The DEAD Box Protein DP103 Is a Regulator of Steroidogenic Factor-1 (original) (raw)

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1Department of Obstetrics and Gynecology and Department of Cell Biology and Physiology Washington University School of Medicine St. Louis, Missouri 63110

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1Department of Obstetrics and Gynecology and Department of Cell Biology and Physiology Washington University School of Medicine St. Louis, Missouri 63110

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1Department of Obstetrics and Gynecology and Department of Cell Biology and Physiology Washington University School of Medicine St. Louis, Missouri 63110

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2Department of Obstetrics and Gynecology (C.D.) University of Bonn Bonn 53105, Germany

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1Department of Obstetrics and Gynecology and Department of Cell Biology and Physiology Washington University School of Medicine St. Louis, Missouri 63110

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1Department of Obstetrics and Gynecology and Department of Cell Biology and Physiology Washington University School of Medicine St. Louis, Missouri 63110

*Address requests for reprints to: Yoel Sadovsky, M.D., Department of Obstetrics and Gynecology, Washington University School of Medicine, 4566 Scott Avenue, Campus Box 8064, St. Louis, MO 63110.

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Revision received:

08 September 2000

Accepted:

02 October 2000

Published:

01 January 2001

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Qinglin Ou, Jean-François Mouillet, Xiaomei Yan, Christoph Dorn, Peter A. Crawford, Yoel Sadovsky, The DEAD Box Protein DP103 Is a Regulator of Steroidogenic Factor-1, Molecular Endocrinology, Volume 15, Issue 1, 1 January 2001, Pages 69–79, https://doi.org/10.1210/mend.15.1.0580
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Abstract

The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus. It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system. We sought to elucidate the mechanisms that govern the transcriptional activity of SF-1. We demonstrate here that a previously uncharacterized domain, located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function. Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1. Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, we cloned an SF-1 interacting protein that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases. DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103. DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues. Functionally, DP103 exhibits a native transcriptional repression function that localizes to the C-terminal region of the protein and represses the activity of wild-type, but not mutant, SF-1. Together, the physical and functional interaction of DP103 with a previously unrecognized repression domain within SF-1 represents a novel mechanism for regulation of SF-1 activity.

Introduction

Normal development and differentiation depend on intricate regulatory loops through which hypothalamic and pituitary hormones regulate the production and release of steroid hormones. Steroidogenic factor-1 (SF-1), a member of the nuclear receptor superfamily of transcription factors, is a major regulator of endocrine and reproductive function through its influence on development and differentiation of hormone-producing tissues (1, 2). Developmentally, SF-1 is essential for formation of female and male gonads, the adrenal gland, and the ventromedial hypothalamus (1). Absent development of these tissues in SF-1 null mice is associated with abnormal gonadotrope differentiation, phenotypic male-to-female sex reversal, low serum levels of corticosteroids, and, consequently, early neonatal death (3–5). In addition, SF-1 synergizes with SOX9 in regulation of Mullerian Inhibitory Substance expression in Sertoli cells (6, 7). In adult tissues SF-1 is highly expressed in steroid-producing cells within the adrenal glands, testis, and ovary, where it regulates the expression of diverse genes that are essential for steroid hormone biosynthesis, such as cytochrome P450 hydroxylases, ACTH receptor, and steroidogenic acute regulatory protein (1). SF-1 is also expressed in pituitary gonadotropes, where it activates the promoter forα -gonadotropin, LH-β, and GnRH receptor (2, 8). Together, these data indicate that SF-1 activity is imperative for intact embryonic development, sex determination, and endocrine differentiation and is a central regulator of the hypothalamic-pituitary-adrenal/gonadal axis.

SF-1 is defined as a nuclear receptor based on structural and functional homology with members of this family of proteins (9, 10). The action of many receptors from this family is modulated by an activating ligand and a homo- or heterodimeric partner. Furthermore, several regulatory domains within these proteins interact with coactivators or corepressors, which influence the activity of the nuclear receptor through protein-protein interaction (11, 12). While SF-1 regulates the expression of proteins that exhibit variable expression during endocrine and reproductive function, the mechanisms that modulate the activity of SF-1 are poorly understood. Unlike many other nuclear receptors, SF-1 is defined as an orphan receptor because a direct ligand, which is essential for its activity, has not yet been identified. Whereas 25-OH cholesterol enhances the transcriptional activity of SF-1, its role as a physiological ligand of SF-1 is uncertain (13, 14). Furthermore, SF-1 binds to its DNA response element as a monomer and thus is not a target for modulation by a DNA binding heterodimerizing partner (15). Lastly, unlike many steroid receptors that harbor an N-terminal ligand-independent activation domain (AF-1) and a C-terminal, ligand-dependent activation domain (AF-2), SF-1 lacks a functional AF-1 domain and depends entirely on domains C-terminal to the DNA binding domain (DBD) for its activity (2, 16). We and others have previously dissected the interaction of SF-1 with the coactivator SRC-1, which potentiates the activity of SF-1 utilizing the highly-conserved AF-2 hexamer at the C terminus of the protein and the proximal interaction domain at residues 226–230 (17, 18). Similarly, we have determined that SF-1 utilizes a distal repression domain to interact with DAX-1, which, like SF-1, is germane for reproductive development and represses the activity of SF-1 in vitro (19, 20). Additional proteins, including early growth response protein 1 (Egr-1), Wilm’s tumor-1 (WT-1), pituitary homeobox 1 (Ptx1), glucocorticoid receptor-interacting protein 1 (GRIP1), and multiprotein bridging factor 1 (MBF1) modulate the activity of SF-1, but the mechanism of their influence on SF-1 is unclear (21–25).

In our pursuit of mechanisms that modulate the transcriptional activity of SF-1, we report here the identification of a previously unknown repression domain within SF-1. Using this repression domain in a yeast two-hybrid system we cloned a novel regulator of SF-1, which represses the transcriptional activation function of SF-1 by direct interaction. Using a BLAST database search we determined that this regulator of SF-1 is a mouse homolog of DP103 (also known as Gemin3), a recently cloned member of DEAD box-containing RNA helicases (26). Although DP103 directly interacts with the survival motor neuron (SMN) protein, as well as with the Epstein Barr Virus proteins EBNA2 and EBNA3C, its function is unknown (26–28). We found that DP103 is expressed predominantly in the testis and is also expressed in other tissues as well as in cell lines that express SF-1. DP103 interacts with the proximal repression domain (PRD) of SF-1 and represses its transcriptional activity.

Results

Identification of the PRD within SF-1

The transcriptional regulatory domains of SF-1 reside C-terminal to its DBD (Fig. 1A). To further explore the function domains within SF-1, we generated a series of N-terminal truncations of SF-1, fused to GAL4 DBD (Fig. 1B), and tested their transcriptional activity in JEG3 cells. Using the GAL4 reporter plasmidΔ GKI, we found that the transcriptional activity of GAL4-SF-1120−462 was 5-fold higher when compared with GAL4 alone, as expected (Fig. 1C). A similar activity was observed using N-terminal truncations down to residue 192. Surprisingly, the activity of GAL4-SF-1202−462 was 10-fold higher than the activity of GAL4-SF-1120−462 (Fig. 1C). Further truncations that deleted the proximal interaction domain at residue 226 (17) markedly diminished the activity of SF-1. These data pointed to the presence of a repression domain located between aa 193–201 of SF-1, which we termed PRD. We generated several point mutations within PRD to identify the residues that are required for transcriptional repression. As shown in Fig. 1C, mutations in residues 194–197 (KSEY) exhibited the most dramatic enhancement of transactivation, resembling the transcriptional activity of GAL4-SF-1202−462. We therefore used mSF-1-AAEY in subsequent analyses. To confirm the repressive function of PRD in the context of native SF-1, we mutated KSEY residues to AAEY in CMV-SF-1 vector. Using a Western analysis we confirmed that the expression of SF-1 was unchanged by the PRD mutation (not shown). As shown in Fig. 1D, the transcriptional activity of mutated native SF-1 was higher than the activity of wild type SF-1 in either JEG3 or CV-1 cells. Together, these data indicate that PRD exhibits a repression function within SF-1.

Figure 1.

SF-1 Harbors a Repression Domain at aa 193–201 A, A diagram depicting key regulatory domains of SF-1, including the PRD. B, A series of N-terminal truncations of SF-1 (the number denotes the aa residues), fused to GAL4 DBD. Five different mutations (underlined) within the PRD in GAL4-SF-1 are shown. C, The transcriptional activity of chimeric proteins (2 ng) composed of either truncated or PRD-mutated SF-1, fused to GAL4 DBD, transfected into JEG3 cells along with 0.5 μg of the reporter plasmid ΔGKI. D, The transcriptional activity of wild-type or AAEY-mutant SF-1 (0.1μ g), transfected into either JEG3 or CV-1 cells, along with 0.5 μg of the SF-1-responsive reporter plasmid S25. Results are expressed as RLU, normalized to β-galactosidase activity, and represent three independent experiments performed in duplicate.

We next examined the autonomous transcriptional capacity of PRD, independent of SF-1 context. Using a GAL4x5-tkLuc reporter, we found that fusion of SF-1 residues 169–220 to GAL4 DBD markedly reduced (56%) the transcriptional activity of GAL4 in JEG3 cells (Fig. 2A). Similar data were obtained with the reporter ΔGKI (not shown). Importantly, either AAEY or KSAA mutations of PRD abrogated this repression (Fig. 2A). We further assessed whether or not PRD can confer a repression function to a different nuclear protein, estrogen receptor (ER). For this purpose, we created a chimeric protein of SF-1’s PRD fused to the AF-2 domain of ER, downstream of GAL4 (GAL41−147-SF-1169−220-ER283−595). Whereas estradiol stimulated the transcriptional activity of the chimeric protein, the activity was further potentiated (7.6-fold) when the SF-1 169–220 segment harbored the AAEY mutation (G4-mSF-1-PRD/ER, Fig. 2B). These results confirm the transcriptional repression function of SF-1’s PRD and indicate that this repression function is independent of SF-1 context. Thus, it is likely that PRD acquires its repression function via interaction with cellular coregulatory proteins, and not via intramolecular interactions within SF-1.

Figure 2.

SF-1’s PRD Exhibits Intrinsic Transcriptional Repression Function A, PRD containing GAL4-SF-1169−219 (0.2 μg) was cotransfected into JEG3 cells along with 0.5 μg of the reporter vector GAL4x5-tkLuc. Wild-type denotes aa KSEY, whereas AAEY or KSAA denote mutations in aa 194–197 of SF-1. B, The transcriptional activity of a chimeric protein (0.1 μg) composed of SF-1’s PRD (aa 169–219) fused to the AF-2 domain of ER (aa 283–595) downstream from GAL4 DBD, cotransfected into JEG3 cells along with 0.5 μg of the reporter vector ΔGKI. Estradiol (E2, 10−8m) was added 24 h after transfection, and ethanol (0.2%) was used as control. Results are expressed as RLU normalized toβ -galactosidase activity and represent three independent experiments, performed in duplicate.

Cloning and Expression of SF-1 Regulatory Protein

To identify a putative corepressor of SF-1, we used a PRD-spanning region of SF-1 (residues 109–280) as a bait in a two-hybrid screen for an interacting protein within a rat ovary cDNA expression library. Of 16 positive clones, we identified one candidate that interacted with wild-type PRD, but not with an AAEY-mutant PRD. This clone encoded 406 aa upstream of a stop codon and a poly-A tail. Using this clone as a probe in a Northern analysis, we detected an approximately 3-kb transcript in mouse testis or ovary. To obtain a full-length cDNA, we used this clone to screen a mouse testis cDNA library and identified a 2,910-bp clone that encodes an open reading frame of 825 aa. The identified amino acid sequence exhibits high homology at its N terminus region with proteins from the DEAD-box family of putative ATP-dependent RNA helicases (reviewed in Ref. 29). These proteins are conserved at their N-terminal region, which harbors eight discrete domains, including a DEAD (Asp-Glu-Ala-Asp) box element (Fig. 3A). Recently, a new member of this family termed DP103 and a human ortholog of our newly identified SF-1 regulator was cloned by Grundhoff et al. (26) from a human B cell lymphocyte line (BJAB) (26). The function of DP103 in lymphocytes is presently unknown. DP103 is identical to Gemin3, a protein of unknown function that interacts with SMN protein (27, 28). The function of DP103/Gemin3 in the nervous system remains unknown. A Western analysis of JEG3 cell lysate, transfected with mouse DP-103 fused to a FLAG-tag and detected using an anti-FLAG antibody, revealed a 98-kDa protein (Fig. 3B), which was consistent with the size of human DP103, and was detected using immunocytochemistry in both the nucleus and cytoplasm (not shown).

Figure 3.

The Structure and Size of DP103 A, A diagram of DP103, denoting domains in the N-terminal region that are conserved in the DEAD-box containing RNA helicase eIF4A-II, as well as other members of putative ATP-dependent RNA helicases (for details, see Ref. 29). The location of the first aa in each domain is shown. Sequences in the C-terminal region, which interact with SF-1, are not conserved among members of this family of proteins. B, Immunoblot of JEG3 cells, which were transiently transfected with a FLAG-DP103 expression vector (2 μg) and detected in cell extract by an antibody against FLAG.

Using Northern analysis of mouse tissues, we identified the highest expression of DP103 in the testis (Fig. 4A). In situ hybridization demonstrated highest expression in the periphery of the seminiferous tubules as well as in intertubular regions, which contain Leydig cells, but not in mature sperm. This pattern parallels SF-1 expression (Fig. 4C). DP103 is also expressed, albeit at a lower level, in the steroid-producing ovary and adrenal, as well as in the placenta, brain, and kidney (Fig. 4A). Interestingly, DP103 expression is dramatically higher in the adult testis when compared with P10 testis, suggesting a role in adult testicular function. While DP103 is expressed in the adult ovary, its level does not seem to fluctuate during the estrous cycle. The expression of DP103 in cell lines correlates with SF-1 expression (Fig. 4B), exhibiting a strong signal in the adrenocortical cells Y1, Leydig MA-10, and gonadotrope LβT2. DP103 is also expressed in trophoblast Rcho cells, and at a weak level in JEG3, Hela, DC3, and NIH3T3 lines.

Figure 4.

Expression of DP103 in Tissues and Cell Lines Total RNA (25 μg), isolated from either tissues or cell lines, was hybridized with a probe for either DP103 or SF-1, as described in Materials and Methods. 18S subunit was used to control the quantity of loaded RNA. A, DP103 expression in mouse tissues. B, DP103 expression in cell lines, including CV-1 (monkey kidney fibroblasts), Y1 (mouse adrenocortical), JEG3 (human choriocarcinoma), Hela (human cervical adenocarcinoma), MA-10 (mouse Leydig), LβT2 (mouse gonadotropes), DC3 (rat ovarian granulosa), NIH3T3 (mouse embryonic fibroblasts), Rcho (rat choriocarcinoma), JAR, and Bewo (both human choriocarcinoma cells). To ensure that the detection level in human cell lines does not reflect species specificity of the murine probe, we hybridized the membrane to a probe derived from a human DP103 ortholog (26 ) and obtained an identical result (not shown). C, In situ hybridization of SF-1 and DP103 expression in adult mouse testis, performed as described in Materials and Methods (magnification× 400). No signal was detected using respective sense probes (not shown). S denotes seminiferous tubules, and black arrows demarcate the tubular margin. White arrows point to Leydig cells containing intertubular region (bar = 50 μm).

DP103 Interacts with SF-1 and Regulates Its Activity

To test for physical interaction between DP103 and PRD domain of SF-1, we performed an immunoprecipitation experiment using a FLAG-DP103-transfected JEG3 extract and in vitro expressed full-length 35S-labeled SF-1. Precipitation by anti-FLAG affinity gel revealed a strong SF-1 signal with wild-type PRD (Fig. 5A). A 4.3-fold weaker signal was detected when SF-1 was mutated (AAEY at PRD), supporting the role of PRD in the physical interaction between SF-1 and DP103 in vitro. This interaction was recapitulated in yeast, where SF-1109−280, which contains the PRD, interacted with DP103, yet a similar AAEY-mutated fragment failed to interact (Fig. 5B). Finally, we confirmed the interaction of DP103 and the PRD domain in mammalian cells, using GAL4-SF-1120−462 and DP103-VP16 hybrids cotransfected into CV-1 cells along with the reporter plasmid ΔGKI. We found a strong interaction (6-fold) between GAL4-SF-1120−462 and DP103 (Fig. 5C). In contrast, this two-hybrid interaction was diminished when SF-1 was either mutated at PRD (AAEY) or harbored an internal PRD deletion (Δ187–220), thus supporting a physical interaction of SF-1’s PRD and DP103.

Figure 5.

DP103 Physically Interacts with the PRD Domain of SF-1 A, Immunoprecipitation of wild-type or AAEY-mutant 35S-labeled SF-1 by FLAG-DP103. FLAG-DP103 was generated by a nuclear extract from JEG3 cells transiently transfected with pCMV2-FLAG-DP103 (15 μg). SF-1 was transcribed, translated, and 35S-labeled using TnT. 35S-SF-1 and nuclear extract containing transfected FLAG-DP103 were incubated and precipitated by anti-FLAG affinity gel, and SF-1 detected by autoradiography. Input contained 20% of 35S-labeled SF-1. When corrected to input expression, the binding of mSF-1AAEY to DP103 was 23% of SF-1wt binding. B, The interaction of DP103 with either wild-type or AAEY-mutated SF-1109–280 fragment in yeast, as described in Materials and Methods. Results represent two independent experiments. C, The interaction of the two hybrids GAL4-SF-1 (1 ng) and DP103-VP16 (0.4 μg), transiently transfected into CV-1 cells along with the reporter plasmid ΔGKI (0.5 μg). Results, which represent three independent experiments performed in duplicate, are expressed as fold activation over control in which the empty pVP16 plasmid was used and normalized to β-galactosidase activity.

To determine the transcriptional function of DP103, we initially assessed its activity when tethered to DNA. As shown in Fig. 6A, expression of GAL4 fused to full-length DP103 in CV-1 cells repressed the activity of GAL4 reporter (GAL4x5-tkLuc) in a concentration-dependent manner. Because DP103 directly interacts with SF-1 through the PRD, we tested for the ability of DP103 to repress the activation function of SF-1. Using a GAL4 reporter and GAL4-SF-1120−462 in CV-1 cells, we found that cotransfection of DP103 resulted in a 60% reduction in the transcriptional activity of GAL4-SF-1 (Fig. 6B). As expected, the activity of GAL4-SF-1 mutated at residues AAEY of the PRD was markedly higher. Importantly, the activity of the mutated SF-1 was not repressed but slightly enhanced after cotransfection of DP103 (Fig. 6B). Similarly, DP103 did not repress the activity of GAL4-ER283−595. Together, these results indicate that the repression effect of DP103 is abrogated when the PRD is mutated or absent. Interestingly, because of the proximity of PRD to serine-203 of SF-1, which was shown important for SF-1 activity (25), we tested whether or not a mutation of serine-203 to alanine alters the influence of DP103 on SF-1. As expected, the activity of SF-1S203A was markedly lower than the activity of wild-type SF-1, yet the repression effect of DP103 on SF-1 was unchanged (not shown). We further confirmed the effect of DP103 on native, full-length SF-1 using the wild-type or AAEY-mutant SF-1 (both expressed using pCMV-SF-1), in the presence or absence of DP103. As expected, DP103 diminished the activity of wild-type, but not AAEY-mutated SF-1 (Fig. 6C). Next, we analyzed the effect of DP103 on the transcriptional activity of SF-1 using the SF-1-responsive P450scc-luciferase reporter plasmid, which harbors SF-1 binding elements (30, 31). When transfected into Y1 adrenocortical cells, which express SF-1 endogenously, we found that DP103 repressed the activity of the P450scc promoter in a concentration-dependent fashion (Fig. 6D). Importantly, DP103 had no effect on the P450scc-luciferase reporter when transfected into JEG3 cells, which do not express SF-1 (Fig. 6E). However, DP103 diminished the activity of P450scc-luciferase when enhanced by cotransfected SF-1. This effect was abolished when the P450scc-luciferase reporter was mutated at the two SF-1 binding sites (30, 31). Taken together, we conclude that DP103 exhibits a native transcriptional repression function, and that its interaction with SF-1 in a PRD-dependent manner represses the transcriptional activity of SF-1.

Figure 6.

DP103 Represses the Transcriptional Activity of Wild-Type SF-1 A, The transcriptional activity of GAL4-DP103 transfected into CV-1 cells along with 0.5 μg of the reporter plasmid GAL4x5-tkLuc. Maximal transcriptional repression was 87%. B, The effect of DP103 on the transcriptional activity of wild-type GAL4-SF-1120−462, GAL4-SF-1120−462mAAEY, or GAL4-ER283−595 (in the presence of 10−8m estradiol). Twenty nanograms of GAL4 fusion proteins and 2 μg of pcDNA3-DP103 (or pcDNA3 empty vector) were cotransfected into CV-1 cells, along with 0.5 μg of the reporter plasmid ΔGKI. C, The effect of DP103 on the transcriptional activity of native, wild-type, or AAEY-mutant SF-1. Either SF-1 construct, expressed from CMV-SF-1 (1 ng), was transfected into CV-1 cells along with 3 μg of pcDNA3-DP103 and 0.5 μg of the reporter plasmid S25. The empty expression vectors CMV-Neo or pcDNA3 were used as control for SF-1 or DP103, respectively. D, The effect of DP103 on the activity of the SF-1-responsive P450scc-luciferase reporter. A total of 3 μg of pcDNA3-DP103 and pcDNA3 empty vector were cotransfected into Y1 cells, along with 0.5 μg of the reporter plasmid P450scc-luciferase. E, The effect of DP103 on the activity of the P450scc-luciferase reporter in JEG3 cells. A total of 3 μg of pcDNA3-DP103 and pcDNA3 empty vector were cotransfected along with 0.1 μg of SF-1 where indicated, as well as 0.5 μg of P450scc-luciferase reporter plasmid, which contained two wild-type or mutated SF-1 binding elements, described previously (31 ). Results are expressed as RLU, normalized to β-galactosidase activity and represent at least two independent experiments, performed in duplicate.

DP103 interacts with EBNA2, EBNA3C, or SMN via its nonconserved C terminus. We therefore fused either the N-terminal (aa 1–410) or the C-terminal fragments of DP103 to the activation domain of VP-16 and determined their interaction with wild-type or AAEY-mutated G4-SF-1 in a mammalian two-hybrid experiment. As shown in Fig. 7A, the interaction of VP16-DP103410−825 with SF-1wt was similar to that seen with full-length DP103. In contrast, VP16-DP1031−410 did not interact with SF-1, and both DP103 fragments did not interact with SF-1 AAEY mutant. Moreover, when we fused these DP-103 fragments to GAL4 and tested for their transcriptional function, we found that the C-terminal fragment of DP103 repressed the activity of the GAL4 reporter gene, whereas the N-terminal fragment had no effect (Fig. 7B). This difference did not reflect an altered expression level, as both fragments were expressed at a level similar to the expression of full-length GAL4-DP103 (Fig. 7C). Together, these findings provide further support to the specific effect of DP103 and indicate that DP103 interacts with SF-1 and represses its activity utilizing domains located at its nonconserved C-terminus.

Figure 7.

The C-Terminal Region of DP103 Interacts with SF-1 and Harbors a Repression Domain A, The interaction of either wild-type or mutant GAL4-SF-1 (1 ng) with DP103 full-length, N terminus (aa 1–410), or C terminus (aa 411–825) fused to VP16, transiently transfected (0.4 μg) into CV-1 cells along with the reporter plasmid ΔGKI (0.5 μg). Results, which represent two independent experiments performed in duplicate, are expressed as fold activation over control in which the empty pVP16 plasmid was used and normalized to β-galactosidase activity. B, The transcriptional activity of GAL4 fused to either DP103-VP16 full-length N terminus (aa 1–410) or C terminus (411–825), transfected into CV-1 cells along with 0.5 μg of the reporter plasmid GAL4x5-tkLuc. C, Immunoblotting of the GAL4-DP103 fusion proteins analyzed in Fig. 7B, detected using an anti-GAL4 antibody as described in Materials and Methods.

Discussion

SF-1 plays a pivotal role in reproductive development and function (1, 2, 16). Dynamic regulation of SF-1’s activity seems essential to account for the diverse spatial and temporal influences of SF-1 on a wide array of target genes, and protein coregulators are likely to play a central role in modulating SF-1 activity during that process (2, 16). In this manuscript we examined a previously unidentified domain within SF-1, located at residues 193–201. Upon deletion or point mutation of key residues within this domain, the transcriptional activity of SF-1 is markedly increased, compared with that of wild-type SF-1. The repression function of the PRD is retained when it is transferred to other transcriptional factors, indicating that the PRD’s function is independent of SF-1 context. Interestingly, sequence analyses revealed that the nine-residue PRD is conserved among nuclear receptors that are closely related to SF-1, such as LRH-1 (FTF) and xFF1R (32, 33). The function of PRD in these proteins has not been examined.

Using the region of SF-1 that spans PRD as bait in a yeast two-hybrid system, we cloned a protein of 825 aa from an ovarian cDNA library. This protein is a mouse ortholog of human DP103, a member of DEAD box family proteins (29, 34). Members of this family are defined by at least eight evolutionary conserved motifs (one of which is the Asp-Glu-Ala-Asp, or DEAD motif) that are necessary for RNA helicase activity. DEAD box proteins play an important role in processes related to RNA metabolism, including pre-mRNA splicing, mRNA transport, and ribosome biogenesis and translation initiation (29, 34). Thus, they are germane for cell development, differentiation, and proliferation. Although it is likely that RNA helicase is a key function of DEAD box proteins, it is noted that members of this family contain regions that share little or no sequence homology with other members in the family. Human DP103 was recently cloned by Grundhoff et al. (26) based on its interaction with Epstein-Barr virus nuclear antigens EBNA2 and EBNA3C. In addition, DP103 (also termed Gemin3), directly interacts with SMN protein (27, 28). Mutations in SMN are responsible for different forms of the neurodegenerative disease, spinal muscular atrophy. The function of DP103 in regulation of these proteins is unknown. In contrast, we demonstrated that DP103 regulates the transcriptional activity of SF-1. Interestingly, the relevance of a different RNA helicase to a steroid receptor was demonstrated by Endoh et al. (35), who identified an RNA helicase (p68) as a transactivator for human ERα, acting through the AF-1 domain. DP103, on the other hand, is a repressor of wild-type SF-1, as shown using either a GAL4 fusion of SF-1 or native SF-1. DP103 also represses a reporter plasmid when tethered to DNA through fusion with GAL4 DBD. Furthermore, when overexpressed in Y1 adrenocortical cells, DP103 represses the activity of the SF-1-dependent P450scc promoter, which regulates the transcription of a rate-limiting enzyme in corticosteroid biosynthesis. The direct interaction of SF-1 with the nonconserved C-terminal region of DP103 resembles the interaction of DP103 with either EBNA proteins or SMN. This suggests that the C-terminal region of DP103 is capable of forming a protein complex, which exhibits transcriptional functions distinct from the conserved RNA-regulatory domain in the N terminus of DP103 (29).

Although DP103 is expressed at a low level in diverse tissues, highest expression is found in the testis. Additionally, DP103 is expressed in the ovary and adrenal gland, which also depend on SF-1 expression for morphogenesis and differentiation (1, 16). Correspondingly, the level of DP103 is high in steroidogenic cell lines Y1 and MA-10, as well as in LβT2 gonadotropes, all of which express functional SF-1. Whereas the level of DP103 in the ovary was unchanged during the estrous cycle, its expression in the testis was considerably enhanced in the adult, when compared with P10. Taken together, the expression pattern of DP103 and its interaction with SF-1 suggest that DP103 may play a role in differentiation of steroid-producing tissues. DP103-dependent repression of SF-1 activity may play a role in down-regulation of SF-1 target genes during development and function of reproductive and steroid-producing tissues. Studies are currently underway to assess these possibilities.

Intriguingly, we noted that DP103 somewhat enhanced transcription when coexpressed with mutant SF-1 (see Fig. 6, B, C, and E). These data suggest that DP103 diminishes the activity of wild-type SF-1 through interaction with PRD, yet an additional interaction with a different domain of SF-1, which may result in enhanced transcriptional activation, cannot be excluded. Recent evidence indicates that other coregulators of nuclear receptors, such as NSD1 and mZAC1b, can act as coactivators or corepressors in a promoter and cell type-dependent manner (36, 37). The mechanism underlying this bipolar effect is presently unknown. Similarly, the mechanism used by DP103 to influence gene activity remains to be determined and may involve modulation of RNA processing. It is noted that RNA helicase activity is dispensable for the transcriptional influence of the DEAD box protein p68 on ER. Moreover, p68 recruits the coactivator CREB-binding protein (CBP) to its complex with ER (35). These findings support a function of DEAD box proteins, which is independent of RNA helicase activity.

We and others have previously identified several SF-1 domains that modulate its activity through interaction with coregulators. These include the C-terminal AF-2 hexamer, interacting with SRC-1 (17) and in turn, CBP (18), the distal repression domain, interacting with DAX-1 that binds nuclear receptor corepressor (N-Cor) (19, 20); and the proximal interaction domain that interacts with both SRC-1 and DAX-1 (17, 19). Additional segments of SF-1, including serine203 (interacting with GRIP1) and the proline-rich region near the DBD play an important role in regulating SF-1 activity (25, 38). Whereas additional proteins, such as Egr-1 and Sp1 (21, 39, 40), synergistically interact with SF-1, the mechanism of this interaction is currently unknown. Unlike these proteins, DP103 is a regulator of SF-1 and interacts with SF-1 through a previously unidentified repression domain. Although our results do not elucidate the specific function of DP103 in development and differentiation of steroidogenic tissues, the identification of a repression domain within SF-1, which interacts with DP103, suggests the involvement of this protein in modulating SF-1 function. Further dissection of DP103 function is likely to shed light on the mechanisms that modulate the transcriptional activity of the orphan receptor SF-1, as well as on the role of DP103 in other tissues.

Materials and Methods

Plasmids and Cloning of Mouse DP103

We used PCR to generate fusion proteins of GAL4 DBD (aa 1–147) in pM2 vector and carboxy-terminal fragments of SF-1 (residues 120–462, 187–462, 192–462, 202–462, 220–462, and 230–462) and 169–219, as previously described (17). The plasmid pBS-SF-1 was used as a template for PCR-based site-directed mutagenesis within the PRD (IKSEYPEPY) of SF-1, performed as previously described (21). Each mutant and chimeric protein was sequenced using the dideoxynucleotide sequencing method on a model 373A DNA sequencer (PE Applied Biosystems, Norwalk, CT). Mutated SF-1 fragments were fused to GAL41−147, and mutated SF-1 clones were cloned back into pCMV-Neo (17). GAL4-SF-1Δ187–220 was generated by reverse PCR amplification of GAL4-SF-1 using forward and reverse primers that correspond to nucleotides at residue 220 and 187, respectively, followed by ligation. The chimeric protein GAL4-SF-1169−219-ER283−595 was generated by amplification of SF-1169−219 with flanking _Bam_HI/_Spe_I sites and ER283−595 with flanking _Spe_I/_Sal_I sites, and subsequent three-way cloning downstream from GAL41−147. The vector pBS-ER (a gift from S. Adler, Washington University, St. Louis, MO) was used as a template. We used two GAL4 reporter genes:Δ GKI, composed of five GAL4 binding sites upstream of an E1B promoter (a gift from P. Webb and P. Kushner, University of California, San Francisco), or GAL4x5-tkLuc, composed of five GAL4 binding sites upstream of a thymidine kinase promoter (a gift from J. Milbrandt, Washington University). The rat P450scc-luciferase reporter construct was subcloned from the rat P450scc −894/+37-GH reporter construct (a gift from J. S. Richards, Baylor College of Medicine, Houston, TX), as previously described (31). The SF-1 reporter S25 was previously described (17).

The cloning of DP103 was performed according to the Matchmaker yeast 2-hybrid protocol (CLONTECH Laboratories, Inc., Palo Alto, CA). A murine SF-1 bait spanning aa 109–280 was cloned downstream of GAL4 DBD in a pAS1 vector [a gift from J. Milbrandt (41)]. A similar fragment harboring the AAEY-mutation was used as a negative control. The bait, along with a rat ovary cDNA library (CLONTECH Laboratories, Inc.), was sequentially transformed into HF7C yeast, and 16 positive colonies were selected by growth in HIS-minus medium and β-galactosidase assay. To eliminate false positive clones, we transformed either the bait or the mutant bait into yeast strain HF7C (MATa), and positive clones into yeast strain Y187 (MATα). Mating was performed according to the CLONTECH manual, and one clone (ARO19), which interacted with wild-type but not AAEY mutant, was identified. To clone full-length DP103, a 400-bp fragment between _Pst_I and _Sca_I of ARO19 was used to probe aλ -phage mouse-testis cDNA library (CLONTECH Laboratories, Inc.). Five independent clones were obtained, two of which were sequenced and identified as ARO19 matches with an open reading frame of 825 aa and a poly-A tail, designated as full-length DP103.

To generate a DP103 expression vector, we subcloned the full-length DP103 from pTripIEX into the _Bam_HI/_Xba_I sites in pcDNA3 vector (Invitrogen, San Diego, CA). The fusion protein FLAG-DP103 was generated by cloning full-length DP103 downstream from FLAG at _Kpn_I/_Xba_I sites of pFLAG-CMV2 vector (Sigma, St. Louis, MO). DP103-VP16, DP103411–825-VP16 (N-terminal), and DP103411–825-VP16 (C-terminal) were generated with DP103 fragments downstream from the VP16 activation domain at _Bam_HI/_Xba_I sites in a pVP16 vector (CLONTECH Laboratories, Inc.). GAL4-DP103, GAL4-DP1031–410, and GAL4-DP103411–825 were generated by cloning DP103 fragments between _Bam_HI/_Xba_I sites downstream from GAL4 DBD.

Cell Culture and Transfection

JEG3 and CV-1 cells were maintained in culture as previously described (17). Y1 cells were cultured in DMEM, supplemented with 10% FBS and antibiotics. Cells were transfected with a total of 1 μg or 5μ g plasmid DNA per plate in either 12- or 6-well plates, respectively, using the calcium phosphate coprecipitation technique as described previously (17). Transfections were performed in duplicate and repeated at least three times. Results were expressed as relative luciferase units (RLU), normalized to β-galactosidase activity. In experiments in which estradiol was used, the cells were cultured in phenol-red free medium with serum that contains negligible levels of 17β-estradiol.

Expression

Using TRI-REAGENT (Molecular Research Center, Inc., Cincinnati, OH) we isolated total cellular RNA from either mouse (Bl/6 or Bl6/129 strains) tissues or diverse murine or human cell lines. RNA samples (25 μg) were separated by electrophoresis. A mouse DP103 probe, which corresponds to aa 216–516, was generated using 900 bp _Hin_dIII fragment from pTripIEX-DP103. The SF-1 probe, its labeling, RNA transfer, hybridization, and detection were previously described (42).

For immunoblotting of FLAG-DP103 we transfected 2 μg of pFLAG-CMV2-DP103 into JEG3 cells plated on a 100-mm plate. After 48 h the nuclear extract was isolated and mixed with anti-FLAG-M2 affinity gel suspension (Sigma) followed by TBS (25 mm Tris, pH 7.4, 150 mm NaCl) washings (Sigma). The protein was separated on an 8% polyacrylamide gel, and then transferred to an Immobilon-P polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA) at 150 mA overnight. The membrane was probed with a mouse anti-FLAG antibody (1:360 dilution, Sigma) followed by peroxidase-labeled antimouse secondary antibody (1:1,000 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody staining was visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington heights, IL). For immunoblotting of GAL4 fusion proteins, we transfected 3 μg of G4 alone, G4-DP103, G4-DP103 N, and G4-DP103 C into CV-1 cells plated on a six-well plate. After 48 h the nuclear extract was isolated with sample buffer (12.5 mm Tris-HCl, pH 6.8, 8% vol/vol glycerol, 0.4% SDS, 1% vol/vol 2-mercaptoethanol, and 0.004% bromophenol blue). The protein was separated as described above, and the membrane was probed with mouse anti-GAL4 DBD antibody (1:1,000 dilution, Santa Cruz Biotechnology, Inc.) followed by peroxidase-labeled antimouse secondary antibody (1:1,000 dilution, Santa Cruz Biotechnology, Inc., Torrance, CA), and developed as described above.

For in situ hybridization we collected mouse tissues, and embedded them in OCT compound (Sakura, Torrance, CA). Cryostat sections (10 μm) were postfixed in 4% paraformaldehyde followed by two PBS washes. Fragments of DP103 (which corresponds to aa 515–618) or SF-1 (which corresponds to aa 1–205) were cloned into pBSK plasmid. Sense and antisense digoxigenin-labeled riboprobes were synthesized using DIG RNA labeling mix (Roche, Indianapolis, IN). Slides were prehybridized for 2 h and hybridized overnight at 58 C in 5× sodium chloride-sodium citrate buffer with 50% formamide and 40 μg/ml heat-denatured ssDNA, followed by stringency washes. Levamisole solution (Zymed Laboratories, Inc., South San Francisco, CA) was used to inhibit endogenous alkaline phosphatase according to the manufacturer’s protocol. Signals were detected using DIG Nucleic Acid Detection Kit (Roche).

Immunoprecipitation

To generate FLAG-DP103 protein, we transfected JEG3 cells with 15 μg of pFLAG-CMV2-DP103 in 100-mm plates and isolated nuclear extracts 48 h later. SF-1 and mutSF1AAEY were 35S-Met labeled in vitro using a TNT kit (Promega Corp., Madison, WI). We mixed 10 μl of labeled wild-type or mutant SF-1 with the FLAG-DP103-containing extracts and then added to 20 μl anti-FLAG-M2 affinity gel suspension (Sigma). After washes the proteins were separated using 8% polyacrylamide gel and exposed to Kodak X-OMAT film at− 80 C overnight.

Data Deposition

The sequence of mouse DP103 reported in this paper has been deposited in the GenBank database under Accession no. AF220454, and a partial sequence of rat DP103 under Accession no. AF220455.

Acknowledgments

We thank S. Adler, P. Webb, P. Kushner, K. Parker, and J. S. Richards for plasmids, and E. Sadovsky and L. Rideout for technical assistance. We also thank J. Milbrandt for plasmids and for insightful discussions.

This work was supported by NIH Grants HD-34110 and HD-37571 and Howard Hughes Medical Institute Pilot Research Projects Award (to Y.S.), and DFG DO 653/1 (to C.D.).

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Copyright © 2001 by The Endocrine Society

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