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Molecular Medicine Program (M.A.L., D.J.P.) and Centre for Cancer Therapeutics (D.P., B.C.V.), Ottawa Health Research Institute, Ottawa, Ontario, Canada K1H 8L6; Facultad de Medicina Veterinaria y Zootecnia (N.P.), Universidad Autónoma del Estado de México, Toluca, México D.F., Mexico; Centre de Recherche en Reproduction Animale (B.D.M.), Faculté de Médecine Vétérinaire, Université de Montréal, St.-Hyacinthe, Quebec, Canada J2S 7C6; Departments of Cellular and Molecular Medicine, Obstetrics and Gynecology (D.P., B.C.V.), and Medicine, and Biochemistry, Microbiology, and Immunology (D.J.P.), University of Ottawa, Ontario, Canada K1H 8M5
Address all correspondence and requests for reprints to: Dr. David J. Picketts, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. E-mail: dpicketts{at}ohri.ca.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Despite advances in the identification of genes involved in the luteinization process, the precise mechanisms underlying their regulation remain poorly understood. Furthermore, our understanding of epigenetic regulation of these genes during ovarian cell differentiation is confined to a few investigations of the modification of histone tails by phosphorylation and acetylation (14) and consequent association with the StAR promoter (15, 16, 17, 18).
Conformational and posttranslational changes of chromatin are important mediators of differentiation because they promote the changes in expression (both activation and repression) of genes that characterize the differentiated phenotype. In the case of luteinization, the extensive tissue remodeling involves renewed expression of some genes, particularly those associated with steroidogenesis, and silencing of others, specifically, those related to the cell cycle (19). In other cell models, it has been shown that the mobilization of nucleosomes is catalyzed by the superfamily of ATP-dependent chromatin remodeling complexes, multiprotein machines that use the energy from ATP hydrolysis to mobilize nucleosomes to bring about regulation of specific genes (20). These complexes are diverse, both in composition and in function, with the common feature being the presence of a SNF2 (sucrose nonfermenting 2 gene) domain within one subunit (21). The SNF2 domains fall within three categories, the SWI2/SNF2 family, the imitation switch (ISWI) family, and the Mi-2 family, the latter distinguished by additional chromatin motifs (20).
The ISWI protein was originally identified in Drosophila and was shown to participate in three distinct complexes, ATP-utilizing chromatin assembly and remodeling factor (ACF), chromatin-accessibility complex (CHRAC), and nucleosome remodeling factor (NURF) (22, 23, 24, 25). Both ACF and CHRAC function to assemble and spatially distribute nucleosomes, whereas NURF was shown to be involved in the specific regulation of target genes (reviewed in Ref. 26). There are two mammalian ISWI homologs, SNF2H and SNF2L (27, 28, 29). SNF2H was found to be prominent in the mammalian equivalents of the ACF and CHRAC complexes and is believed to play a role in nucleosome assembly (30, 31). In contrast, the SNF2L protein is a component of a mammalian NURF complex that is prevalent in the brain where it promotes the in vitro terminal differentiation of neurons (32).
Our recent investigation of the murine orthologs, SNF2H2 and SNF2L, demonstrated expression in a number of tissues in a pattern suggestive of a role for SNF2H in proliferating cell populations and SNF2L in the regulation or maintenance of a differentiated phenotype (28). Both genes were highly expressed in the adult mouse ovary and transcripts for both were abundant in the granulosa cells of preovulatory follicles. The SNF2L signal increased markedly in the developing CL at a time when SNF2H was reduced. Given the remarkable distribution of these gene products in ovarian tissue, we were interested in their relationship to the processes of ovulation and its sequel, the formation of the CL. Here, we demonstrate that human chorionic gonadotropin (hCG; an LH analog) induction of ovulatory changes provokes SNF2L expression and that the SNF2L protein physically interacts with the PR and with the StAR promoter, suggesting that chromatin remodeling initiated by the mammalian ISWI proteins contributes to both ovulation and luteinization.
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RESULTS |
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). Although expression of SNF2H appeared to be ubiquitous at postnatal d 12, by d 14 and 16, the localization of SNF2H mRNA became more pronounced and restricted to the highly proliferative granulosa cells of developing follicles (Fig. 1A). The intensity of the signal was greater in the smaller, rapidly proliferating, preantral follicles than in the larger antral follicles indicating that antral follicles, although expressing SNF2H, have relatively fewer proliferating granulosa cells. However, given that in situ hybridization is not easily quantifiable, confirmation of such an observation would require other methods. Sense probes for both SNF2H and SNF2L were used as controls and showed very little background hybridization (Fig. 1A and data not shown). This suggests that SNF2H, but not SNF2L expression may be required in the response of granulosa cells to signals that promote follicle cell proliferation and follicle growth.
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). In ovaries from mice treated with pregnant mare serum gonadotropin (PMSG) for 48 h to induce synchronous follicular growth to the antral stage, there was little evidence of SNF2L transcripts in the granulosa cells of preovulatory follicles (Fig. 1B). Nonetheless, treatment with hCG to induce ovulation, resulted in a dramatic increase in SNF2L RNA by 8 h after administration, such that all preovulatory follicles displayed high levels of the signal (Fig. 1B). Examination of ovaries taken at 8 h after hCG by Northern blot confirmed the increased expression of the SNF2L transcript (Fig. 2A). The quantitative increase of approximately 1.5-fold (normalized to actin) was less robust than the apparent increase in preovulatory follicles (Fig. 1B), due to the use of whole ovaries in the Northern analysis. By 18 h after hCG treatment, which corresponds to approximately 6 h after ovulation, expression of SNF2L remained high in the differentiating granulosa cells of ovulated follicles and the incipient CL (Fig. 1B). The strong signal in the CL persisted through at least 24 and 36 h after hCG treatment (Fig. 1B).
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); however, Northern blots indicated that there was a reduction of approximately 1.5-fold in whole ovaries (Fig. 2A). At 6 h after ovulation (18 h after hCG), expression of SNF2H remained prominent in the developing CL, whereas it underwent a clear decline at 24 and 36 h (Fig. 1B).
Treatment with hCG Induces SNF2L Protein Levels
To determine whether the SNF2L protein was expressed in a pattern resembling its cognate mRNA in granulosa cells after hCG treatment, we generated antibodies that specifically detected SNF2L (supplemental data, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org.). Western blot analysis using this antiserum revealed that there was a 3-fold increase in the relative abundance of SNF2L protein in granulosa cells harvested from ovaries from PMSG-treated mice at 8 h after the ovulatory stimulus (Fig. 2B). We also examined the abundance of StAR and PR, markers of granulosa cell differentiation in the rodent ovary (2). Both showed concurrent increases in expression after gonadotropin treatment (Fig. 2B), indicating that the up-regulation of the SNF2L protein occurred in cells that were committed to the differentiation program.
We then investigated whether SNF2L regulation relative to PR and StAR expression was recapitulated in the process of cell differentiation in primary cultures of rat or porcine granulosa cells, or in a human ovarian granulosa cell line. In primary cultures of rat granulosa cells, addition of the cAMP analog, dibutyryl cAMP (dbcAMP) induced a rapid (<3 h) induction in StAR protein levels with a concomitant induction of SNF2L (Fig. 2C). This result is in agreement with observations of granulosa cells cultured from porcine ovaries (Fig. 2D) in which dbcAMP stimulation also caused a significant increase in SNF2L protein levels.
To assess whether cells expressing increased levels of SNF2L protein were also the cells that expressed StAR, we examined the primary rat granulosa cells for StAR and SNF2L protein expression by coimmunofluorescence. SNF2L protein was present in the nucleus in freshly isolated cells in a relatively uniform pattern with some cells containing nuclear speckles, whereas StAR protein was not detectable, as expected (Fig. 3D). Upon FSH and hCG stimulation for 24 h, SNF2L protein expression in the nucleus became more prominent, with a distinctive and intense punctate pattern in most cells, conspicuously in cells that also expressed the StAR protein (Fig. 3, A, B, and E–G). The increased prominence of SNF2L in the nucleus observed upon differentiation is similar to results obtained when primary neuronal cultures are induced to differentiate and may represent clustering of the protein at specific subnuclear sites such as target genes (Picketts, D., unpublished observations). These findings are consistent with the idea that SNF2L protein regulates StAR gene expression.
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). This increase was significant (0 vs. 24 h, one-way ANOVA; P = 0.0133) and was accompanied by accumulation of progesterone in the media (data not shown), suggesting that the cells had attained luteal potential in vitro. A slight increase in SNF2L protein was evident in the 3–24 h period after treatment with dbcAMP (Fig. 4A); however, this increase did not reach significance (Fig. 4B), most likely because of the variable basal levels of SNF2L protein we observed in this cell line. Nonetheless, the data gathered using granulosa cells derived from several species indicate that SNF2L expression is induced by gonadotropin stimulation and during granulosa cell differentiation.
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, lanes 4 and 7). No significant amount of PR-A nor PR-B was detected when the prebleed serum or the SNF2H antibody was used for immunoprecipitation (Fig. 4C, lanes 3, 5, and 6). These results indicate that, in human granulosa cells, there is an interaction between SNF2L and PR-A that occurs in both differentiated and undifferentiated granulosa cells. It remains to be determined whether this interaction promotes gene repression or activation at specific target loci in response to hormone treatment. Moreover, whether such an interaction is necessary for ovulation must await the generation of SNF2L knockout mice.
SNF2L Regulates Expression of StAR
Because our previous studies have suggested that SNF2L protein plays an important role in terminal differentiation of neurons, we next asked whether SNF2L protein regulated terminal differentiation of granulosa cells. The best characterized gene in terminally differentiated granulosa cells is that encoding StAR in which there is pronounced up-regulation as luteinization ensues. The promoter and regulatory elements of this gene have been well documented rendering it a good candidate for chromatin immunoprecipitation (ChIP) assay for involvement of chromatin remodeling directed by the SNF2L protein.
SNF2L ChIP assays were performed using primary cultures of pig granulosa cells to obtain sufficient quantities of cell extracts. Extracts were immunoprecipitated with the SNF2L antibody or with sheep IgG as a negative control. After immunoprecipitation, either a 400-bp region of the StAR proximal promoter or a 500-bp fragment within the StAR open reading frame was amplified by PCR. The results of these experiments (Fig. 5A) show that SNF2L specifically associates with the proximal promoter of the StAR gene but not at a region further downstream corresponding to a coding sequence. Moreover, binding of SNF2L to the promoter was increased 2-fold (2.24 ± 0.13) in cells after treatment with cAMP for 24 h (Fig. 5, A and B). The increased occupancy of SNF2L on the StAR promoter suggests that ISWI chromatin remodeling is a step in the activation of the StAR gene by the cAMP/protein kinase pathway.
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, compare lanes 1 and 2) that was specific, because siRNA GFP oligonucleotides had no effect on either SNF2L or StAR expression. Similar inhibition of SNF2L expression was seen in cells treated with siRNA to SNF2L followed by treatment with dbcAMP for 3 and 6 h (89.5 ± 7.8% at 3 h; 90.2 ± 8.2% at 6 h; two-way ANOVA; treatment is significant at P = 0.0018). The siRNA-induced reduction in SNF2L expression was associated with a significant suppression of StAR protein expression (Fig. 5C, compare lanes 3 and 4, and lanes 5 and 6, and see Fig. 5D; two-way ANOVA; treatment significant at P = 0.0163), whereas SNF2H expression was unaffected. Taken together, these results suggest that gonadotropin stimulation increases binding of SNF2L at the StAR promoter and that this interaction is required for proper activation of this gene, most likely for nucleosome remodeling toward a chromatin environment conducive to transcription. |
DISCUSSION |
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This study was prompted by our earlier findings that SNF2L transcripts were prominent in the CL (28). By means of a more thorough spatial and temporal expression study, we have now extended this work by demonstrating that both SNF2L protein and RNA expression increased during follicular maturation, ovulation, and development of the mouse CL. This change was rapid, occurring approximately 8 h after hormone treatment, timing that concurs with terminal differentiation (34). Moreover, preliminary microarray studies monitoring rapid changes in gene expression during porcine luteinization have similarly shown an early increase in SNF2L expression (Gadsby, J., personal communication). In contrast, SNF2H levels were highest during early stages of follicular growth. This finding provides support for the view that SNF2H and SNF2L may have diverged sufficiently to perform distinct ISWI functions (28). SNF2H has been identified as a member of several protein complexes with remodeling activity and it appears to play an important role in DNA replication through highly condensed chromatin, nucleosome assembly and spacing, and in chromatin condensation (26). Moreover, SNF2H knockout mice die during the periimplantation stage of embryogenesis due to an inability of early blastocyst-derived cells to proliferate (35). Our observations are consistent with a role for the SNF2H protein in regulating proliferation, because SNF2H transcripts were most prevalent in the granulosa cells of small preantral follicles, cells that have recently been shown to be actively proliferating upon staining with phosphohistone H3 antibodies (36).
In contrast, SNF2L maintains a distinct expression pattern from SNF2H throughout mouse development and has been suggested to be an important regulator of transcription associated with cell differentiation and/or maturation (28). Indeed, we have recently shown a role for the SNF2L-containing complex, NURF, in the regulation of engrailed genes during neuronal differentiation (32). In addition, ectopic expression of SNF2L in proliferating neuroblasts induces their differentiation (32). It may be that the ratio of SNF2H to SNF2L contributes to growth and differentiation of particular cell types, because we observed that SNF2H levels slightly increased in granulosa cells when SNF2L was reduced by siRNA knockdown (Fig. 5C).
After in vivo hCG treatment, we observed a rapid increase (within 8 h) in SNF2L expression, suggesting that it is a regulatory target of the hormone signal. It is well known that the LH receptor transduces early intracellular signals via G protein-mediated synthesis of cAMP and the subsequent activation of protein kinase A (PKA) (34). PKA modulates transcriptional activity through the phosphorylation of transcription factors (37); and histones (14). Major targets include the cAMP response element-binding protein (CREB) and the cAMP response element modulators (38). Interestingly, the SNF2L proximal promoter contains two well-conserved cAMP response element consensus sites, suggesting that cAMP-mediated SNF2L activation may be induced by the preovulatory LH surge. Indeed, examination of SNF2L expression during differentiation of granulosa cells in vitro across a number of culture systems after 24 h of hormone treatment was sufficient to observe a consistent increase in protein levels in primary cultures and a human SVOG-4o cell line. Nonetheless, there were differences in the timing and the intensity of the response, suggesting that other signaling pathways may mediate the response. Alternatively, the differences may arise endogenously from the morphologic and temporal variation known to exist in the process of luteinization among species (1).
The ChIP assay demonstrated that SNF2L interacts with the region of the StAR proximal promoter that contains conserved consensus binding sites for a number of factors previously shown to transactivate the StAR gene. Among these can be found CCAAT/enhancer-binding protein ß (C/EBPß) and steroidogenic factor-1 (SF-1), both of which have been shown to be important for StAR transcription in a number of species (39, 40, 41). In addition, other studies have demonstrated a requirement for CREB-binding protein (CBP), SP1, GATA-4, and CREB in the activation of the StAR gene (reviewed in Refs. 17 and 42). Our results suggest that chromatin remodeling also plays an important role in the regulation of the StAR gene, because, when SNF2L expression is compromised by siRNA inactivation, StAR gene expression is attenuated. What role might SNF2L have at the StAR promoter? Studies using MA-10 Leydig cells have shown that, in response to dbcAMP treatment, there is a rapid increase in SF-1 and C/EBPß binding observed at the StAR promoter (17, 18). Moreover, cAMP induces modifications of the N-terminal tail of H3, including rapid hyperacetylation at K9 and K14, dimethylation of K4 and loss of K9 dimethylation (15, 18). As depicted in Fig. 6, the LH surge may activate SNF2L through PKA, thereby inducing one or more of several sequelae. The SNF2L protein may interact with CBP or CREB to facilitate H3 modifications through mobilization of nucleosomes. The protein may also use its chromatin remodeling function to enhance binding of transcription factors (e.g. C/EBP, CREB, and SF-1) that are essential transactivators of the StAR promoter. Further studies are required to delineate the precise function of SNF2L at this and other promoters in the ovarian context.
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In summary, our study provides the first evidence of regulation of ISWI expression and ISWI-dependent regulation of target genes necessary for ovarian function. Furthermore, it relates the expression of SNF2L to the processes of terminal differentiation that represent the ultimate fate of the ovarian follicle.
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MATERIALS AND METHODS |
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Granulosa Cell Cultures
Immature female Sprague Dawley rats were injected sc for 3 d, from 19–21 d of age, with 1 mg/d diethylstylbesterol (Sigma, St. Louis, MO) to stimulate follicle growth. Granulosa cells were harvested from dissected ovaries by follicle puncture using a 25-gauge needle and washed twice in DMEM/F12 culture medium containing antibiotics. Cells were plated at a density of 8 x 105 cells/ml on 60-mm plates in DMEM/F12 containing 2% fetal bovine serum and antibiotics. Cells were incubated in a humidified 95% air/5% CO2 incubator at 37 C. Once cells adhered to plates (
3–4 h), FSH (275 mIU/ml; Sigma) was added for 24–48 h, followed by treatment with 1 mM dbcAMP (Roche, Basel, Switzerland) or hCG (10 IU/ml; Sigma) for selected times.
The cell line SVOG-4o, derived from human ovarian granulosa cells immortalized with simian virus 40 early genes, was the generous gift of Dr. N. Auersperg (University of British Columbia, Vancouver, British Columbia, Canada) and was cultured in MCDB105:199 medium with 10% fetal calf serum, 2 mM glutamate, and 400 µg/ml hydroxycortisone and penicillin/streptomycin. SVOG-4o cells were induced to differentiate in the presence of 1 mM cAMP (Roche). For siRNA experiments, cells were grown to confluence, and then transfected with SNF2L-specific siRNA oligonucleotide as described previously (32) or a GFP siRNA oligonucleotide (Dharmacon, Lafayette, CO; p-002102-01-20) using Oligofectamine (Invitrogen, Carlsbad, CA). After transfection, cells were cultured in medium alone for 4 h, and then supplemented with 10% fetal calf serum for 48 h before treatment with 1 mM cAMP for 0, 3, 6, or 24 h. Each experiment was performed in triplicate.
Porcine granulosa cells were aspirated from medium-sized (3–5 mm) follicles from prepubertal pig ovaries and cultured as previously described (47). Cells were pooled (6–8 x 106 cells/ml) in MEM (Invitrogen) containing 1 mg/liter insulin (Sigma), 0.1 mM nonessential amino acids (Invitrogen), 5 x 104 IU/liter penicillin (Invitrogen), 50 mg/liter streptomycin (Invitrogen), 0.5 mg/liter fungizone (Invitrogen), and 10% (vol/vol) fetal bovine serum (Invitrogen). Incubations were carried at 37 C in 95% humidified air with 5% CO2. At initiation of culture, some cultures were treated with 1 µM 8-dibutyryl-cAMP (Sigma), whereas control cultures received medium alone. Cultures were terminated at intervals through 48 h for Western and for 24 h for ChIP analyses.
Protein Analysis
Protein extracts were prepared from freshly isolated mouse granulosa cells or from cultures of rat and human granulosa cells by resuspending washed cellular pellets in appropriate volumes of radioimmunoprecipitation assay (RIPA) buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8)] containing a protease inhibitor cocktail and 0.5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma). Lysates were incubated for 30 min on ice and centrifuged for 10 min at 10,000 x g. Western blots were prepared from protein samples fractionated on SDS-PAGE gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). All blots were blocked with 5% milk in Tris-buffered saline with 0.05% Tween 20. Commercially available primary antibodies included anti-StAR (1µg/ml; Affinity Bioreagents, Golden, CO), anti-progesterone receptor (1:50; Novocastra Laboratories, Newcastle, UK) and anti-ß-tubulin (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA). Primary antibodies for SNF2L (25 ng/ml) and SNF2H (1:3000) were raised in sheep by Affinity Biologicals, Hamilton, Ontario, Canada. The antigens were purified GST fusion proteins to fragments corresponding to amino acids 1–82 of the SNF2LA isoform (27) or amino acids 1–237 of human SNF2H (29). Secondary antibodies were horseradish peroxidase-conjugated antisheep IgG, antirabbit IgG, antimouse, and biotinylated antimouse IgG used with a streptavidin-horseradish peroxidase label. Proteins were detected on blots using chemiluminescence. Blots were performed in triplicate, scanned at 600 dpi and analyzed with Gel-Pro analyzer 4.0 (Media Cybernetics, Silver Spring, MD). The values represent the absolute integrated optical density of the bands corrected for background and were used for statistical analyses described below.
For immunoprecipitation, 500 µg SVOG-4o human granulosa cell protein extract was combined with either prebleed serum, SNF2L or SNF2H antibodies, and protein G-Sepharose in RIPA buffer containing protease inhibitors and 0.5 mM PMSF, and mixed overnight at 4 C on a rotating mixer. Antibody-protein complexes bound to protein G-Sepharose beads were washed extensively in RIPA buffer with protease inhibitors at 4 C and eluted from the beads with the addition of SDS-PAGE sample loading buffer and heating. Samples were separated on SDS-PAGE gels and transferred to Immobilon P for Western blot analysis. Quantification of Western and Northern blots was performed using NIH Image (version 1.63).
For immunofluorescence, coverslips containing rat granulosa cells were washed three times with cold PBS, and then incubated for 5 min on ice in a 3:1 ethanol:methanol solution, followed by four more washes with cold PBS. After fixation, cells on coverslips were blocked for 10 min in 2% BSA in PBS for 1 h at room temperature, and then incubated with the anti-SNF2L (described above) or rabbit anti-StAR antibodies (1:100; gift of Dr. Douglas M. Stocco, Texas Tech University Health Sciences Center, Lubbock, TX) diluted in 2% BSA in PBS for 1 h at room temperature. Cells were then washed with PBS three times followed by 1-h incubation with an appropriate secondary antibody diluted at 1:1500 (antirabbit or sheep IgG Alexa 488 or 594) with 2% BSA in PBS at room temperature in the dark. Coverslips were mounted on slides with Vectashield Mounting Medium for Fluorescence (Vector Laboratories, Burlingame, CA). We examined slides using a Zeiss Axiophot photomicroscope using the x40 objective lens (Fig. 3, A–C) or the x100 objective lens (Fig. 3, D–G). All images were captured using identical exposure times with the mercury bulb set at the same intensity to allow comparison of untreated and treated samples.
ChIP Assays
ChIP assay followed the method of Kuo and Allis (48), with minor modifications. DNA and cell proteins in granulosa cell cultures were cross-linked for 10 min at room temperature by addition of formaldehyde to a final concentration of 1%. Cells were washed and scraped in ice-cold PBS containing protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A; all reagents from Sigma), collected by centrifugation, and resuspended in 200 µl ChIP lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), and protease inhibitors]. Cells were incubated 10 min on ice and disrupted by sonication, and centrifuged (10 min, 20,000 x g at 4 C). The supernatant was then diluted 10-fold in ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris (pH 8.1), 167 mM NaCl, and protease inhibitors; Sigma]. An aliquot of 2 µl lysate was used for purification of total DNA. Each sample was precleared by incubating with 80 µl salmon sperm DNA/protein A-agarose 50% gel slurry (Upstate Biotechnology, Lake Placid, NY) for 60 min at 4 C to reduce nonspecific background. One sample (2 ml) was divided, and each 1-ml subsample was incubated with 5 µg antibody and treated overnight at 4 C with agitation. The antibody used in this experiment was ovine anti-SNF2L. Control precipitation was performed with an equivalent dilution of sheep IgG (Upstate Biotechnology). Immunocomplexes were collected with 60 µl salmon sperm DNA/protein A-agarose for 2 h at 4 C with rotation and were washed once with the each of the following buffers in sequence: low salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl]; high salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl]; LiCl wash buffer [0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)]; TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. Immunocomplex elution was achieved by adding 250 µl elution buffer (1% SDS, 0.1 M NaHCO3). The DNA-protein cross-linking was reversed by incubation at 65 C for 6 h followed by proteinase K treatment. DNA was recovered by purification with the Qiaquik PCR purification column (Qiagen, Valencia, CA). A 0.5-kb fragment from the proximal promoter region of the StAR protein was amplified by PCR in total DNA and immunoprecipitated DNA. The sense primer used was 5'-CCATCCCCTTGCACCACAAC-3', and antisense primer was 5'-TTTCCTGGTAGCGGAGGCAGGCC-3'. PCR products were resolved on agarose gels and visualized by means of an Alpha Imager gel documentation system.
Statistical Analyses
All experiments were performed a minimum of three times. Data obtained from densitometric analyses were log transformed and subjected to one-way (time course) or two-way (time course and treatment) ANOVA, followed by Bonferroni posttests to identify significant differences. Significant differences were inferred at P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address for M.A.L.: Health Canada, Therapeutic Products Directorate, Bureau of Cardiology, Allergy and Neurological Sciences, Tunney’s Pasteur, Ottawa, Ontario, Canada K1A 1B9.
First Published Online June 1, 2006
1 M.A.L. and D.P. contributed equally to this work. 
Abbreviations: ACF, ATP-utilizing chromatin assembly and remodeling factor; CBP, CREB-binding protein; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; CHRAC, chromatin-accessibility complex; CL, corpus luteum; CREB, cAMP response element-binding protein; dbcAMP, dibutyryl cAMP; hCG, human chorionic gonadotropin; ISWI, imitation switch; NURF, nucleosome remodeling factor; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; PMSG, pregnant mare serum gonadotropin; PR, progesterone receptor; RIPA, radioimmunoprecipitation assay; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; siRNA, small interfering RNA; SNF2, sucrose nonfermenting 2 gene; StAR, steroidogenic acute regulatory protein.
2 For simplicity, we use uppercase text to refer to the mammalian ISWI genes and proteins, even in instances in which we refer to the mouse, rat, and porcine orthologs.
Received for publication May 27, 2005. Accepted for publication May 25, 2006.
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