-generated clones from HeLa cells were unmethylated at the CpG site located within the E-box (Fig. 4AGo). Because this DNA regulatory element has repeatedly been identified as crucial for SF1 expression, we speculate that the specific lack of methylation allows binding of a factor that can direct low levels of SF1 expression in HeLa cells, regardless of the overall hypermethylation of the proximal promoter. Of specific interest in this regard is a recent study suggesting that abnormal expression of USF2 and its interaction with the E-box partly accounts for the aberrant expression of SF1 in endometriosis (23). The demethylation/remethylation processes that occur in germ cells complicated the establishment of a correlation between DNA methylation and SF1 expression in the testis. DNA methylation patterns are generally erased during gametogenesis, and complete remethylation in the male is not evident until the pachytene stage of meiosis (34). In line with this, our analyses of embryonic testis demonstrated complete demethylation of nearly all clones examined (Fig. 6Go), and furthermore, a far greater number of clones than would be accounted for by the Leydig and Sertoli lineages were demethylated in adult testes (Fig. 5BGo). To determine the methylation status of the proximal promoter in SF1 expressing vs. nonexpressing cells in the ovary and testis, we performed bisulfite sequencing on cells sorted from mice expressing enhanced green fluorescent protein under the control of 50 kb of the 5' Sf1 promoter region (44). However, these analyses demonstrated that the methylation pattern of the transgenic construct differs from that of the Sf1 gene, and the presence of multiple copies of the transgenic construct in the SF1/enhanced green fluorescent protein mice impeded these analyses (Hoivik, E. A., M. Bakke, and K. L. Parker, unpublished results).

Although we did not analyze the methylation status at the earliest time point of SF1 expression in the adrenogonadal primordium (at E9.0 in mice), we hypothesize that the methylation pattern of the proximal promoter is established at this stage and that it is maintained thereafter. Thus, as depicted in Fig. 9Go, the proximal promoter will be demethylated in cells that are programed to express SF1 continuously or in an interrupted manner and methylated in other cells. In agreement with this idea, the down-regulation of SF1 levels that occurs in the ovary at the time of sexual differentiation is not associated with a change in methylation status (Fig. 6Go). Instead, the change in SF1 expression during ovarian development is more likely to be caused by the action of transcription factors (although we cannot exclude epigenetic modifications elsewhere in the gene). Genetic studies have identified several transcription factors that appear to affect SF1 expression in the developing mouse embryo. SF1 expression is reduced to a minimum in mice carrying a targeted deletion of Lhx9 (17). Similarly, the knockout models for WT-1 (16) and the Pbx1 (45) exhibit undetectable levels of SF1, clearly indicating that these factors act upstream of SF1. Moreover, Pod1 most likely acts as a repressor of SF1 expression because loss of this factor leads to enhanced SF1 expression in the developing testis, followed by ectopic expression of SF1 and aberrant commitment of precursor cells to the steroidogenic lineage (25). Thus, a number of transcription factors, in addition to tissue-specific promoters (26, 27), and epigenetic regulatory mechanisms seemingly work side by side to control the time and cell-specific expression of SF1. Interestingly, in silico analyses suggest the presence of CpG islands within the human and mouse SF1 genes. Typically, CpG islands are associated with regions that are involved in transcriptional regulation (1), and future experiments should answer whether these regions confer epigenetic regulation. According to our preliminary analyses, the previously identified VMH-specific intronic enhancer (26, 27) resides within a CpG island.


Figure 9
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  		FIG. 9. Proposed mechanism for tissue-specific expression of SF1 by DNA methylation. In SF1-negative tissues, DNA methylation of the proximal promoter prevents expression of SF1. In contrast, the proximal promoter is hypomethylated in SF1-expressing tissues. We hypothesize that the methylation pattern is established at the earliest time point of SF1 expression (E9 in the mouse) and maintained thereafter in a clonal manner. Both cell lineages that express SF1 continuously and those that express SF1 periodically maintain a demethylated pattern. Various transcription factors presumably interact with the hypomethylated promoter, controlling the level of SF1 expression. Unmethylated CpG sites are indicated by open lollipops, whereas methylated CpG sites are shown as filled lollipops. Key time points are indicated. Expression profiles are based on a published report (49 ).

 
In conclusion, our results clearly indicate that epigenetic mechanisms control the expression of SF1 during embryonic development, in adult tissues, and in cell lines. In this study we have not investigated the mechanisms that direct the cell-specific methylation pattern of the proximal promoter. But because SF1 is a transcription factor associated with embryonal lineage commitment, the SF1/Sf1 gene provides an interesting candidate for future studies that aim to reveal the mechanisms whereby CpG methylation contributes to cell type-specific gene expression during development.


   Acknowledgments
 
We thank Torild Ellingsen for excellent technical assistance.


   Footnotes
 
This work was supported by the National Program for Research in Functional Genomics in Norway in the Research Council of Norway, the Norwegian Cancer Society, and Helse Vest.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: A2780, Human ovarian carcinoma cells; ChIP, chromatin immunoprecipitation; Dnmt, DNA methyl transferase; E, embryonic day; HeLa, human cervix epitheloid carcinoma; Hepa, hepatoma liver cells; H295R, human adrenocortical carcinoma cells; HEK-293, human embryonic kidney cells; HRP, horseradish peroxidase; Luc, luciferase; MA-10, mouse Leydig tumor cells; MeCP2, methyl-CpG-binding domain protein 2; MSC-1, mouse Sertoli cells; NIH-3T3, mouse embryonic fibroblast cells; polII, RNA polymerase II; SDS, sodium dodecyl sulfate; SF1, steroidogenic factor 1; SMAT-1, mouse prepubertal Sertoli cells; USF, upstream stimulatory factor; VMH, ventromedial hypothalamic nucleus.

Received January 23, 2008.

Accepted for publication July 11, 2008.


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