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Translin Coactivates Steroidogenic Factor-1-Stimulated Transcription

Department of Obstetrics, Gynecology, and Reproductive Sciences (S.H.M., S.R.B., C.D., J.-L.V., P.B.B.), The Center for Reproductive Sciences, University of California, San Francisco, California 94143; and Department of Obstetrics and Gynecology, and Center for Research on Reproduction and Women’s Health (N.B.H.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Synthia H. Mellon, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, 513 Parnassus Avenue, Box 0556, San Francisco, California 94143-0556. E-mail: mellon{at}cgl.ucsf.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Transcription of the rat P450c17 gene in Leydig cells requires steroidogenic factor-1 (SF-1) (NR5A1), nerve growth factor-inducible protein B (nurr77), COUP-TF, and SET. The –447/–419 region of this promoter contains two binding sites for orphan nuclear receptors that are required for activation by SF-1, nerve growth factor-inducible protein B, and cAMP. We identified a novel factor, steroidogenic factor-inducer of transcription-2, that binds to this –447/–419 region. We have now purified steroidogenic factor-inducer of transcription-2 from mouse Leydig MA-10 cells and identified it by mass spectrometry as translin, a 27-kDa protein that exerts many functions. By itself, translin cannot activate a P450c17-promoter/reporter construct in HeLa cells; however, translin increased SF-1-stimulated transcription 2-fold, indicating cooperativity between SF-1 and translin. Mutation of both SF-1 binding sites in the –447/–419 sequence eliminated activation by SF-1 and translin. Translin did not augment SF-1-stimulated transcription from all SF-1-responsive elements, suggesting that the activation is specific for the sequence of the SF-1 response element. Gel shift analysis of double- and single-stranded DNA showed that translin binds to single-stranded DNA, but its transcriptional activation is independent of DNA binding. The hinge region of SF-1 is necessary for activation by translin; deletion of hinge amino acids 170–225 in SF-1 eliminates translin’s ability to augment SF-1-dependent transcription. A translin-like protein, called translin-associated factor X, can substitute for a translin moiety; translin homomers and translin/translin-associated factor X heteromers activated SF-1-stimulated transcription equally. Thus, we have identified a new factor that works together with SF-1 to augment gene transcription in a DNA-specific fashion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE ARRAY OF STEROIDS produced by a tissue depends on which steroidogenic enzymes are expressed in that tissue. One steroidogenic enzyme, P450c17, has two major activities, 17-hydroxylase, producing 17-hydroxy C21 steroids and C17,20 lyase, producing C19 steroids. These two activities are regulated by posttranslational modification (1, 2) and by allosteric factors (3, 4); hence, steroid production is regulated both transcriptionally and posttranslationally (reviewed in Ref. 5). Hydroxylase and lyase activities are necessary for the development and maintenance of reproductive function (5), and appear to be important for production of neurosteroids that regulate neural differentiation and function (6) and for early embryonic development (7).

Transcriptional regulation of P450c17 is both species-specific and tissue-specific, and involves transcription factors that vary among mammalian species and among steroidogenic tissues (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). P450c17 is expressed in the gonads of all vertebrates, in the adrenals of primates (21) and ungulates (22), but not rodents (23, 24), and it is expressed in placenta of rodents (25, 26), but not primates (21). P450c17 expression is also regulated developmentally. The mammalian fetus expresses P450c17 in the testis but not the ovary (10, 21, 27), and it is required for male sexual differentiation (21, 28). It is also expressed transiently in the neonatal rat liver (29) in the developing rodent nervous system (30). In the mouse embryo, its expression appears to be essential for development beyond embryonic day 7 (7), although human beings lacking P450c17 activity develop to term.

P450c17 of all species is regulated by steroidogenic factor-1 (SF-1) in those steroidogenic tissues that express SF-1 (12, 13, 14, 18, 31, 32, 33, 34). Rat P450c17 transcription is also regulated by developmentally regulated factors (9, 10), by tissue-specific factors (11, 12), and by multiple orphan nuclear DNA-binding receptors (12, 13, 14).

We have identified and characterized several transcriptionally regulated regions in the rat P450c17 gene in detail (9, 12, 13, 14). We found that the region of the rat P450c17 gene at –447/–419 is regulated by SF-1 together with two novel factors we termed steroidogenic factor-inducer of transcription-1 and -2 (StF-IT-1 and StF-IT-2) (12, 35). We subsequently identified StF-IT-1 as a phosphoprotein called SET, which inhibits cell cycle (36, 37) and is an inhibitor of protein phosphatase 2A (2, 38) in addition to regulating transcription (9, 10). We have now identified StF-IT-2 as the protein known as testis brain RNA-binding protein (TB-RBP) (39, 40) also known as translin (41), and we now show that translin activates SF-1-mediated transcription in a DNA-specific manner.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Purification and Identification of StF-IT-2
We previously identified StF-IT-2 as a nuclear protein in mouse Leydig MA-10 cells by EMSAs using the –447/–419 segment of the rat P450c17 gene promoter (12, 35). To purify StF-IT-2, we used homogenates from MA-10 cells as an abundant source of protein. Initial steps at StF-IT-2 purification included using whole-cell vs. nuclear extracts, ammonium sulfate precipitation, sizing columns, reversed-phase columns, and heparin columns. In addition, various gradients of elution buffers were used on these columns, and on the fast protein liquid chromatography and HPLC columns that were ultimately used.

Our established protocol for purification of StF-IT-2 began with fast protein liquid chromatography of MA-10 whole-cell extracts; fractions containing StF-IT-2 were identified by EMSAs (gel shift assays), combined, and purified further by HPLC on a MonoQ column (Fig. 1). SDS-PAGE showed that the fractions containing StF-IT-2 DNA binding activity were enriched for a protein of approximately 25 kDa. The approximately 25,000 molecular mass protein from fraction 29, which contained the majority of the StF-IT-2 binding activity, was excised, digested with trypsin, purified by HPLC, and analyzed by tandem mass spectrometry (MS/MS) (Table 1). The sample contained three different proteins, each of which could be identified by three or more peptides. Two were proteins of approximately 25 kDa: translin (26.2 kDa) and 14-3-3 (27.7 kDa); the other was nexin 2 (58 kDa), which was not considered further, because its molecular mass did not match that identified by SDS-PAGE. The peptide masses identified by mass spectrometry encompassed 37% coverage of the sequence of translin and 24% coverage of the sequence of 14-3-3 but only 7% of nexin 2.

View larger version (102K): [in this window] [in a new window]   Fig. 1. Purification of StF-IT-2 A, Gel shift analysis of aliquots of fractions from the final Mono-Q HPLC column. StF-IT-2 is enriched in fractions 28–31. B, Silver-stained SDS polyacrylamide gel from aliquots of the final MonoQ HPLC column. A protein of approximately 25 kDa was enriched in fractions 28–31, where the majority of the StF-IT-2 binding activity was identified in A. The remainder of fraction 29 was purified on a preparative SDS polyacrylamide gel, stained with Coomassie blue, and the 25-kDa protein band was excised and analyzed by MS/MS, after trypsinization and HPLC purification. SM, Starting material.

The family of 14-3-3 proteins are approximately 30-kDa acidic proteins, so-named because of their purification profile (55, 56, 57). They are a highly conserved, ubiquitously expressed protein family composed of at least seven isoforms. They bind to phosphoserine and phosphothreonine (58) and hence can interact with a wide variety of proteins yielding many different functions (59, 60, 61, 62, 63). These functions include modulating enzymatic activity, modulating protein localization, preventing dephosphorylation, promoting protein stability, and inhibiting and mediating protein interactions.

Translin Antibodies Supershift StF-IT-2:DNA Complex
To determine whether translin or 14-3-3 were StF-IT-2, we performed gel shift assays using antibodies against each of these proteins (Fig. 2). Using the –447/–419 rP450c17 oligonucleotide as probe, MA-10 cell extracts produced two protein:DNA complexes. The more rapidly migrating complex was an SF-1:DNA complex, identified previously by antibody supershift assays (35) and competition assays (12), and the more slowly migrating complex was StF-IT-2:DNA. Addition of an antibody against mouse translin/TB-RBP caused a specific supershift of the St-F-IT-2:DNA complex obtained from MA-10 cell protein (Fig. 2, lane 4) and from fraction 28 of the MonoQ column (Fig. 2, lane 7). Addition of preimmune serum (Fig. 2, lane 5), or antibodies against either 14-3-3 or 14-3-3ß (Fig. 2, lanes 11 and 12) did not alter the migration of this complex. Immunodepletion of MA-10 extracts with the antibody against translin, but not with preimmune serum, decreased the intensity of the StF-IT-2:DNA complex (data not shown). Thus, the StF-IT-2:DNA complex contains translin and does not contain 14-3-3 or 14-3-3ß.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Antibody Supershift of StF-IT-2;DNA Complexes with a Translin Antibody Extracts from MA-10 cells (left panel, lanes 1–5; right panel, lanes 9–12) or fraction 28 (middle panel, lanes 6–8) from the StF-IT-2 purification (Fig. 1) were incubated with the –447/–419 oligonucleotide (lanes "Probe"). Two protein:DNA complexes, SF-1:DNA and StF-IT-2:DNA, are formed with MA-10 cell extracts (left panel, lane 2), indicated by arrows. A single complex corresponding to StF-IT-2:DNA formed with fraction 28 (middle panel, lane 6). The SF-1:DNA and StF-IT-2:DNA complexes are competed by 100-fold excess –447/–419 oligonucleotide (lane 3, +cold). Addition of an antibody against translin supershifts the StF-IT-2:DNA complex, and forms another complex, StF-IT-2' (lanes 4 and 7). Addition of preimmune serum (lanes 5 and 8) or addition of antibodies against 14-3-3 or 14-3-3ß (right panel, lanes 11 and 12) has no effect.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Luciferase Activity in HeLa Cells A, HeLa cells transfected with a wild-type –447/–419 p36Luc construct (447/–419, 1 µg DNA) alone, with a translin pCDNA expression vector (+translin), with an SF-1 pCDNA expression vector (+SF-1), or with both a translin and SF-1 expression vector (+Translin/SF-1) and assayed 48 h after transfection. B, HeLa cells transfected with mutant –447/–419 p36Luc reporter constructs. HeLa cells were transfected with wild-type, mutant 1 (Mut 1), mutant 2 (Mut 2), or mutant 1/2 (Mut1/2) p36Luc reporter constructs in the presence of nothing (basal), or expression vectors for translin, SF-1, or both SF-1 and translin, and assayed 48 h after transfection. Transfection efficiencies were controlled using the dual Renilla/firefly luciferase assay system. Data are shown as mean ± SE from three experiments.

Translin Binds to Single-Stranded DNA
Translin can bind to single-stranded RNA (39, 47, 48, 49) and to single-stranded DNA sequences found at breakpoints of chromosomal translocations (41). To determine whether translin bound to the –447/–419 region of the rat P450c17 gene as single- and/or double-stranded DNA, we assessed the binding of translin prepared by an in vitro transcription/translation system to labeled sense and antisense oligonucleotides and to the annealed double-stranded –447/–419 oligonucleotide. Gel shift assays showed that translin could bind to the gel-purified, double-stranded oligonucleotide (Fig. 4A, DS Kinase), but the degree of binding was much greater with either single-stranded oligonucleotide probe (Fig. 4A; only single-stranded sense strand probe, SS-S, is shown). These data suggest that translin can bind to either double- or single-stranded DNA. However, these experiments were done with probes labeled with [-³²P]ATP and polynucleotide kinase, and such kinased probes may contain a small amount of single-stranded DNA. Therefore, we also synthesized a double-stranded probe that was labeled using [-³²P]dCTP and the Klenow fragment of DNA polymerase, which cannot label a single-stranded probe. Incubation of this probe with translin did not yield any protein:DNA complex (Fig. 4A, DS Klenow). By contrast, when the double-stranded, Klenow end-labeled probe was incubated with MA-10 extracts that contain SF-1 and translin, only SF-1 yielded its protein:DNA complex, indicating that SF-1 could bind to this double-stranded probe, as expected, whereas translin could not (Fig. 4B). These data suggest that translin only binds to single-stranded DNA.

View larger version (86K): [in this window] [in a new window]   Fig. 4. Translin Binds to Single-Stranded DNA A, Gel shift assay of recombinant translin using double-stranded and single-stranded –447/–419 rP450c17 oligonucleotides as probes. Recombinant translin was incubated with double-stranded oligonucleotides that were end-labeled using [-32P]dATP and kinase (lanes labeled "DS Kinase") or with double-stranded oligonucleotides that were end-labeled using [-32P]dCTP and Klenow fragment of DNA polymerase (lanes labeled "DS Klenow"). Recombinant translin was also incubated with a single-stranded, sense strand of the –447/–419 oligonucleotide that was end-labeled using -32P and kinase (lanes "SS-S"). Lanes "Pr" contain each specific probe and no recombinant translin; lanes marked 1 and 3 indicate the number of microliters of recombinant translin used in the binding assay. Arrowhead marked "Tr" indicates the translin:DNA complex. A translin:DNA complex is seen using a kinase-labeled double-stranded DNA probe or using a single-stranded DNA probe; no translin:DNA complex is seen when the double-stranded probe was end-labeled using Klenow DNA polymerase. B, Gel shift assay comparing DNA binding using extracts from MA-10 cells vs. DNA binding using recombinant translin. DS Kinase lanes contain double-stranded –447/–419 oligonucleotide probes end-labeled using [-32P]dATP and kinase; DS Klenow lanes contain double-stranded –447/–419 oligonucleotide probes end-labeled using [-32P]dCTP and Klenow fragment of DNA polymerase. Lanes "Pr" contain each specific probe alone, with no MA-10 extract or recombinant translin. The arrowhead marked "Tr" indicates the translin:DNA complex; the arrowhead marked "SF-1" indicates the SF-1:DNA complex, seen only with MA-10 extracts. No translin:DNA complex is seen using either MA-10 extracts or recombinant translin and the double-stranded oligonucleotide probe end-labeled using Klenow DNA polymerase. C and D, Competition gel shift assays, using recombinant translin and single-stranded –447/–419 sense strand as probe. Single-stranded competitor oligonucleotides, both sense and antisense strands, were used at 250-fold excess. Mut 1, Mut 2, and Mut 1/2 refer to the oligonucleotides in which one or both SF-1 sites within the –447/–419 oligonucleotide were mutated, shown in Table 2. Sequences of other rP450c17 oligonucleotides (–418/–399 and –84/–55) and of a P450c21 oligonucleotide that contains and SF-1 site (–95/–58) are shown in Table 2.

Because this –447/–419 oligonucleotide contains SF-1 or estrogen response element half-site-like binding sites, we used additional oligonucleotides that contained previously identified SF-1 or similar binding elements as competitors. These oligonucleotides were the –418/–399 and –84/–55 regions of the rat P450c17 gene, as well as the –95/–58 region of the mouse P450c21 gene. Our gel shift data indicate that both the sense and antisense strands of each of these oligonucleotides could compete with the –447/–419 sense strand for translin binding (Fig. 4C).

As competitors for translin binding, we also used –447/–419 oligonucleotides in which the SF-1 sites were mutated (Fig. 4D). In the oligonucleotides, listed in Table 2, each SF-1 site was mutated independently (Mut 1, Mut 2), or together (Mut 1/2). Competition gel shifts demonstrate that, when of either of the SF-1 sites within the –447/–419 oligonucleotide were mutated, the resulting sense and antisense oligonucleotides were able to compete with wild-type –447/–419 sense oligonucleotide for translin binding. In addition, oligonucleotides that contained mutations of both SF-1 sites also competed for translin binding.

Translin’s Activity Does Not Require DNA Binding
To determine whether translin needed to bind to DNA to elicit its effects, we created a previously described variant of translin containing three mutations, R86T, H88N, and H90N; this form of translin cannot bind to nucleic acid (54). We expressed recombinant wild-type and mutant translin in bacteria, and used these proteins in gel shift assays to determine whether mutant translin could bind to the –447/–419 DNA. Gel shift assays confirmed that the mutant translin could not bind to DNA (Fig. 5A). To determine whether the lack of DNA binding affected translin’s ability to stimulate transcription, we subcloned the wild-type and mutant translins into a mammalian expression vector. We cotransfected HeLa cells with either of these translin-expression vectors together with the –447/–419 luciferase reporter and the SF-1 expression vector. Luciferase assays demonstrated that, whereas neither the wild-type nor mutant translin could stimulate P450c17 transcription alone, both the wild-type and mutant translins increased SF-1-stimulated transcription about 2- to 3-fold (Fig. 5B). Taken together, the data suggest that translin does not need to bind to DNA to increase SF-1-stimulated transcription.

View larger version (36K): [in this window] [in a new window]   Fig. 5. DNA Binding and Transcriptional Activation by Mutant Translin A, Gel shift assay. Recombinant mouse translin (wild type) or mutant translin (Mut) were incubated with a double-stranded, kinased 32P-labeled –447/–419 oligonucleotide probe (DS), or with a single-stranded sense (SS-S) or antisense (SS-AS) oligonucleotide probe. Wild-type translin, but not mutant translin, forms a protein:DNA complex with these probes. B, Luciferase activity in HeLa cells after transfection with luciferase reporter constructs (1 µg) –447/–419 p36Luc, and expression vectors containing wild-type translin, mutant translin, SF-1, or a combination of translin (wild type or mutant) plus SF-1. Transfected cells were assayed 48 h after transfection. Transfection efficiencies were controlled using the dual Renilla/firefly luciferase assay system. Results are means ± SE from triplicate wells from three experiments.

View larger version (97K): [in this window] [in a new window]   Fig. 6. Translin Does Not Affect SF-1 Binding to DNA, Assessed by Gel Shift Assays Mouse translin synthesized in vitro in a transcription/translation system, was incubated in increasing concentrations with a 32P-labeled –447/–419 oligonucleotide probe plus empty vector (pCDNA, left lanes) or with a constant amount of SF-1 synthesized in vitro in a transcription/translation system (SF-1, right lanes). The translin:DNA and SF-1:DNA complexes are indicated. A nonspecific protein:DNA complex is indicated as "ns."

View larger version (38K): [in this window] [in a new window]   Fig. 7. GST Pull-Down Assays A, SF-1 binds to translin. [35S]SF-1 was incubated with GST (lane GST) or GST-translin (lane GST-translin) bound to the glutathione-Sepharose matrix, and specifically bound proteins separated on 12% SDS polyacrylamide gels. The lane [35S]SF-1 contains 10% of the [35S]SF-1 applied to the beads. Similarly, [35S]translin was incubated with GST (lane GST) or with GST-translin (lane GST-translin) bound to the glutathione-Sepharose matrix, and specifically bound proteins were separated on 12% SDS polyacrylamide gels. The lane [35S]translin contains 10% of the [35S]translin applied to the matrix. Arrows indicate the 35S-labeled SF-1 and the 35S-labeled translin. B, Regions of SF-1 that bind to translin. Various regions of SF-1 used in the pull-down assays were cloned as indicated by the amino acid numbers below the cartoon; the regions of SF-1 included in these constructs are indicated by horizontal lines below the SF-1 line cartoon, on the left. Each of these constructs was labeled with [35S]methionine in vitro, and incubated with GST-translin bound to the glutathione-Sepharose matrix. The lanes input contain 10% of the 35S-labeled SF-1 fragments applied to the beads. DBD, DNA binding domain (amino acids 1–79); LBD, ligand binding domain (amino acids 225–451); AF2, activating function 2 domain (amino acids 451–462). C, Translin activation of SF-1 hinge mutants. Three deletional hinge mutants of SF-1 were cloned into p36Luc and transfected with the –447/–419 luciferase reporter into HeLa cells in the presence of nothing or translin expression vector. SF-1 mutants had deletions in amino acids 101–169 (SF-1 101–169), amino acids 101–225 (SF-1 101–225), or amino acids 170–225 (SF-1 170–225). D, Translin activation of SF-1 serine 203 mutant. A serine 203 to alanine mutant of SF-1 (Ser 203 Mut) was transfected with the –447/–419 luciferase reporter into HeLa cells in the presence of nothing or translin expression vector. Transfected cells were assayed 48 h after transfection. Transfection efficiencies were controlled using the dual Renilla/firefly luciferase assay system. Results are means ± SE from triplicate wells from three experiments.

To determine whether the hinge region of SF-1 is important for translin activation, we created three SF-1 mutants: two SF-1 mutants deleted the proline-rich region and lacked either amino acids 101–169 (SF-1 101–169) or lacked amino acids 101–225 (SF-1 101–225), and one mutant contained this proline-rich region but lacked amino acids 170–225 (SF-1 170–225). Each of these constructs was transfected into HeLa cells with the –447/–419 luciferase reporter, plus or minus the translin expression vector (Fig. 7C). Transfection data demonstrate that both the SF-1 101–169 and SF-1 101–225 could not activate transcription of the –447/–419 luciferase reporter, suggesting that the proline-rich region of SF-1 is required for SF-1’s transcriptional activity. However SF-1 170–225 could stimulate transcription from the –447/–419 luciferase reporter, although this stimulation was about half of that seen with the full-length SF-1. Translin cotransfected with SF-1 170–225 could not activate transcription of the –447/–419 luciferase reporter. Translin also could not activate the SF-1 mutants lacking the proline-rich region. These data suggest that the hinge region of SF-1 between amino acids 170 and 225 are important for translin’s ability to augment SF-1 transcriptional activation.

The 170–225 region of the SF-1 hinge contains serine 203 that has been shown to be important for SF-1’s transcriptional activity (69). We created a seine 203 to alanine mutant of SF-1 (Ser 203 Mut) in our SF-1-expression vector, and transfected this mutant construct together with the –447/–419 luciferase reporter construct into HeLa cells (Fig. 7D). Ser 203 Mut SF-1 could stimulate luciferase activity from the P450c17 reporter construct about 5-fold (Fig. 7D). Translin could also increase this SF-1-stimulated transcription 2-fold further. These data demonstrate that serine 203 does not play a role in mediating the effects of translin.

Translin Activation of SF-1-Stimulated Transcription Is Dependent upon DNA Sequence
To determine whether translin augments the transcription of other genes that are regulated by SF-1, we cloned several different SF-1-responsive elements into the p36Luc reporter construct. These included another region of the rat P450c17 gene, –84/–55 (13, 14), the rat P450aro gene, –87/–68 (70, 71, 72), and the mouse P450c21 gene, –95/–58 (65, 73). These three elements are proximal SF-1 regulatory elements, and each is located within 100 bp of the TATA box of each gene. When these luciferase reporter constructs were transfected into HeLa cells, they had very low basal activity (Fig. 8A). Cotransfection of these reporter constructs with an SF-1-expression construct increased luciferase activity 4-, 13-, and 4-fold, respectively. When we cotransfected the reporter constructs containing the SF-1 binding domains from the rat P450c17 –84/–55, rat P450aro, or mouse P450c21 with SF-1 and translin expression vectors, luciferase activity was not significantly different from the activity obtained with the SF-1 expression vector alone (Fig. 8A). Transfection with the luciferase reporter constructs plus translin gave luciferase activities no different from basal activity. Thus, translin had little effect on SF-1-stimulated transcription of these three SF-1-responsive elements.

View larger version (37K): [in this window] [in a new window]   Fig. 8. Translin Activation of SF-1-Dependent Elements A, Four SF-1-responsive regions were cloned into p36Luc and transfected into HeLa cells in the presence of nothing, or expression vectors for translin or SF-1. The SF-1-responsive elements are the –84/–55 region of the rat P450c17 gene, the –87/–68 region of the rat P450aro gene, and the –95/–58 region of the mouse P450c21 gene. These are compared with the –447/–419 region of the rat P450c17 gene. B, A p36Luc construct containing two –84/–55 SF-1 elements was used as reporter in cell transfection assays, as described in A. C, A p36Luc construct containing five mouse P450c21 elements (–95/–58) was used as a reporter in cell transfection assays, as described in A. Transfected cells were assayed 48 h after transfection. Transfection efficiencies were controlled using the dual Renilla/firefly luciferase assay system. Results are means ± SE from triplicate wells from three experiments.

We also used a concatamer of the P450c21 element containing five repeated SF-1-responsive elements, which had been used originally to demonstrate SF-1-dependent transcription (65, 73). This DNA was cloned into p36Luc, and transfected into HeLa cells. SF-1 stimulated luciferase activity 200-fold, substantially greater than the 4-fold stimulation obtained when a single SF-1 site was used. Furthermore, whereas translin alone had no effect, cotransfection of translin and SF-1 expression vectors further augmented SF-1-stimulated transcription 2.5-fold. Therefore, translin appears to discriminate among SF-1-response elements, and only activates transcription from a subset of these elements.

Translin-Associated Factor X (TRAX) Also Augments SF-1-Dependent Transcription
TRAX is a 33-kDa protein that is 28% identical with translin and has been shown to interact with translin (66, 74, 75, 76, 77, 78). Both translin and TRAX have putative leucine zipper regions, found in the carboxyl terminus of translin and in the mid-region of TRAX (41, 46). Although these two proteins are known to interact, TRAX, unlike translin (79), does not form homomultimers. TRAX alone does not bind directly to nucleic acids, although heterodimers of translin and TRAX can bind to DNA (76). Although we did not identify TRAX in our MS/MS analysis of StF-IT-2, we determined whether TRAX was found in MA-10 cells, whether TRAX could also be part of the StF-IT-2:DNA complex, and whether TRAX could augment transcription.

To determine whether TRAX was part of the StF-IT-2:DNA complex, the –447/–419 oligonucleotide probe was incubated with MA-10 cell extracts in the presence of anti-TRAX antibodies (Fig. 9A). TRAX antibodies, but not preimmune serum, supershifted the protein:DNA complex that was identified as StF-IT-2, in a manner similar to that seen when the translin antibody was used (Fig. 2B). However, TRAX does not bind directly to the –447/–419 DNA. Unlike translin, which binds to the –447/–419 DNA, recombinant TRAX prepared in bacteria does not bind to this DNA (Fig. 9B). Thus, because the TRAX antibody could supershift the StF-IT-2:DNA complex, the data suggest that translin and TRAX probably form heteromers in MA-10 cells, and these heteromers bind to DNA.

View larger version (39K): [in this window] [in a new window]   Fig. 9. TRAX Is Part of the StF-IT-2:DNA Complex A, Gel shift assay using MA-10 cell extracts, incubated with a 32P-labeled –447/–419 oligonucleotide. Addition of an antibody against TRAX (66 ) supershifts the StF-IT-2 complex, and forms another complex, StF-IT-2'. Addition of preimmune serum has no effect. B, Gel shift assay using recombinant TRAX and translin prepared in bacteria. Recombinant TRAX or translin were incubated with a 32P-labeled –447/–419 rP450c17 double-stranded (DS) or single-stranded (SS) oligonucleotide probe; TRAX does not bind to DNA because there is no protein:DNA complex, whereas translin prepared in a similar fashion binds to the –447/–419 oligonucleotide. C, Luciferase activity in HeLa cells. HeLa cells were transfected with the –447/–419 p36Luc reporter construct together with expression vectors for translin, TRAX, SF-1, or a combination of the three vectors. Transfections with translin or TRAX alone contained 0.15 µg of plasmid; transfections with both translin and TRAX contained either 0.075 µg of each plasmid [+SF-1/(Trans/Trax)/2], or 0.15 µg of each plasmid (+SF-1/Trans/Trax). Transfected cells were assayed 48 h after transfection. Results are means ± SE from triplicate wells from three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Translin and TRAX can now be added to the growing list of coactivators and corepressors that can regulate SF-1-mediated transcription. These include Dax-1, glucocorticoid receptor-interacting protein 1 (GRIP1), silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), SRC-1, and WT-1 (Wilms’ tumor), C/EBPß , CREB, SREBP1a, and RIP 140 (80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90). SF-1 is centrally involved in the adrenal and gonadal regulation the genes that encode steroidogenic enzymes (91, 92, 93, 94), and hence SF-1 is a key factor determining production of mineralocorticoids, glucocorticoids, and sex steroids. SF-1 also plays a central role in adrenal and gonadal differentiation, hypothalamic function, and sexual development (91, 95, 96, 97, 98, 99). Disruption of the SF-1 gene in mice results in absence of adrenal glands, gonads, and the ventromedial hypothalamic nucleus, which leads to loss of the pituitary cell types. Identifying the target genes controlled by SF-1 and the molecular partners with which it interacts to modulate expression of these genes is essential for understanding how SF-1 exerts its pleiotropic effects.

Translin is a 228-amino acid protein that has many cellular roles. It is involved in DNA rearrangements through binding to single-stranded DNA sequences at breakpoints (44), cell proliferation (50), mRNA transport and/or stabilization and translational regulation (51, 52), and as a linker protein that binds specific mRNAs to microtubules (39, 53). Translin’s interactions with mRNAs have been found in the nucleus, in cytoplasm, and between male germ cells, implicating translin as a protein that is involved in RNA transport between cells (52). Translin binds - and -tubulins, consistent with a role in microtubule organization, stabilization, and/or chromosome segregation. In neurons, inhibition of translin results in a lack of mRNA sorting for those mRNAs bound by translin, including ligatin and calcium- or calmodulin-dependent protein kinase II (100). The role of nuclear receptor coactivators in RNA processing, as well as in a variety of other activities has recently been reviewed (101). Several splicing-related coactivators such as p68, p72, CAPER, and PGC-1, have RNA binding motifs. One unique coactivator, SRA, is an RNA that may be important structurally for coactivation (102). Because translin is an RNA binding protein, it may likewise act as a coactivator of SF-1 through RNA binding.

The domains of SF-1 that interact with DNA and with other proteins have been identified (69, 80, 81, 84, 91, 103, 104). As with other members of the nuclear hormone receptor family of transcription factors, SF-1 contains a zinc finger motif that is responsible for DNA binding. Immediately downstream from the zinc-finger motif lies a basic region (termed the Ftz-F1 box) that facilitates binding of SF-1 to specific DNA sequences, and which is similar to the A box seen in other nuclear hormone receptors. SF-1 also contains an LBD at the C terminus. Recently, potential phospholipid ligands for SF-1 have been identified through crystallization studies (105, 106, 107). The LBD is usually the dimerization site for those nuclear hormone receptors that act as homodimers or heterodimers, but SF-1 acts as a monomer (104). Transactivation by SF-1 appears to depend on a region in the LBD, termed the AF2 region, and a region consisting of the Ftz-F1 box and an adjacent proline cluster (103). However, our previous data suggest that transactivation by SF-1 within the –447/–419 region of the P450c17 gene does not require the AF2 region (35). In addition to the AF2 region, an N-terminal AF1 region crucial for ligand-independent transactivation by other nuclear hormone receptors is needed for maximal SF-1-mediated transcription and interaction with nuclear receptor cofactors (103). This interaction requires phosphorylation of serine Ser 203 located in the hinge region (69). Our GST-pulldown assays and SF-1 deletional mutants suggest that translin interacts with the hinge region of SF-1. This region is important for stabilizing the ligand binding domain (108) and interacts with other proteins (86). Phosphorylation of Ser 203 in the hinge region enhances the interaction of GRIP1 and SMRT with the AF1 and AF2 regions of SF-1 (69), whereas sumoylation of lysines within the hinge region increase interactions with DEAD-box proteins, and result in transcriptional repression (85, 86). Mutation of serine 203 in our studies did not affect translin’s ability to stimulate SF-1-mediated transcription, indicating that the mechanism by which translin augments SF-1’s activity is different from that of other coactivators like GRIP1 and SMRT.

The mechanism by which translin augments SF-1-stimulated transcription is unclear, but it appears to depend on the DNA sequence to which SF-1 binds. Our previous data suggested that there are sequence-specific differences in SF-1 binding to DNA, and we could assess these differences by the degree to which SF-1 could bend DNA (35). Furthermore, the different putative ligands recently identified for SF-1 may play distinct roles in SF-1 regulation of gene expression, and may also determine the nature of the coactivators/corepressors that interact with SF-1. Recent studies identifying novel ligands for SF-1 have indicated that exogenously added C12–C16 fatty acids increased interaction between SF-1 and TIF2 coactivators (106). Such ligand-dependent differences in coactivator/corepressor recruitment have also been demonstrated for other nuclear hormone receptors (109, 110, 111, 112, 113). Thus, translin may discriminate between the SF-1:DNA interactions that may be mediated by different ligands.

The role of translin in the SF-1-mediated transcription of the rat P450c17 gene is not known. Mice in which the translin gene has been ablated are viable, small at birth, and have behavioral and reproductive abnormalities (114). Translin knockout mice have slightly elevated concentrations of FSH and slightly reduced concentrations of LH, progressively reduced sperm counts in male mice, and reduced fertility and litter size in female mice that are not due to obvious abnormalities in reproductive organs. Plasma or gonadal steroid concentrations were not reported, but are likely to be within normal ranges, because alterations may have been reflected in abnormal gonadotropin concentrations. Gene array analysis of testis mRNA from wild-type and translin knockout mice did not detect differences in expression of steroidogenic enzyme mRNAs, suggesting that changes, if any, would be less than 2-fold (N. Hecht, unpublished data). Recent behavioral analysis of translin knockout mice indicate that these mice exhibit sex-specific differences in learning and memory, locomotor activity, anxiety-related behavior, as well as tonic-clonic seizures induced by handling (115). Some of these behavioral abnormalities may be related to alterations in monoamine neurotransmitter levels in several forebrain regions that are seen in the translin knockout mice, and may be modified by gonadal hormones.

Ablation of translin also causes reduction in TRAX protein but not mRNA, suggesting that these two proteins may stabilize one another (114, 116). By use of small interfering RNA, we were unable to reduce translin protein expression in MA-10 cells to any great extent, suggesting that translin is both abundantly expressed and relatively stable in mouse Leydig MA-10 cells (our unpublished data). Our previous results suggested that SF-1 and translin were coexpressed in the developing gonad and in mouse Leydig MA-10 cells suggesting that the –447/–419 region of the rat P45017 gene could be coactivated by both of these factors during development as well as in the adult gonad (35).

Thus, there are many coactivators and corepressors regulating SF-1 transcriptional activity in a variety of tissues, contributing to the pleiotropic effects of SF-1. Translin and TRAX are new additions to this family.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Preparation of Soluble Cellular and Tissue Extracts
Animal experimentation was performed in accordance with protocols and guidelines approved by the Committee for Animal Research at the University of California, San Francisco. Cellular extracts from mouse MA-10 Leydig cells and rodent tissues were prepared as previously described (13, 117). Tissues were homogenized in homogenization buffer [60 mM KCl, 15 mM NaCl, 15 mM HEPES (pH 7.8), 14 mM ß-mercaptoethanol, 0.3 M sucrose, plus proteinase inhibitors; Sigma, St. Louis, MO] using a tissue homogenizer (Fisher Scientific, Houston, TX) and kept on ice. The soluble fraction of proteins was isolated by centrifuging the homogenate at 15,000 x g for 20 min at 4 C to remove debris and mitochondria, and then centrifuging the postmitochondrial supernatant at 100,000 x g (S100) for 60 min at 4 C. The soluble supernatant fraction was collected and kept at –70 C until needed. Protein concentrations were determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL).

Purification of StF-IT-2
The S100 fraction was adjusted to 0.5 M ammonium sulfate by adding solid ammonium sulfate under constant stirring. The high salt S100 fraction was then loaded onto a 30-ml phenyl Sepharose 6 fast flow column (Amersham Biosciences, Uppsala, Sweden), which had been previously equilibrated with 50 mM phosphate buffer containing 0.5 M ammonium sulfate (pH 7.0). The protein elution was monitored at 280 nm.

To determine which fractions contained the StF-IT-2 protein, gel shift assays were performed as described previously (13). Fractions containing StF-IT-2 were then pooled and further purified on a 1 ml MonoQ column (Amersham Biosciences). Before loading, the pooled fractions were dialyzed against 10 mM Tris buffer (pH 8.0), and the column was equilibrated in the same buffer. Increasing NaCl concentrations linearly to 0.4 M, in 20 bed volumes of 10 mM Tris (pH 8.0), eluted retained proteins. Eluted proteins were detected at 280 nm. Fractions were analyzed for StF-IT-2 by gel shift assays. Eluted proteins were also characterized by separation on a 10% polyacrylamide gel by electrophoresis followed by a Coomassie blue staining. The approximately 30-kDa proteins from fraction 29 were excised from the polyacrylamide gel, and sent to the Proteomic Core Facility at The Rockefeller University (New York, NY) for analysis. Protein from this band was treated with trypsin, purified by HPLC, and analyzed by MS/MS.

Gel Shift Assays
Gel shift assays were performed as described previously (13). Oligonucleotides corresponding to the –447/–419 region of the rat P450c17 gene promoter and mutants of this region (12) were used in these studies, and are presented in Table 2. Sense and antisense oligonucleotides were mixed in a 1:1 molar ratio, boiled, and reannealed slowly at room temperature before end-labeling using [-³²P]ATP and T4 polynucleotide kinase, or labeling with [-³²P]dCTP and Klenow DNA polymerase, and mixed with the cellular extracts (150,000 cpm/reaction) in the presence of 100 µg/ml polydeoxyinosinic deoxycytidylic acid, 50 µg/ml salmon sperm DNA, 5 mM dithiothreitol, and 1 mg/ml BSA, and incubated at room temperature for 45 min. For antibody supershift assays, 1–3 µl of antiserum was added to the gel shift reaction mix (75, 118, 119). Antisera were affinity-purified polyclonal rabbit antibodies directed against recombinant mouse TB-RBP (75) or were polyclonal rabbit antibodies directed against the C-terminal amino acids of translin or TRAX (118, 119), or commercially available antibodies against 14-3-3 proteins (Santa Cruz Biotechnology, Santa Cruz, CA). One third of the total reaction was loaded onto a 6% nondenaturing polyacrylamide gel and run in 0.5x Tris-borate-EDTA as running buffer, to separate the free labeled probe from probe bound by protein. The gel was then dried and exposed to a phosphorimager screen or x-ray film at –70 C using an intensifying screen.

Transient Transfections and Reporter Gene Assay
Luciferase reporter constructs were prepared using the minimal mouse prolactin promoter linked to the firefly luciferase gene in the vector p36Luc (64). Double-stranded oligonucleotides were cloned into this vector using BamHI restriction sites. All clones were sequenced to ensure proper orientation and to ensure that only a single copy of each oligonucleotide was cloned into the vector. Cells were transfected with the reporter gene constructs containing wild-type or mutant –447/–419 regions using a lipofection reagent (Fugene 6; Roche Diagnostics, Indianapolis, IN) and cells were assayed for luciferase activity 48 h later. All transfections used pRLTK-Renilla luciferase as internal standard for transfection efficiency. Firefly and Renilla luciferases were assayed using a dual-luciferase reagent and protocol according to manufacturer’s instructions (Promega, Madison, WI).

Preparation of Translin and TRAX
Mouse translin (40) was cloned into the pTarget mammalian expression vector (Promega). Mouse TRAX cDNA was obtained by RT-PCR amplification of MA-10 cell RNA, and was cloned into the pCDNA3.1 mammalian expression vector for use in transfections (Invitrogen, Carlsbad, CA). These cDNAs were also used to create recombinant protein using an in vitro transcription/translation system (Promega). These cDNAs were also cloned in the EcoRI/BamHI sites of pET42A (Novagen, Madison, WI; emdbiosciences.com) to obtain recombinant protein in bacteria, and to obtain GST-fusion proteins. Bacterially expressed GST-fusion proteins were purified on a glutathione-Sepharose 4B matrix. The GST was cleaved from the fusion protein with Factor Xa (Promega), and protein was eluted from the matrix.

Glutathione-Sepharose 4B Column Chromatography
GST-translin and GST-TRAX were created in pET42A, an N-terminal GST-fusion vector. Recombinant protein was prepared from BL21DE3pLysS bacteria that were transfected with the fusion proteins; proteins were induced by isopropyl ß-D-thiogalactoside treatment of bacteria for 3 h. After induction, bacteria were precipitated, resuspended in 300 µl PBS, and sonicated to lyse cells, and the lysate was cleared by centrifugation at 10,000 x g. The supernatant was then mixed with glutathione-Sepharose 4B matrix. Protein was bound in PBS containing 0.5% Triton X-100, for 30 min at room temperature, and the matrix was washed five times in binding buffer containing 10% glycerol.

For pull-down assays, proteins were labeled in vitro with [³⁵S]methionine using a transcription/translation system, and then added to the GST-protein matrix, incubated 1 h, and washed five times with binding buffer to remove nonspecific binding. Specifically bound proteins were resuspended in SDS gel loading buffer, boiled 5 min, separated on 12% SDS polyacrylamide gels, and exposed to x-ray film and intensifying screens at –70 C overnight.

Preparation of Full-Length and Fragments of Mouse SF-1
Full-length mouse SF-1 cDNA (65) was cloned into the EcoRI site of pBlueBacHis2B, and were used to infect Sf9 insect cells at a multiplicity of infection of 10:1. Cells were grown at 27.5 C for 36–48 h, sonicated, and recombinant protein containing a 6-His-tag at the amino terminus was purified over a cobalt affinity resin (TALON column; Clontech, Palo Alto, CA).

Full-length mouse SF-1 cDNA was also cloned into the EcoRI/BamH1 site of pCDNA3.1 (Invitrogen). Plasmid DNA was used in a coupled transcription/translation reaction (Promega) to generate recombinant protein, or was used directly as a eukaryotic expression plasmid in cell transfections. Fragments of SF-1 protein were created by PCR amplification of full-length mouse SF-1 cDNA. The oligonucleotides used to create these peptides and internal SF-1 mutants are listed in Table 2. SF-1AF2 was created by site-directed mutagenesis (QuikChange; Promega), mutating amino acid 451 from Asn to a stop codon. Serine 203 mutant was also created by changing the serine to an alanine by site-directed mutagenesis

	ACKNOWLEDGMENTS

 
We thank Dr. Holly Ingraham (University of California, San Francisco, CA) for the antibody against SF-1, Dr. Jay M. Baraban (Johns Hopkins University, Baltimore, MD) for antibodies against translin and TRAX, and Dr. Keith Parker (University of Texas Southwestern, Dallas, TX) for the pCMV-SF-1 expression vector. We also thank Arpi Siyahian and Yesmina Zavala for assisting with the transcriptional analyses.

	FOOTNOTES

 
This work was supported by National Institutes of Health (NIH) Grant HD27970 (S.H.M.). P.B. was supported by Pediatric Endocrinology NIH Training Grant T32 DK07161.

Present address for P.B.B.: IBM Healthcare and Life Sciences, Research Triangle Park, North Carolina 27709.

S.H.M., S.R.B., C.D., J.-L.V., N.B.H., and P.B.B. have nothing to declare.

First Published Online October 19, 2006

Abbreviations: AF2, Activation function-2; GRIP1, glucocorticoid receptor-interacting protein 1; GST, glutathione-S-transferase; LBD, ligand binding domain; MS/MS, tandem mass spectrometry; SF-1, steroidogenic factor-1; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; StF-IT-1, steroidogenic factor-inducer of transcription-1; TB-RBP, testis brain RNA-binding protein; TRAX, translin-associated factor X.

Received for publication August 31, 2005. Accepted for publication October 12, 2006.

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