Role of the LXXLL-Motif and Activation Function 2 Domain in Subcellular Localization of Dax-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1)
Kaname Kawajiri,
Togo Ikuta,
Taiga Suzuki,
Masatomo Kusaka,
Masami Muramatsu,
Kenji Fujieda,
Masayoshi Tachibana and
Ken-ichirou Morohashi
Research Institute (K.K., T.I., M.T.), Saitama Cancer Center, Ina-machi, Kita-adachi, Saitama 362-0806, Japan; Core Research for Evolutional Science and Technology (CREST) (K.K., T.S., K.-I.M.), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan; Department of Developmental Biology (T.S., M.K., K.-I.M.), National Institute for Basic Biology, and Department of Molecular Biomechanics (M.K., K.-I.M.), School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan; Department of Biochemistry (M.M.), Saitama Medical School, Moroyama-machi, Iruma-gun, Saitama 350-0451, Japan; and Department of Pediatrics (K.F.), Asahikawa Medical College, Asahikawa 078-8510, Japan
Address all correspondence and requests for reprints to: Professor Ken-ichirou Morohashi, Ph.D., Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan. E-mail: moro{at}nibb.ac.jp.
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ABSTRACT
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Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax-1, NR0B1) is an orphan nuclear receptor that represses transcription by Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1, NR5A1). Observations on human diseases and the phenotypes of mice, in which the corresponding genes have been disrupted, have elucidated essential roles of these two nuclear receptors in differentiation of steroidogenic tissues. However, little is known about how the functions of these factors are regulated. Here we have examined their subcellular localization and have clarified the molecular mechanisms regulating subcellular localization of Dax-1. Prompted by the finding that nuclear localization of Dax-1 correlates with the presence of Ad4BP/SF-1 in the early stages of pituitary development, we have tested the possibility that interaction between the two factors is essential for the nuclear localization of Dax-1. In vitro studies with cultured cells demonstrated that an interaction involving the LXXLL motifs in the N-terminal repeat region of Dax-1 plays a key role in its subcellular localization. In addition, we found that a mutant form of DAX-1 (L466R), from a patient with adrenal hypoplasia congenita, was defective in nuclear localization in spite of having an intact N terminus. Taken together, the results reveal that the subcellular localization of Dax-1 is influenced by the presence of Ad4BP/SF-1, and that two regions of Dax-1 have important roles for this process.
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INTRODUCTION
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DAX-1 (NR0B1) (1) [dosage-sensitive sex reversal-adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] has been positionally cloned from the dosage-sensitive sex reversal and AHC region located at Xp21 (2, 3). Structurally, DAX-1/Dax-1 is related to nuclear receptors (3, 4). The C terminus of Dax-1 contains the ligand binding domain (LBD) common to all members of the nuclear receptor superfamily. However, its N-terminal half lacks a Zn-finger domain; instead, it is composed of three short repeats each containing an LXXLL-related motif (NR box) (5). This motif was originally identified in nuclear receptor coactivators and is critical for interaction with nuclear receptors (6, 7, 8, 9).
Dax-1 is predominantly expressed in the adrenal cortex, gonads, hypothalamus, pituitary, and prostate (4, 10, 11, 12, 13), and its expression profile is quite similar to that of Ad4BP/SF-1 (NR5A1) (1) [Ad4 binding protein (14)/steroidogenic factor 1 (15)]. All the tissues except the prostate are affected in mice deficient in the Ad4BP/SF-1 gene (16, 17, 18, 19), and functional and structural failures have been reported in many of these tissues in AHC patients (2, 3). These findings have led to the view that the two nuclear receptors cooperate in gonadal and adrenocortical differentiation. Indeed, it is well known that Dax-1 interacts with Ad4BP/SF-1 and represses its transcriptional activity (20, 21, 22, 23, 24). Such transcriptional interactions between the two factors require their simultaneous presence in the nucleus, and, as anticipated, their colocalization has been revealed by immunohistochemical analyses of the appropriate tissues (12, 25, 26). In agreement with the in vivo observation, an in vitro study with cultured cells showed that nuclear localization of Ad4BP/SF-1 is driven by a typical nuclear localization signal (NLS) (26). In contrast, Dax-1 has been shown to be present in the cytoplasm as well as the nucleus (13, 23, 27), reflecting its ability to shuttle between the two compartments (27). However, the molecular mechanism underlying nuclear import and export of Dax-1 remains to be clarified.
In general, nuclear import of proteins larger than 4060 kDa requires a specific targeting sequence designated a NLS, which is characterized by a single stretch or a bipartite sequence of basic amino acids (28, 29). NLS sequences are recognized by cargoes (importins), and the resulting complexes are transported into the nucleus via a nuclear pore complex. Nuclear entry of transcription factors such as nuclear receptors is regulated by a variety of mechanisms, which orchestrate a substantial repertoire of gene expression (30).
In the present study, we provide evidence that subcellular localization of Dax-1 is influenced by manners dependent on its LXXLL motifs and the activation function 2 (AF2) core. Even though each LXXLL motif in Dax-1 has the potential to interact with and thus import with Ad4BP/SF-1 into the nucleus, one of the LXXLL motifs is especially involved in its subcellular localization. The AF2 core in Dax-1 also contributes to its subcellular localization, probably through a distinct mechanism.
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RESULTS
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Subcellular Localization of Dax-1 in Association with Ad4BP/SF-1
Our immunohistochemical studies using developing pituitaries yielded an unusual Dax-1 staining pattern: Dax-1 was distributed diffusely in both nucleus and cytoplasm in all cells of the Rathkes pouch at embryonic day 10.5 (E10.5) (Fig. 1A
). The cytoplasmic distribution of Dax-1 was confirmed by overlay staining (Fig. 1E
) of the nucleus with 4,6-diamidino-2-phenylindole (DAPI) (Fig. 1D
) and Dax-1 with the antibody (Fig. 1C
). Another unexpected finding was that Ad4BP/SF-1 was not yet expressed at the stage of Rathkes pouch (Fig. 1B
). On the contrary, both Dax-1 (Fig. 1F
) and Ad4BP/SF-1 (Fig. 1G
) were localized in the nuclei at E18.5. The nuclear localization of Dax-1 was again confirmed by overlay staining with the antibody and DAPI (Fig. 1
, HJ). As described previously, both Dax-1 (31) and Ad4BP/SF-1 (16, 19) are expressed in gonadotrophs of the adult anterior pituitary. Because all the trophs differentiate during the fetal stage (32), it is likely that the Dax-1-immunoreactive cells at E18.5 are gonadotrophs. However, it should be noted that Dax-1, a potential gonadotroph marker, is expressed in all cells of Rathkes pouch at E10.5, before the specific trophs differentiate.

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Figure 1. Subcellular Localization of Dax-1 and Ad4BP/SF-1 in the Developing Pituitary of Mouse Fetuses
Localization of Dax-1 (A, C, F, and H) and Ad4BP/SF-1 (B and G) at E10.5 (AE) and E18.5 (FJ) was determined immunohistochemically with the specific antibodies. Dax-1 immunofluorescent staining (C and H) and nuclear DAPI staining (D and I) are merged in E and J. Bar, 20 µm in A, B, F, and G; 10 µm in C, D, E, H, I, and J.
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Because Dax-1 appears to lack a conventional NLS (3, 4), it seemed possible that its nuclear localization was achieved by interaction with Ad4BP/SF-1. To examine this possibility, the native form of Dax-1 was expressed in HeLa cells, and the cells were stained with the antibody to Dax-1. As shown in Fig. 2A-a
, transiently expressed Dax-1 was localized in diffuse fashion in both cytoplasm and nucleus. However, when Dax-1 was coexpressed with Ad4BP/SF-1 (+Ad4/SF-1), it accumulated in the nucleus (Fig. 2A
, b). Proteins of up to 4060 kDa are thought to be able to enter and leave the nucleus by passive diffusion through nuclear pore complexes (28, 29). Because the molecular mass of Dax-1 is less than 60 kDa, it was difficult to judge whether the nucleocytoplasmic distribution of Dax-1 in the absence of Ad4BP/SF-1 is regulated by an active transport or merely by a passive transport. Thus, we constructed two fusion proteins with the N terminus of Dax-1 linked to 6x[His]-Tag (His-Dax) and ß-galactosidase (ß-Gal-Dax), respectively, and thereby examined whether these fusion proteins behaved like native Dax-1. Because the 6x[His]-Tag is too small to influence nuclear translocation via the nuclear pore, whereas the molecular mass of ß-Gal is large enough (120 kDa) to prevent passage through the pore by diffusion, these two fusion proteins, in combination, have been widely used as reporters for investigating the subcellular localization of proteins. When the Dax-1 fusion proteins were expressed in the absence of Ad4BP/SF-1, they were distributed throughout both cytoplasm and nucleus (Fig. 2A
, c and e), whereas, as anticipated, they accumulated in the nucleus when coexpressed with Ad4BP/SF-1 (Fig. 2A
, d and f). These observations strongly suggested that the nucleocytoplasmic localization of Dax-1 in the absence of Ad4BP/SF-1 is due not to a passive, but to an active, transport and export (27), and that nuclear localization of Dax-1 is enhanced by Ad4BP/SF-1 efficiently.

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Figure 2. Nuclear Localization of Dax-1 in Association with Ad4BP/SF-1
A, Subcellular localization of native Dax-1, Dax-1 fused with 6x[His]-Tag, and Dax-1 fused with ß-Gal. Native Dax-1 (Dax) (a and b), Dax-1 fused with 6x[His]-Tag (His-Dax) (c and d), and Dax-1 fused with ß-Gal (ß-Gal-Dax) (e and f) were expressed in HeLa cells in the absence (a, c, and e) or presence (b, d, and f) of Ad4BP/SF-1, and subcellular localization was determined. B, Correlation of nuclear localization of Dax-1 with that of Ad4BP/SF-1. Subcellular localization of ß-Gal fusions with full-length Ad4BP/SF-1 (ß-Gal-Ad4/SF-1) (a) and its NLS region (amino acids 79126) (ß-Gal-Ad4/SF-1[NLS]) (b) was determined. ß-Gal-Dax and His-Dax were expressed in the presence of Ad4BP/SF-1[ NLS] (c and d) and the subcellular localization was determined. Subcellular localization of His-Dax was determined in the presence of Ad4BP/SF-1 containing an NLS of SV40 large T antigen (T-NLS) in place of the cognate NLS (Ad4/SF-1[T-NLS]) (e). Dax-1 containing T-NLS (His-Dax[T-NLS] was transfected and the subcellular localization was determined (f). C, Subcellular localization of Dax-1 in the presence of ER , Sox9, and GATA4. ß-Gal-Dax was cotransfected with expression vectors for ER (a), Sox9 (b), and GATA4 (c), and the subcellular localization was determined. Dax-1 is visualized with antibodies to Dax-1 (panel A, a and b) and to the His-Tag (panel A, c and d; panel B, df) or by ß-Gal activity (panel A, e and f; panel B, ac; panel C, ac).
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To confirm this, we investigated effects of an NLS in Ad4BP/SF-1 on the subcellular localization of Dax-1. Since the NLS of Ad4BP/SF-1 had been reported to be localized between the Zn-finger and the LBD (26), we constructed ß-Gal fusion carrying either full-length Ad4BP/SF-1 (ß-Gal-Ad4/SF-1) or the NLS region alone (amino acids 79126) (ß-Gal-Ad4/SF-1[NLS]). Both ß-Gal-Ad4/SF-1 and ß-Gal-Ad4/SF-1[NLS] were detected in nuclei (Fig. 2B
, a and b), whereas Ad4BP/SF-1 lacking the NLS (Ad4/SF-1[
NLS]) showed no nuclear localization (data not shown). When ß-Gal-Dax and His-Dax were coexpressed with Ad4/SF-1[
NLS], the two Dax-1 fusion proteins distributed diffusely in both nucleus and cytoplasm as expected (Fig. 2B
, c and d). To confirm that the nucleocytoplasmic distribution of Dax-1 was tightly coupled with the loss of nuclear localization of Ad4BP/SF-1[
NLS], Ad4/SF-1[T-NLS] carrying an NLS from SV40 large T antigen instead of the endogenous NLS was constructed. As shown in Fig. 2B-e
, nuclear localization of Dax-1 was rescued by Ad4/SF-1[T-NLS]. We also found that when Dax-1 was fused with the NLS of SV40 large T antigen at its N terminus (His-Dax[NLS]) it accumulated in nuclei even in the absence of Ad4BP/SF-1 (Fig. 2B
, f).
To investigate whether proteins that are structurally related to Ad4BP/SF-1 have a similar effect on the nuclear localization of Dax-1, the structurally related nuclear receptors [estrogen receptor (ER)
and ERß] were cotransfected with Dax-1. ER
(Fig. 2C
, a) and ERß (data not shown) facilitated Dax-1 nuclear localization in the presence of a ligand. However, Sox9 and GATA4, neither of which are members of the nuclear receptor superfamily, did not facilitate Dax-1 nuclear localization (Fig. 2C
, b and c).
The LXXLL Motif of Dax-1 Is Required for Nuclear Localization with Ad4BP/SF-1
To determine the region of Dax-1 responsible for nuclear import with Ad4BP/SF-1, ß-Gal fusions of truncated or mutated forms of Dax-1 were expressed in HeLa cells. As shown in Fig. 3A
, we divided Dax-1 into two portions, the N-terminal repeats (amino acids 1209) and C-terminal LBD (amino acids 210472), and investigated their potential for nuclear localization. In the presence of Ad4BP/SF-1, the N-terminal fusion protein, ß-Gal-Dax[1209], accumulated in the nucleus like full-length Dax-1 (ß-Gal-Dax). In contrast, the C-terminal fusion protein, ß-Gal-Dax[210472], did not accumulate in the nucleus. Subsequently, we investigated whether each of the repeated units of Dax-1 was able to direct nuclear localization. Three constructs, ß-Gal-Dax[169], ß-Gal-Dax[70137], and ß-Gal-Dax[138209], each containing one of the repeated units, were prepared. When transfected in the absence of Ad4BP/SF-1, they were distributed diffusely in both cytoplasm and nucleus. In the presence of Ad4BP/SF-1, staining was almost entirely nuclear in the case of ß-Gal-Dax[169] and ß-Gal-Dax[138209]. However, less complete nuclear localization was observed in the case of ß-Gal-Dax[70137] (Fig. 3B
).

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Figure 3. Contributions of LXXLL Motifs to Subcellular Localization of Dax-1
A, Subcellular localization of ß-Gal fused with full-length Dax-1 (ß-Gal-Dax), its N terminus (ß-Gal-Dax [1209]), or its C terminus (ß-Gal-Dax[210472]). ß-Gal-Dax, ß-Gal-Dax[1209], and ß-Gal-Dax[210472] were transfected into HeLa cells in the absence (-) or presence (+) of Ad4BP/SF-1 and the subcellular localization was determined. Shaded columns with arrows represent the three repeat units containing an LXXLL motif (motifs 13, indicated by solid bars). B, Subcellular localization of ß-Gal fused with each of the three repeats. ß-Gal-Dax [169], ß-Gal-Dax[70137], and ß-Gal-Dax[138209] containing the first, second, and third repeat, respectively, were transfected in the absence (-) or presence (+) of Ad4BP/SF-1, and the subcellular localization was determined. C, Subcellular localization of ß-Gal fused with wild-type (Wt) and mutant forms (Mut) of LXXLL motifs. ß-Gal-motif 1, ß-Gal-motif 2, and ß-Gal-motif 3 contain amino acids 1119, 7886, and 146154, respectively. LL and ML in the LXXLL motifs were replaced with AA in the mutant forms. These fusion proteins were expressed in the absence (-) or presence (+) of Ad4BP/SF-1 and the subcellular localization was determined. D, Subcellular localization of whole Dax-1 carrying mutations at each LXXLL motif. Mutations in the LXXLL motifs were shown in red (indicated on the left). Wt and mutant forms of Dax-1 were expressed in the presence of Ad4BP/SF-1, and the subcellular localization was determined. More than 200 cells expressing Dax-1 fused with ß-Gal were observed and were classified into two groups, one showing predominantly nuclear localization and the other either predominantly cytoplasmic localization, or equal distribution between nucleus and cytoplasm. Bars show ratios of cells showing predominantly nuclear localization. The mean ± SD values from three independent experiments are shown. *, P < 0.001 compared with the Wt. E, Interaction between the first LXXLL motif of Dax-1 and Ad4BP/SF-1. GST fusion proteins, GST-motif 1 [Wt] carrying amino acids 529 (LYSLL) of Dax-1 and GST-motif 1 [Mut] carrying the identical region of Dax-1 with two amino acids substitution (LYSAA), were synthesized in E. coli and purified. Ad4BP/SF-1 was synthesized in vitro in the presence of [35S]methionine. The radiolabeled Ad4BP/SF-1 was incubated with GST-motif 1 [Wt] or GST-motif 1 [Mut] and GST-pull-down assay was performed. Inputs (10%) of radiolabeled proteins synthesized from empty pCMX (-) or pCMV-Ad4BP/SF-1 (Ad4BP/SF-1) are shown.
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As described previously, Dax-1 has three LXXLL motifs in each N-terminal repeat, and these particular motifs are responsible for interaction with ER
and ERß (5). Moreover, we have shown recently that the LXXLL motifs in Dax-1 are also necessary for interaction with Ad4BP/SF-1 (24). It was therefore investigated whether interaction through the LXXLL motifs is involved in the nuclear localization of Dax-1. We prepared ß-Gal fusion constructs (ß-Gal-motif 1, ß-Gal-motif 2, and ß-Gal-motif 3) containing the three separate LXXLL motifs. As indicated in Fig. 3C
, ß-Gal-motif 1 and ß-Gal-motif 3 accumulated efficiently in nuclei in the presence of Ad4BP/SF-1, whereas ß-Gal-motif 2 accumulated less completely, in agreement with our observations with the whole repeats (Fig. 3B
). Furthermore, mutant forms of ß-Gal-motif 1, ß-Gal-motif 2, and ß-Gal-motif 3, in which the last two leucine (or methionine) residues of the motifs [LXXL(M)L] were replaced by alanine (LXXAA), failed to accumulate in the nucleus even when cotransfected with Ad4BP/SF-1.
The function of the LXXLL motifs was further investigated in the whole Dax-1 molecule. We replaced each LXXLL motif by LXXAA and examined the ability of the resulting constructs to become concentrated in the nucleus (Fig. 3D
). The numbers of cells expressing these forms of Dax-1 in predominantly the nucleus or predominantly in the cytoplasm were determined. Approximately 75% of cells expressing wild-type Dax-1 showed nuclear localization, and this proportion decreased to 25% with the mutant motif 1. However, mutations in the other motifs (motif 2 and motif 3) had no effect on the frequency of nuclear localization (Fig. 3D
). The same outcome was observed when the 6x[His]-Tag was fused to wild-type and mutant forms of Dax-1 (data not shown).
To examine whether Dax-1 interacts directly with Ad4BP/SF-1 through the LXXLL motif, we prepared a glutathione-S-transferase (GST) fusion protein containing amino acids 529 of Dax-1 that include the first motif and subjected it to pull-down assay with in vitro synthesized Ad4BP/SF-1. As shown in Fig. 3E
, the in vitro synthesized Ad4BP/SF-1 was recovered with the GST fusion protein (GST-motif 1 [Wt]), but not with GST-motif 1 [Mut] in which the LXXLL motif was replaced by LXXAA. However, the protein was not recovered efficiently with GST-motif 1 [Wt]. This low recovery through the LXXLL motif is consistent with our recent observation, in which interaction between Ad4BP/SF-1 fused with maltose binding protein and a whole Dax-1 molecule carrying intact or mutated LXXLL motifs was investigated (24).
Involvement of the AF2 Core of Dax-1 in Nuclear Localization
A number of mutations have been reported in the human DAX-1 gene. Some are predicted to produce truncated forms, whereas others cause amino acid substitutions. We investigated the intracellular distribution of some of the mutants and found one that had an unexpected localization. As in the distribution profile of mouse Dax-1 described above, human DAX-1 fused with 6x[His]-Tag (His-DAX[WT]) or ß-Gal (ß-Gal-DAX[WT]) is diffusely distributed over the cytoplasm and nucleus in the absence of Ad4BP/SF-1. When cotransfected with Ad4BP/SF-1, it accumulated in the nucleus (Fig. 4A
, upper panel). Interestingly, DAX-1 with a single amino acid substitution at 466 (33) (His-DAX[L466R] and ßGal-DAX[L466R]) had a predominantly cytoplasmic localization in the absence of Ad4BP/SF-1, and even in its presence did not show exclusive nuclear localization (Fig. 4A
, lower panel); its frequency of nuclear localization was approximately 20%, substantially lower than wild type (75%) (Fig. 4B
).

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Figure 4. Role of the C-Terminal AF2 Core of Dax-1 in Nuclear Localization
A, Subcellular localization of human DAX-1 derived from a normal and AHC patient. Wild type (WT) of DAX-1 and a mutant form, DAX-1[L466R], were fused with 6x[His]-Tag (His-Dax[WT] and His-Dax[L466R]) as well as ß-Gal (ß-Gal-Dax[WT] and ß-Gal-Dax[L466R]). These forms of DAX-1 were expressed in HeLa cells in the absence (-) or presence (+) of Ad4BP/SF-1 and the distribution was examined. B, Reduced nuclear localization of DAX-1 carrying L466R mutation. Three experiments described in panel A were performed. More than 200 cells expressing Dax-1 fused with ß-Gal were observed and were classified into two groups, one showing predominantly nuclear localization and the other either predominantly cytoplasmic localization, or equal distribution between nucleus and cytoplasm. Bars show ratios of cells showing predominantly nuclear localization. The mean ± SD values from three independent experiments are shown. *, P < 0.001 compared with the WT. C, Constructs of mouse Dax-1 mutants with amino acid substitutions in the AF2 core. Amino acids M464, E466, L468, and C469 were replaced with alanine (A) to produce M464A, E466A, L468A, and C469A as indicated. D, Effect of mutations in the AF2 core on nuclear localization of Dax-1. ß-Gal fusions with wild-type and mutant Dax-1 (M464A, E466A, L468A, and C469A) were transfected in the absence (-) or presence (+) of Ad4BP/SF-1. Bars show ratios of cells showing predominantly nuclear localization. The mean ± SD values from three independent experiments are shown. *, P < 0.001 compared with the WT.
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Because amino acid 466 of human DAX-1 is located in the AF2 core, a highly conserved region of LBD, we attempted to define further the role of the core in nuclear localization. A series of Dax-1 mutants, Dax-1 (M464A), Dax-1 (E466A), Dax-1 (L468A), and Dax-1 (C469A), was fused with ß-Gal (Fig. 4C
). As with wild-type Dax-1, in the absence of Ad4BP/SF-1 all the mutants were localized in the cytoplasm or diffusely throughout both cytoplasm and nucleus. In the presence of Ad4BP/SF-1, the frequencies of nuclear localization of Dax-1 (M464A), Dax-1 (E466A), and Dax-1 (L468A) were approximately 25%, 23%, and 16%, respectively, in each case much lower than that of wild type (74%). On the other hand, the efficiency of nuclear localization of Dax-1 (C469A) (71%) was not significantly decreased.
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DISCUSSION
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Intracellular Distribution of Dax-1
It is well established that Dax-1 has an exclusively nuclear location in tissues such as the gonads, adrenal cortex, pituitary, ventromedial hypothalamic nucleus (12, 25, 31, 34), and prostate (27). In the present study we have obtained in vivo evidence that Dax-1 is present in both cytoplasm and nucleus in the fetal pituitary for the first time. We have also shown that Ad4BP/SF-1 is not yet expressed at that time and that Dax-1 accumulates in the nucleus after the onset of Ad4BP/SF-1 expression. This change strongly suggested that the presence of Ad4BP/SF-1 affects subcellular localization of Dax-1, and we have shown with cultured cells that coexpression of Ad4BP/SF-1 shifts Dax-1 to the nucleus.
In vitro studies with cultured cells have revealed a nucleocytoplasmic distribution of Dax-1 (13, 27, 35). In the present study, we also showed that Dax-1 distributed both in the nucleus and cytoplasm. However, it has been unclear whether the nucleocytoplasmic distribution is due to passive diffusion or active transport because, considering the molecular size, Dax-1 might be able to pass through nuclear pore complexes by passive diffusion (28, 29). To address it, we investigated distribution of ß-Gal-Dax-1 fusion protein of which molecular mass is large enough to prevent passive diffusion, and showed that the fusion protein exhibits nucleocytoplasmic distribution as well as native Dax-1. These observations indicated that nucleocytoplasmic distribution of Dax-1 is attributable to active transport but not passive diffusion, and showed a good agreement with the results by Lalli et al. (27), in which it was revealed that Dax-1 has a potential to shuttle between the two compartments. However, because Dax-1 has no typical NLS in its primary structure, the fine mechanisms underlying the active transport of Dax-1 remained to be clarified.
Comparing subcelluar localization of Dax-1 among those studies, it was found that the ratios of cells showing nuclear, nucleocytoplasmic, and cytoplasmic distribution are inconsistent. Similar to the present study, Holter et al. (13) reported that approximately 70% cells showed cytoplasmic or nucleo-cytoplasmic distribution of DAX-1, whereas Lehmann et al. (35) reported that more than 90% cells showed nuclear localization. Although there is no appropriate explanation for the discrepancy, it is reasonable to assume that subcellular localization of Dax-1 is determined by coordinated actions of import into and export from the nucleus. Therefore, the histochemical observations seem to detect a certain state of equilibrium between the two actions, which is possibly regulated by a variety of stimuli and factors. In the present study, we revealed that Ad4BP/SF-1 is one of the factors that affect the equilibrium of Dax-1 subcellular localization toward the nucleus.
Role of the LXXLL Motif in Nuclear Import of Dax-1
It has been reported that the LXXLL motifs of Dax-1 are responsible for interaction with ER
and ERß and that, as a result of this interaction, Dax-1 represses ER
- and ERß-mediated transcription (5). Likewise, our recent work has shown that Dax-1 interacts with Ad4BP/SF-1 via the same LXXLL motifs, resulting in repression of the function of Ad4BP/SF-1 (24). It therefore appeared reasonable to assume that interaction through the LXXLL motifs contributes to nuclear localization of Dax-1, and, as expected, molecular dissection studies revealed that Ad4BP/SF-1 is able to promote translocation of Dax-1 to the nucleus through the LXXLL motifs. In addition, interaction through the motif was revealed by in vitro pull-down assays, possibly indicating that Dax-1 nuclear translocation with Ad4BP/SF-1 is driven by direct interaction through the LXXLL motif. Unexpectedly, however, the interaction seems weak based on the inefficient recovery in the pull-down assay. Thus, it might be possible that an unknown factor is necessary for stabilization of interaction between Ad4BP/SF-1 and Dax-1. Alternatively, the observed influence of Ad4BP/SF-1 on Dax-1 nuclear localization might be due to indirect interaction between the two proteins.
Recently, we found that certain nuclear receptors, such as liver receptor homolog 1, ER
, estrogen-related receptor 2, and Drosophila FTZ-F1, interact with the LXXLL motifs in Dax-1, while hepatocyte nuclear factor 4 and retinoid-related orphan receptor-
do not (24). It is therefore possible that the former set of nuclear receptors stimulate nuclear localization of Dax-1. Indeed, ER
and ERß also enhance Dax-1 nuclear localization. In contrast, however, it was recently reported that androgen receptor did not promote nuclear localization of Dax-1 despite physical interaction between the two proteins (13). Interestingly, Dax-1 reduced the nuclear localization of androgen receptor by tethering it in the cytoplasm. The molecular basis of cytoplasmic tethering remains to be clarified. Taken together, nuclear receptors may be classified into three groups on the basis of their interaction with Dax-1 and its subsequent effect on intracellular distribution. The first group consists of nuclear receptors that do not interact with Dax-1, and thus are unlikely to have any effect on its distribution. Nuclear receptors of the second group are able to interact with Dax-1 and, as a consequence, shift the distribution of Dax-1 to the nucleus, while the last group remains in the cytoplasm after interacting with Dax-1. It is interesting to note that, according to this classification, Dax-1 causes repression by two distinct mechanisms: it represses the function of members of the second group in the nucleus by recruiting corepressors, such as nuclear receptor corepressor (20) and Alien (36), whereas it represses those of the third group by interfering with nuclear import.
Distinct Properties among LXXLL Motifs
It was interesting to note that nuclear accumulation of Dax-1 driven by the first and third LXXLL motifs was more efficient than that mediated by the second motif. Showing a good agreement with this result, our recent study indicated that interactions through the first and third motifs are stronger than that through the second motif (24). These observations strongly suggested that the two motifs contribute equally to Dax-1 nuclear localization. Interestingly, however, when each LXXLL motif was disrupted in the whole Dax-1 molecule, nuclear accumulation was affected only with mutation only at the first motif and not at the second and third motifs, indicating that the first motif is predominantly involved in nuclear accumulation along with Ad4BP/SF-1. Indeed, the LXXLL motifs in Dax-1 were shown to be different in requirement of the AF2 core for interaction with Ad4BP/SF-1; the interaction through the second and third motifs required the AF2 domain of Ad4BP/SF-1, whereas that through the first motif did not necessarily require it (24). Although the molecular basis of the interaction through the first motif is unknown, the feature of the first LXXLL motif might closely correlate with the distinct contribution to the nuclear localization of Dax-1. Moreover, as described below, a sort of nuclear receptor interacts with Dax-1, and, in fact, ER
and ERß have the ability to translocate Dax-1 into the nucleus. Because the expressions of the nuclear receptors are overlapped with that of Dax-1 in some tissues, it is possible that a particular type of nuclear receptor, including Ad4BP/SF-1, regulates intracellular localization of Dax-1.
Role of the AF2 Core in Dax-1 Nuclear Translocation
It has been reported that LBD plays an important role for nuclear localization of certain nuclear receptors. For instance, nuclear localization of the vitamin D receptor was affected by deletion of the AF2 core (37). Moreover, it is of interest that a region of the LBD is involved in the nuclear translocation of constitutive active nuclear receptor (or constitutive androstane receptor) (38) and progesterone receptor (39). Our findings implicate the AF2 core of Dax-1, in addition to the LXXLL motifs, in regulating the subcellular localization of Dax-1. However, ß-Gal-Dax[210472] did not accumulate into the nucleus even in the presence of Ad4BP/SF-1. This observation suggested that the region of Dax-1 by itself does not interact with Ad4BP/SF-1. In fact, our previous results with yeast two-hybrid assay strongly suggested the absence of interaction through this region (24). Therefore, it is unlikely that the effect of mutations in AF2 core on nuclear localization of Dax-1 is due to loss of direct interaction. Structure of LBD including AF2 is well conserved among the members of the nuclear receptor superfamily and has been known to be essential for functions of nuclear receptors. Actually, mutations of AHC patients mostly mapped at the LBD of DAX-1. Interestingly, Lehmann et al. (35) indicated that many of these mutations resulted in abnormal intracellular distribution of DAX-1. Similarly, our present study showed that mutations at the AF2 core reduced nuclear localization of Dax-1 in the presence of Ad4BP/SF-1. Taken together, these observations strongly suggested that intact structure of the LBD is crucial for the nuclear localization of Dax-1, although the fine mechanism remains to be clarified.
As has been shown for other mutations of DAX-1 (20, 21, 22), DAX-1[L466R] has reduced activity as a transcriptional repressor of Ad4BP/SF-1 (data not shown). Because almost all of these mutations lead to truncation or amino acid substitution of the LBD, their reduced repressor activity could be interpreted as due to defective functioning of the LBD. It would not be unreasonable to suppose that the L466R mutation also affects LBD function as the repressor. However, since repression must depend on nuclear localization, it is more likely that the primary cause in this case is a defect in nuclear localization. In this regard, Lehmann et al. (35) reached conclusions similar to the present study. The activity of biologically important proteins is frequently regulated at the step of nucleocytoplasmic trafficking (30) and disruption of this process can cause disease. Our present study indicates that a defect in subcellular localization of DAX-1 can be responsible for AHC.
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MATERIALS AND METHODS
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Immunohistochemical Analysis
Pituitary glands were dissected from mouse fetuses (E10.5 and E18.5). Guinea pig antibody to mouse Dax-1 (31) and a rabbit antiserum to bovine Ad4BP/SF-1 (40) were used as primary antibodies. Biotinylated antiguinea pig IgG and antirabbit IgG were used as the secondary antibodies. Antigen-antibody complexes were detected using a Histofine kit (Nichirei Co., Tokyo, Japan) or TSATM Biotin System (NEN Life Science Products, Boston, MA) according to the manufacturers recommendations.
Plasmids
cDNAs for mouse Ad4BP/SF-1 (15), Dax-1 (31), Sox9 (41), and GATA4 (42), and for human ER
(43) and ERß (44) were used. Full-length cDNAs, and various cDNA segments produced by PCR, were subcloned into pCMX. A modified pSV-ß-Gal (45) vector and the pSRHis expression vector (46) were used to yield the in-frame fusion genes, ß-Gal-Dax and His-Dax. ß-Gal-motif 1, 2, and 3 are pSV-ß-Gal constructs that include a nine-amino acid sequence containing an LXXLL motif (LXXL/ML) and two additional amino acids at each end. Mutant forms (LXXAA) of these vectors were also constructed. The QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to produce mutant forms of Dax-1/DAX-1 according to the manufacturers instructions. Each mutant was confirmed by sequencing.
Subcellular Localization
HeLa and CV-1 cells were maintained in DMEM supplemented with 10% fetal calf or bovine serum at 37 C in a 5% CO2 atmosphere. Electroporation of HeLa cells was carried out with 15 µg of pSV-ß-Gal fused with various Dax-1 sequences (ß-Gal-Dax), with or without 10 µg of a vector expressing Ad4BP/SF-1 (pCMX-Ad4BP/SF-1). After 48 h incubation, the cells were fixed with 0.2% glutaraldehyde, stained with 0.2% 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (45), and the subcellular localization of the fusion proteins was examined microscopically. pSRHis-Dax (His-Dax) and pCMX-Dax (Dax) (0.5 µg each) were transfected into HeLa cells using LipofectAMINE PLUS (Life Technologies, Inc., Gaithersburg, MD) in the presence or absence of 0.5 µg of pCMX-Ad4BP/SF-1. Cellular expression of Dax-1 was detected by staining with anti-6x(His) and anti-Dax-1 antibodies (31), followed by incubation with FITC-conjugated antirabbit and antiguinea pig antibodies, respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The subcellular localization of Dax-1 was determined by fluorescence microscopy.
Protein-Protein Interaction Assays
For GST-pull-down assays, GST-motif 1 [Wt] carrying wild-type Dax-1 (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) (LYNLL) or a mutant version GST-motif 1 [Mut] (LYNAA), was expressed in Escherichia coli, and the fusion proteins were purified using glutathione-Sepharose (Amersham Pharmacia Biotech, Buckinghamshire, UK). In vitro transcription-translation of pCMX-Ad4BP/SF-1 was carried out with the T7 Quick Coupled Transcription/Translation System (Promega Corp., Madison, WI) in the presence of [35S]methionine. Purified GST-motif 1 was incubated with the radiolabeled Ad4BP/SF-1 for 90 min at 0 C. Glutathione-Sepharose (10 µl) was then added, and the radiolabeled Ad4BP/SF-1 bound to the GST-motif 1 was eluted with 10 mM glutathione after vigorous washing. The eluate was electrophoresed on a denatured 10% polyacrylamide gel containing 1% sodium dodecyl sulfate, followed by autoradiography to detect the radiolabeled Ad4BP/SF-1.
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ACKNOWLEDGMENTS
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We thank Dr. O. Gotoh for critical reading of the manuscript, and N. Sinoda, Y. Miyaura, and F. Sinonaga for technical assistance.
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FOOTNOTES
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and Health Sciences Research Grants from the Ministry of Health Labour and Welfare. This study was also supported by the Special Coordination Funds for Promoting Science and Technology of the Science and Technology Agency of the Japanese Government, as well as the Cell Science Research Foundation.
Abbreviations: Ad4BP, Adrenal-4 binding protein; AF2, activation function 2; AHC, adrenal hypoplasia congenital; DAPI, 4,6-diamidino-2-phenylindole; Dax-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; ER, estrogen receptor; ß-Gal, ß-galactosidase; GST, glutathione-S-transferase; His, 6x [His]-Tag; LBD, ligand binding domain; NLS, nuclear localization signal; SF-1, steroidogenic factor 1.
Received for publication October 30, 2002.
Accepted for publication February 21, 2003.
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