SF-1 and Dax-1 bind to SRA.
TRα1 binds to SRA via a novel RNA binding domain, designated C1C2 (72
), distal from the zinc fingers. A sequence alignment revealed modest similarity between TRα1C1C2 and SF-1 amino acids 69 to 113 (“C1C2”), which lie in a region distal from the SF-1 zinc fingers and include the so-called FTZ-F1 box (Fig. ). Due to this similarity, we tested whether SF-1 also binds to SRA. Indeed, we found that GST-SF-1 binds [32
P]SRA in vitro at least as well as TRα1 does (Fig. ). However, the deletion of the putative “C1C2” region of SF-1 (generating SF-1Δ“C1C2”) reduced SRA binding by only 30%, indicating that this domain does not fully account for the SF-1 interaction with SRA. We therefore used a series of SF-1 deletion and truncation mutants to map the SF-1 RNA binding domain. As shown in Fig. , the SF-1 ligand binding domain (amino acids 219 to 462) is not involved in SRA binding because this domain by itself has almost no binding activity (Fig. , bar 3) and the deletion of this domain from SF-1 (generating ΔLBD) does not impair SRA binding (Fig. , bar 4). A further C-terminal truncation of the ΔLBD construct so that it extends only through the DNA binding domain and FTZ-F1 box (amino acids 1 to 128, in construct ΔLBDHP) eliminated SRA binding (Fig. , bar 5), suggesting that the RNA binding domain may lie between the FTZ-F1 box and the ligand binding domain (amino acids 129 to 218). However, this possibility was not confirmed, as amino acids 112 to 218, designated SF-1 H1, showed very limited SRA binding (Fig. , bar 6). Extending this construct in the N-terminal direction to include the FTZ-F1 box (producing the SF-1 H2 construct, amino acids 79 to 218) fully restored SRA binding (Fig. , bar 7), and further truncation at the C terminus to produce the SF-1 H3 construct, amino acids 79 to 193, also preserved SRA binding (Fig. , bar 8). In addition, the deletion of the H3 domain (generating SF-1ΔH3) eliminated SRA binding (Fig. , bar 9). Thus, the SF-1 SRA binding domain encompasses residues 79 to 193 and includes the FTZ-F1 box. This domain overlaps with but is larger than the homologous 41-amino-acid RNA binding domain of TRα1.
RNA binding proteins usually bind different RNA homopolymers with different affinities (61
). We therefore tested the ability of nonradiolabeled poly(A), poly(C), poly(G), or poly(U) to compete with radiolabeled SRA for binding to SF-1 (Fig. ). The data indicate that poly(U) competed most strongly and that poly(A) showed essentially no competition, suggesting that SF-1 may have a preference for U-rich sequences and no binding to A-rich sequences within a target RNA.
Dax-1 is also an RNA binding protein (35
), and given that its RNA targets are not well defined, we investigated whether Dax-1 might bind to SRA. Indeed, GST-Dax-1 binds [32
P]SRA (Fig. , bar 2), and the N-terminal three-and-a-half repeat region (designated N3R) (Fig. , bar 3) is largely responsible for its RNA binding since the ligand binding domain has virtually no RNA binding activity (Fig. , bar 4). Recent data show that Dax-1 N3R contains LXXLL motifs that bind to SF-1 (59
). To test if the LXXLL motifs also play a role in binding to SRA, in vitro RNA binding assays were performed with GST-Dax-1 proteins harboring deletions of these motifs. The specific deletion of any of the three N3R LXXLL motifs or the deletion of all three (without altering the other N3R amino acids) did not diminish the interaction of Dax-1 with SRA, indicating that the LXXLL sequences are not required for the Dax-1-SRA interaction (data not shown). In addition, the deletion of the Dax-1 AF2 domain amino acids 463 to 468 (generating Dax-1 ΔAF2) and the naturally occurring AHC mutations R269P and ΔV271 did not impair SRA binding (Fig. , bars 5 to 7), indicating that these mutations within the Dax-1 ligand binding domain do not have secondary effects on the interaction of N3R with SRA.
SF-1 coactivation properties of SRA, Dax-1, and Dax-1 mutant proteins.
SRA was initially identified as an RNA coactivator for steroid receptor transactivation and was shown to function in a p160 family coactivator complex to enhance target gene transcription (36
). These observations and the findings that SF-1 and Dax-1 bind to SRA in vitro prompted us to investigate the functional relevance of SF-1-SRA-Dax-1 interactions in steroidogenic gene transcription. To this end, JEG-3 cells, which do not express endogenous SF-1, were transfected with the SF-1-targeted promoter of the ACTH receptor (Mc2R) linked to a luciferase gene (the Mc2R-luc construct). Transfection was performed either with or without SF-1, SRA, and increasing doses of wild-type or mutant Dax-1. As shown in Fig. , bars 1 to 8, neither SRA nor Dax-1 affected luciferase expression in the absence of cotransfection with SF-1. SF-1 induced luciferase activity ~2-fold (Fig. , bar 9 versus bar 1), an effect that was marginally inhibited by low-dose Dax-1 (from 10 ng of the Dax-1 expression plasmid) (Fig. , bar 10 versus bar 9). Surprisingly, high-dose Dax-1 (from 100 ng of plasmid) significantly increased luciferase activity above the level seen with SF-1 alone (Fig. , bar 12 versus bar 9). The deletion mutant N3R and the AHC mutants R269P and N422I displayed severely reduced coactivation (Fig. , bar 12 versus bars 25, 31, and 43), although Dax-1 LBD and the AHC mutant ΔV271 retained modest coactivation (Fig. , bar 12 versus bars 19 and 37). The cotransfection of cells with SRA and wild-type Dax-1 vectors further stimulated luciferase expression (Fig. , bar 16 versus bar 12). (The effect of SRA overexpression is not as strong as the effect of the knockdown of endogenous SRA, depicted subsequently in Fig. and .) However, SRA in combination with the Dax-1 deletion mutant N3R or AHC mutants R269P and N441I was severely defective in reporter gene transactivation (Fig. , bar 16 versus bars 28, 34, and 46), whereas SRA in combination with Dax-1 LBD or ΔV271 achieved modest gene coactivation, albeit less than that achieved with wild-type Dax-1 (Fig. , bar 16 versus bars 22 and 40).
FIG. 2. Dax-1, the noncoding SRA, and the p160 coactivator TIF2 coactivate SF-1-dependent transcription. (A) JEG-3 cells were transfected with Mc2R-luc and expression vectors for SF-1, SRA, Dax-1, and its deletion or AHC mutant forms (N3R, LBD, R269P, ΔV271, (more ...)
FIG. 5. The knockdown of endogenous SRA impairs the ability of Dax-1 to function as an SF-1 coactivator. (A) siRNA knockdown of endogenous SRA. JEG-3 cells were transfected with either a nontargeting control siRNA or siRNA directed against SRA. Forty-eight hours (more ...)
FIG. 8. The knockdown of endogenous SRA in Y1 cells interferes with the expression of steroidogenic genes and impairs the interaction of SF-1 and TIF2. (A) Knockdown of endogenous SRA in Y1 cells. Y1 cells were infected with a retrovirus expressing an shRNA directed (more ...)
Since SRA and SF-1 both are known to form complexes with p160 family coactivators such as TIF2, we also tested whether the coexpression of Dax-1 with TIF2 would have additive or synergistic effects on the SF-1 induction of Mc2R-luc. Neither TIF2 nor Dax-1 influenced Mc2R-luc expression in the absence of SF-1 (Fig. , bars 1 to 8). The inhibition of SF-1 transactivation was again seen with low-dose Dax-1, such that luciferase expression in the presence of TIF2 plus 10 ng of the Dax-1 expression vector was only 50% of that seen with TIF2 alone (Fig. , bar 14 versus bar 13). Conversely, the coexpression of high-dose Dax-1 and TIF2 synergistically enhanced Mc2R-luc expression up to 2.2-fold compared to that in the presence of Dax-1 alone (Fig. , bar 16 versus bar 12). Similar synergistic effects of Dax-1 and SRC-1 were also observed (data not shown). We next studied the same Dax-1 mutants used for the analysis presented in Fig. . Dax-1 LBD was essentially as active as wild-type Dax-1 in coactivation (Fig. , bar 22 versus bar 16), N3R and AHC mutant R269P displayed significantly reduced coactivation (Fig. , bars 28 and 34 versus bar 16), and ΔV271 and N442I behaved similarly to wild-type Dax-1 (Fig. , bars 40 and 46 versus bar 16). Thus, the overall effects of the Dax-1 mutants were qualitatively similar in terms of coactivation with SRA and TIF2, except for Dax-1 N422I, which showed no synergy with SRA but was fully active in conjunction with TIF2.
We also obtained synergistic effects of Dax-1 and TIF2 on the SF-1 induction of StAR-luciferase. Relative to an average baseline luciferase expression level in the presence of SF-1 of 100 ± 5, expression with Dax-1 was 105 ± 3, that with TIF2 was 201 ± 16, and that with Dax-1 plus TIF2 was 281 ± 24.
Although Dax-1 is an atypical orphan nuclear receptor, its putative ligand binding domain does contain a putative AF2 hexamer (residues 463 to 468) homologous to the AF2 domains of conventional NRs such as SF-1 (Fig. ). For some NRs such as SF-1 and TRs, the AF2 domain is required for transcriptional activation, but for others such as the androgen receptor, it plays a relatively minor role. To examine the importance of the Dax-1 AF2 domain in the coactivation of SF-1, we deleted it to create Dax-1 ΔAF2. In our standard cotransfection paradigm with SF-1, TIF2, and Mc2R-luc, Dax-1 ΔAF2 retained about 75% of the activity of wild-type Dax-1 (Fig. , bar 14 versus bar 8), indicating that the AF2 domain plays only a minor role in the Dax-1-TIF2 coactivation of SF-1.
To test whether the Dax-1 mutants were expressed at levels similar to that of wild-type Dax-1 in the above-described transfections, we subjected lysates from transfected JEG-3 cells to immunoblotting using a Dax-1 antibody (Fig. ). This study demonstrated that wild-type Dax-1, its AHC mutant forms (R269P, ΔV271, and N442I), and the ΔAF2 mutant are expressed at comparable levels. However, we could not detect the truncated proteins N3R and LBD (calculated sizes, 22 and 30 kDa). This result was likely due to the Dax-1 antibody's not recognizing N3R and LBD, since Myc epitope-tagged versions of N3R and LBD are expressed at levels similar to that of full-length Dax-1 (see Fig. ).
FIG. 6. Dax-1 is recruited to SF-1 target promoters, but the Dax-1 mutants N3R and R269P exhibit decreased promoter occupancy. (A) Expression of Dax-1-Myc and its mutant versions. Y1 cells were transfected with equal amounts of vectors expressing Myc-tagged wild-type (more ...)
TRα1 was used to test whether Dax-1 coactivation is specific for SF-1 or occurs with other nuclear receptors. Transfection with high-dose Dax-1 without or with SRA yielded no coactivation activity on T3-induced luciferase but instead showed a slight inhibitory effect (Fig. , bar 6 versus bar 2 and bar 8 versus bar 4). These data suggest that the coactivation properties of Dax-1 cannot be generalized for all nuclear receptors.
Dax-1 binds to TIF2 in vitro and in mammalian cells.
Dax-1 and TIF2 bind to each other in vitro, as shown by GST pull-down assays (Fig. ). The N-terminal fragment of Dax-1 (N3R) binds TIF2 as well as wild-type Dax-1 does, whereas Dax-1 LBD binds less well even though LBD has stronger transactivation properties than N3R in the reporter gene assays (Fig. ). In addition, Dax-1 ΔAF2 and the AHC mutants R269P and ΔV271 bind TIF2 as well as wild-type Dax-1 does.
FIG. 3. Dax-1 interacts with TIF2 in vitro and in JEG-3 cells. (A) Interaction of GST-Dax-1 and its mutant forms with 35S-TIF2. 35S-labeled in vitro-translated TIF2 was incubated with equal amounts of purified GST-Dax-1 wild-type or mutant proteins adsorbed to (more ...)
The Dax-1-TIF2 interaction was substantiated by coimmunoprecipitation from JEG-3 cells transfected with Dax-1-Myc and TIF2 expression vectors (Fig. ). The Dax-1 deletion (N3R, LBD, and ΔAF2) and AHC (R269P and ΔV271) mutants also coimmunoprecipitated with TIF2, but the interactions were relatively weak, especially for LBD, ΔAF2, and R269P. The Dax-1-TIF2 interaction in living cells was investigated further, as described below.
Abnormal intracellular localization of Dax-1 mutants interacting with TIF2.
We utilized BiFC (22
) in transfected COS-1 cells to confirm the interaction of Dax-1 with TIF2 and to visualize the locations of these proteins in living cells. This technique is based on the reconstitution of YFP from nonfluorescent N-terminal and C-terminal YFP fragments when they are brought together by two interacting proteins fused to the fragments. Recently, this method has been modified to be more specific and sensitive by using two fragments (VN and VC) from the optimized YFP variant Venus (57
). Previous studies have reported that Dax-1 localizes both to the nucleus and to the cytoplasm (20
) and that TIF2 is almost exclusively nuclear (8
). Before the BiFC analysis, we assessed the subcellular localization patterns of the individual wild-type and deletion or AHC mutant Dax-1 proteins expressed from the VC vector (encoding a HA tag) and TIF2 expressed from the VN vector (encoding a Flag tag). Figure shows that Dax-1-VC fluorescence was detected mostly in the cytoplasm, although minor expression in the nucleus was also observed. However, the deletion mutant N3R-VC was localized mainly in the nucleus, LBD-VC was distributed mostly outside of but adjacent to the nucleus, and the AHC mutant R269P-VC was diffusely localized in the cytoplasm. As expected, TIF2-VN was mostly nuclear, although occasionally minor cytoplasmic expression of TIF2-VN was observed (data not shown).
FIG. 4. Mutant Dax-1-TIF2 complexes mislocalize in the cytoplasm in living cells as shown by BiFC analysis. (A) Subcellular localization patterns of Dax-1 or its mutant forms and TIF2. COS-1 cells were transfected with 100 ng of either a plasmid encoding Dax-1 (more ...)
The BiFC analysis is shown in Fig. . The coexpression of Dax-1-VC and TIF2-VN resulted in exclusively nuclear fluorescence, indicating that the interaction of Dax-1 and TIF2 occurs in the nucleus and perhaps suggesting that TIF2 recruits Dax-1 to or stabilizes it in this compartment. In contrast, the majority of fluorescence induced by the interaction of N3R-VC and TIF2-VN was cytoplasmic (in 70% of cells), although 30% of the cells showed both nuclear and cytoplasmic fluorescent foci. The interaction of AHC mutant R269P-VC with TIF2-VN was exclusively cytoplasmic, and no or very weak fluorescence complementation was observed in cells coexpressing Dax-1 LBD-VC and TIF2-VN. No fluorescence was observed in control cells cotransfected with Dax-1-VC and empty VN vectors or empty VC and TIF2-VN vectors. Overall, these results suggest that the Dax-1-TIF2 interaction helps maintain Dax-1 in the nucleus and that this localization requires both the N- and C-terminal portions of the Dax-1 protein. Furthermore, the interactions of N3R and the AHC mutant R269P with TIF2 mislocalize to the cytoplasm, consistent with the poor transcriptional activity of these Dax-1 mutants in the luciferase reporter assay (Fig. ).
Dax-1 transactivation is dependent on SRA.
The observations that Dax-1 binds to SRA and that exogenous SRA enhances the Dax-1 induction of Mc2R-luc raise the question of whether the transactivation properties of Dax-1 depend on endogenous SRA. To test this possibility, we examined the effect of the knockdown of endogenous SRA on Dax-1 induction of Mc2R-luc with or without the cotransfection of cells with p160 coactivators. As shown in Fig. , endogenous SRA was successfully silenced by 70%. The transactivation of Mc2R-luc by Dax-1 was abolished by the knockdown of endogenous SRA (Fig. , bar 4 versus bar 2). Furthermore, the transactivation by coexpressed Dax-1 and TIF2 was nearly abolished after endogenous SRA had been silenced (Fig. , bar 6 versus bar 3). Similar results were obtained when SRC-1 was used in place of TIF2 (data not shown). These observations support the hypothesis that SRA exists in a complex with p160 coactivators and Dax-1 to enhance target gene activation by SF-1.
Dax-1 is recruited to the Mc2R and StAR gene promoters, but the recruitment of N3R and AHC mutant R269P is impaired.
The finding that Dax-1 has the capacity to transactivate Mc2R-luc suggests that Dax-1 may function as a transcriptional activator of genes like the Mc2R gene in steroid hormone metabolism at least in some cellular contexts, which is opposite the conventional model of Dax-1 as a transcriptional repressor (18
). To address this possibility, Myc-tagged wild-type Dax-1 and its deletion or AHC mutant forms (N3R, LBD, and R269P) were transiently expressed at similar levels in Y1 adrenocortical cells (Fig. ). ChIP experiments were performed, confirming that wild-type Dax-1-Myc is recruited to SF-1 binding sites of the endogenous Mc2R and StAR promoters (Fig. , bars 2 and 3). As negative controls, similar real-time PCRs were performed using exonic primers for Mc2R and StAR genes, neither of which yielded significant amplification (Fig. , bars 4 and 5). Dax-1 mutants N3R and R269P were recruited less well than wild-type Dax-1 to the Mc2R promoter (Fig. ), which may explain their deficiencies as SF-1 coactivators (Fig. ). These ChIP data are also consistent with the extranuclear mislocalization of R269P and N3R complexes with TIF2 observed by BiFC (Fig. ). Interestingly, the ChIP data also show that Dax-1 LBD was efficiently recruited to the Mc2R promoter (Fig. ), consistent with its transactivation properties in the Mc2R-luc reporter gene assay (Fig. ).
SRA regulates the transcription of endogenous steroidogenic genes in Y1 mouse adrenocortical cells.
The observation that SRA can function as an SF-1 coactivator by directly binding to SF-1 and Dax-1 suggests that SRA may regulate the expression of genes involved in steroidogenesis. To begin to test this hypothesis, we first asked whether endogenous SRA might be associated with SF-1 and Dax-1 in Y1 mouse adrenocortical cells. To examine this possibility, Y1 cells (which do not express endogenous Dax-1) were transfected with an empty Myc vector or a Dax-1-Myc vector and the cell extracts were immunoprecipitated with anti-Myc agarose beads. In parallel, Y1 cell extracts were immunoprecipitated with either anti-SF-1 antibody or normal IgG as a negative control. To eliminate the potential for contaminating genomic DNA, the immunoprecipitated materials were treated with DNase I before being reverse transcribed and analyzed by real-time PCR for SRA (and for β-actin as a negative control). The results indicate that endogenous SRA coimmunoprecipitates with both Dax-1-Myc (Fig. ) and endogenous SF-1 (Fig. ).
FIG. 7. Dax-1 and SF-1 associate with endogenous SRA in Y1 cells. (A) Dax-1-Myc associates with endogenous SRA in Y1 cells. Y1 cells were transfected with an empty Myc vector (Myc-Vec) or a Dax-1-Myc expression vector. The cell lysates were immunoprecipitated (more ...)
We next asked whether the knockdown of endogenous SRA would interfere with the expression of steroidogenic genes. As shown in Fig. , we efficiently knocked down endogenous SRA in Y1 cells by using a retroviral shRNA, as opposed to a scrambled control shRNA. As shown in Fig. , StAR mRNA expression was induced 3.5-fold by a 3-h exposure to ACTH (Fig. , bar 2 versus bar 1), and this induction was decreased by 37% with SRA knockdown (Fig. , bar 4 versus bar 2). Similar data were obtained after 8 h of exposure to ACTH: ACTH induced StAR mRNA expression 2.7-fold (Fig. , bar 6 versus bar 5), and this induction was decreased by 25% by SRA knockdown (Fig. , bar 8 versus bar 6). StAR protein expression, normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression, also decreased by 40% at 8 h of ACTH treatment (Fig. ).
In contrast to the expression of StAR mRNA, the expression of Mc2R mRNA in Y1 cells did not increase with exposure to ACTH (Fig. ). However, the knockdown of SRA inhibited Mc2R mRNA expression by about 40%, and a modest inhibition of Mc2R protein expression, normalized to GAPDH expression, was also observed (Fig. ). These results indicate that SRA plays a positive role in the expression of the steroidogenic genes for Mc2R and StAR in Y1 adrenocortical cells.
Since the data in Fig. to suggest that SRA forms a complex with p160 coactivators such as TIF2 when inducing the expression of SF-1 target promoters, we asked whether the physical interaction of TIF2 with SF-1 is dependent on SRA. To investigate this issue, we immunoprecipitated SF-1 from Y1 SRA knockdown and control cells and then immunoblotted the precipitates for endogenous TIF2. TIF2 coimmunoprecipitated with SF-1 from control cells but not from SRA knockdown cells (Fig. , upper panel). Since the knockdown of SRA did not influence TIF2 expression (Fig. , lower panel), the data support the hypothesis that SRA facilitates the interaction of SF-1 with TIF2.
SRA is expressed in mouse adrenal glands and testes.
Although SF-1 and Dax-1 are known to be coexpressed in adrenal and gonadal cells, the expression of SRA in the adrenal gland and gonads has not been reported previously. We found that SRA is expressed at a much higher level in mouse adrenals and testes than in the liver (Fig. ), which supports the hypothesis that SRA may be a novel regulator of steroidogenic gene expression in vivo, in coordination with SF-1 and Dax-1.
FIG. 9. Expression levels of SRA and Dax-1 mRNAs in mouse adrenal, testis, and liver tissues. Adrenal, testis, and liver RNAs were isolated from 18-week-old male mice (n = 4). Real-time RT-PCR analysis of SRA, Dax-1, and β-actin for normalization (more ...) Knockdown of endogenous Dax-1 impairs the expression of a subset of steroidogenic genes.
Since the human adrenocortical cell line H295R (53
) and the mouse Leydig tumor cell line MA-10 (4
) express Dax-1, SF-1, and steroidogenic enzymes, these cell lines were used in Dax-1 knockdown experiments to test whether endogenous Dax-1 might function as a coactivator for the expression of steroidogenic genes. Endogenous Dax-1 was efficiently knocked down by shRNA at both the mRNA and protein levels in H295R and MA-10 cells (Fig. ). Although exogenous Dax-1 has been shown to inhibit SF-1-mediated transactivation of steroidogenic genes (24
), we found that, in both H295R and MA-10 cells, the knockdown of Dax-1 inhibited the expression of the steroidogenic genes for CYP11A1 (Fig. and ) and StAR (Fig. and ) at both the mRNA and protein levels (StAR was not detectable in MA-10 cells). In addition, Mc2R mRNA expression was downregulated by Dax-1 silencing in H295R cells (Fig. ). However, the knockdown of Dax-1 had no effect on CYP17A1 mRNA expression in either H295R or MA-10 cells (Fig. ), nor did it affect SF-1 expression (data not shown). These results suggest that endogenous Dax-1 can function as a coactivator to increase the expression of a subset of steroidogenic genes in adrenal and gonadal cells, thus supporting our transfection data that indicate that Dax-1 can function as a coactivator in addition to its previously described role as a corepressor.
FIG. 10. The knockdown of endogenous Dax-1 in H295R cells (A to E) and MA-10 cells (F to I) inhibits the expression of steroidogenic genes. (A) H295R cells were infected with lentivirus expressing an shRNA directed against human Dax-1 (hDax-1) or a scrambled-sequence (more ...)