Function of Steroidogenic Factor 1 Domains in Nuclear Localization, Transactivation, and Interaction with Transcription Factor TFIIB and c-Jun

Lih-Ann Li1, Evelyn F-L. Chiang, Jui-Chang Chen, Nai-Chi Hsu, Ying-Ja Chen and Bon-chu Chung

Institute of Molecular Biology Academia Sinica Nankang, Taipei, Taiwan, Republic of China


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178–201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenic factor 1 (SF-1) plays an essential role at various levels in the development of the hypothalamic-pituitary-adrenal/gonadal axis. Inactivation of the SF-1 gene severely damages the structure of the ventromedial hypothalamic nucleus and the production of FSH and LH in mice. Furthermore, SF-1 null mice lack the adrenal and gonad, and all the knockout mice have the female genitalia irrespective of their genetic sex (1, 2, 3).

SF-1 controls endocrine development by regulating expression of many important genes along the axis. In the ventromedial hypothalamic nucleus and the pituitary gonadotrope, SF-1 controls transcription of the {alpha}-subunit of glycoprotein hormones (4), the ß-subunit of LH (5) and GnRH receptor (6). In the adrenal cortex and gonads, SF-1 activates expression of the genes for steroid hormone biosynthesis (7). SF-1 also regulates the adrenal expression of ACTH receptor (8) and mitochondrial cholesterol transportation protein StAR (steroidogenic acute regulatory protein) (9). In the testicular Sertoli cells, SF-1 governs gene expression of Mullerian-inhibiting substance, which blocks the formation of the Mullerian duct-derived female structure (10). SF-1 appears to be an essential regulator of endocrine and sex development.

SF-1 is a member of the nuclear hormone receptor superfamily of transcription factors. The same as other nuclear hormone receptors, SF-1 contains a zinc finger motif responsible for DNA binding (11, 12). A small basic region situated immediately downstream of the zinc-finger motif, termed Ftz-F1 box (13), facilitates the binding of SF-1 to specific DNA sequence elements. It is functionally similar to the A box in other nuclear hormone receptors (13, 14, 15). SF-1 also contains a ligand-binding domain (LBD) at the C terminus (12, 16). A recent study shows that 25-hydroxycholesterol potentiates SF-1 activity in CV-1 cells (17). However, another study opposes the claim of 25-hydroxycholesterol as an SF-1 ligand (18). The natural ligand for SF-1 remains unclear. Nuclear hormone receptors usually activate transcription as a homodimer or heterodimer, and dimerization occurs at the LBD. SF-1, by contrast, activates gene expression in a monomeric form (19).

Nuclear hormone receptors usually contain two transactivation domains. The region essential for ligand-independent transactivation (activation function 1 or AF1) is located in the N-terminal A/B domain. The region critical for ligand-dependent transactivation (AF2) contains a putative {alpha}-helix at the C-terminal end of the LBD (20, 21, 22). Lacking the AF1 domain, SF-1 has a similar sequence capable of forming an {alpha}-helix at the AF2 region (16). To characterize the function of SF-1 domains, we have performed a series of mutagenesis studies. The mutant studies reported here indicate that the AF2 sequence is indispensable for the gene activation function of SF-1. We also show that the FP region, an SF-1-specific region consisting of the Ftz-F1 box and the following proline cluster, functions in nuclear localization and interaction with TFIIB and c-Jun in addition to the known function in DNA binding (13).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear Localization Signal (NLS)
Nuclear proteins require a short peptide signal to guide themselves into nuclei. The nuclear targeting sequences characterized to date contain a number of basic amino acids, lysine and arginine, and they are often situated adjacent to the DNA-binding domain (DBD) in transcription factors (23, 24). SF-1 also contains a highly basic region after the second zinc finger (Fig. 1AGo). Therefore, we tested whether the NLS of SF-1 was located in this basic region.



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Figure 1. Identification of the NLS of SF-1

A, Sequences of SF-1 examined for NLS. The dots on the top of the sequence denote basic amino acids. F, Ftz-F1 box, P, proline cluster. B, Fluorescent localization of the reporter protein. Individual NLS sequence is fused with the GFP of pEGFP-C1. NLS2 and NLS2.2 lead the fusion protein to the nuclei.

 
Two Lys/Arg-rich peptides, NLS1 and NLS2, were selected from the basic region for the study of SF-1 nuclear localization (Fig. 1AGo). The NLS2 sequence coincides with the Ftz-F1 box, a region that is well conserved among SF-1 and other Ftz-F1 proteins (13). NLS1 and NLS2 were separately fused to the C terminus of green fluorescent protein (GFP). The ability of each peptide to bring the fusion protein to the nucleus was evaluated by the subcellular distribution of GFP. After transfection into human adrenocortical H295 cells, NLS2 guided the GFP reporter into the nuclei, while the GFP-NLS1 fusion protein and the parental GFP remained in the cytoplasm (Fig. 1BGo).

The GFP-NLS2 fusion protein was transported into the nuclei of COS-1 as well as H295 (data not shown), indicating that the nuclear localization function of NLS2 or Ftz-F1 box is independent of cell lineage. The NLS2 peptide was dissected into three shorter overlapping sequences, NLS2.1, -2.2, and -2.3 for further analysis in COS-1 cells (Fig. 1AGo). NLS2.2 guided the majority of the GFP-NLS2.2 fusion protein into nuclei, whereas NLS2.1 and NLS2.3 failed to translocate the GFP reporter (Fig. 1BGo). It indicates that the NLS2.2 sequence (RGGRNKFGPMYKR) is critical for nuclear localization. The NLS2.1 and NLS2.3 sequences cover the basic amino acids residing at the separate ends of NLS2.2, but both lack the nuclear transport capacity, suggesting that the basic residues at both ends of NLS2.2 are required for nuclear localization. The NLS of SF-1 should thus be a bipartite basic motif.

Defective Transactivation Function of SF-1 Mutants
To identify the regions of SF-1 essential for its transactivation function, a variety of SF-1 mutants (Fig. 2AGo) were examined by transfection together with SF-1-tk-CAT, a reporter that has an SF-1-binding site in front of the tk promoter/CAT (chloramphenicol acetyltransferase) gene cassette, into human placental JEG3 cells or adrenocortical H295 cells. When no SF-1 expression plasmid was transfected, there were only background levels of CAT activity in JEG3 cells. The CAT activity, in contrast, was high in H295 cells under the same conditions (Fig. 2BGo). This occurs because H295 cells contain endogenous SF-1, but JEG3 cells do not (25).



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Figure 2. Transactivation Function of Mutant SF-1

A, Structure of wild-type and mutant SF-1. B, Impaired transactivation of SF-1 mutants. Cells were cotransfected with reporter SF-1-tk-CAT (3 µg) and plasmid expressing either wild-type or mutant SF-1 (5 µg for JEG3 cells, 3 µg for H295). Transactivation function of mutants is expressed as relative CAT activity to that found for the wild-type SF-1. The locations of the DBD, LBD, Ftz-F1 box (F), proline cluster (P), activation function 2 region (AF2), point mutation (*), and insertion ({triangledown}) are shown. Restriction sites used for subcloning are also shown.

 
Three SF-1 LBD mutants were generated for functional analysis. LBD Mut1 carries a double-point mutation (E455G, M456Y). LBD Mut2 and Mut3 contain a deletion and a 27-bp insertion at the C-terminal end, respectively (Fig. 2AGo). All the mutations disrupt the AF2 domain (16). Expression of the LBD Mut1, Mut2, and Mut3 in JEG3 cells yielded 12–44% of wild-type SF-1 activity (Fig. 2BGo). These results confirms earlier notion that SF-1 requires an integral AF2 domain for gene activation (25, 26, 27). In addition, the residues E455 and M456 are critical for the structure and activation function of AF2. The three LBD mutants suppressed endogenous SF-1 activity by 62–85% in H295 cells (Fig. 2BGo). Therefore, disruption of the AF2 structure does not only impair the transactivation activity, but also creates a dominant negative feature.

The Bpu-del mutant exhibited profound dominant negative effects in H295 cells. Additional deletion of the LBD and hinge domain in the SalI-del and DBD mutants did not produce extra deleterious effects. However, further deletion truncating the NLS removed most, if not all, of the transactivation activity and dominant inhibition in the BsrG-del mutant (Fig. 2BGo). Nuclear entry and subsequent DNA binding appear to be important for the dominant negative function of the SF-1 mutants examined here.

The dominant negative activity of Bpu-del was further examined by cotransfection with wild-type SF-1 into COS-1 (Fig. 3AGo) and H295 (Fig. 3BGo) cell lines lacking endogenous SF-1. Increasing amounts of the Bpu-del expression plasmid diminished the CAT activity activated by SF-1 (Fig. 3Go, A and B). Western blot analysis showed that increasing amounts of Bpu-del protein were made in parallel with its dominant negative effects on transcription, while the levels of wild-type SF-1 protein stayed constant (Fig. 3AGo). Bpu-del, on the other hand, did not have significant effects on Rous sarcoma virus (RSV)-CAT expression (Fig. 3BGo). This indicated that the dominant negative effect did not result from a nonspecific squelching effect, but was targeted specifically against SF-1.



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Figure 3. Dominant Negative Effects of Bpu-del Mutant

A, Increasing amounts of the Bpu-del expression plasmid were cotransfected with the SF-1 expression plasmid (0.4 µg) and the reporter SF-1-tk-CAT (0.8 µg) into COS-1 cells. Effects of Bpu-del mutant on SF-1 transactivation activity were assayed through the reporter activity, while expression levels of SF-1 and Bpu-del proteins were measured by Western blot analysis of 6 µg of total protein. B, The Bpu-del mutant exhibited little dosage effects on expression of RSV-CAT as compared with SF-1-tk-CAT when cotransfected with 3 µg of either reporter into H295 cells.

 
c-Jun Strengthens SF-1 Transactivation Activity
SF-1 might serve as a common mediator to direct tissue-specific or lineage-specific expression by recruiting various trans-acting factors and, in turn, coordinate development and function along the hypothalamus-pituitary-adrenal/gonadal axis (1, 2, 3). Potential DNA-binding sites for AP1 (activating protein-1) were found in the proximity of an SF-1-binding site in the upstream cAMP-responsive sequence of human CYP11A1 (SCC) gene (28). The earlier studies in our laboratory demonstrated that SF-1 and AP1 synergistically activated a reporter gene through the CYP11A1 upstream cAMP-responsive sequence (I.-C. Guo and B.-C. Chung, manuscript in preparation). The functional interaction of SF-1 with AP-1 was further analyzed in JEG3 cells using SF-1-tk-CAT or SCC55 as reporter. SCC55 contains the CAT reporter gene driven by 55 bp of the human CYP11A1 5'-flanking sequence that includes a TATA box and an SF-1-binding site (29).

SF-1 activated transcription of the CAT gene from both the composite promoter of SF-1-tk-CAT and the human CYP11A1 native promoter SCC55 (29). Expression of c-Jun together with SF-1 raised the transactivation activity of SF-1 remarkably. On the other hand, c-Fos could neither increase the SF-1 activity alone nor significantly amplify the c-Jun enhancement (Fig. 4Go, A and B). Apparently, c-Jun, not c-Fos, could act in synergy with SF-1.



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Figure 4. Functional and Physical Interaction between SF-1 and c-Jun

c-Jun stimulates SF-1-mediated transcriptional activation from SF-1-tk-CAT (A) and SCC55 (B). SF-1-tk-CAT (3 µg) and SCC55 (1.5 µg) were individually cotransfected with various combinations of plasmids expressing SF-1, c-Jun, c-Fos, or their parental vectors (1 µg each effector in panel A, 0.5 µg in panel B) into JEG3 cells. C, c-Jun interacts with SF-1 physically through amino acids 1–169 of c-Jun. SF-1 was cloned into a T3 expression vector and synthesized using a TNT transcription/translation-coupled lysate system in the presence of [35S]methionine. After incubation with the indicated GST-c-Jun fusion proteins in the presence (+) or absence of 25-hydroxycholesterol, the 35S-labeled SF-1 retained by glutathione sepharose beads was analyzed by SDS-PAGE and autoradiography. The lane "input" shows one tenth of the amount of SF-1 used for each test.

 
The SF-1/c-Jun synergy might result from their physical interaction. We examined this possibility by means of an in vitro protein-protein interaction assay. Before incubation with radioactive SF-1, various c-Jun domains were fused to glutathione S-transferase (GST) and bound to glutathione sepharose beads. The fusion protein containing amino acids 1–169 of c-Jun (GST-c-Jun1-79 and GST-c-Jun170-331 did not (Fig. 4CGo). These results indicate that c-Jun80-169 contains region(s) essential for interaction with SF-1 in vitro. Addition of the putative SF-1 ligand 25-hydroxycholesterol had little effects on this interaction (Fig. 4CGo).

The c-Jun-interacting domain of SF-1 was determined in a similar experiment (Fig. 5Go). The GST-c-Jun1-169 protein interacted with the N-terminal amino acids 1–279 (SalI-del) but not the C-terminal amino acids 259–462 of SF-1 (9BD). Deletion of the first 77 amino acids in mutant FP or truncation of residues after amino acid 110 in mutant DBD did not eliminate the interaction of SF-1 with c-Jun. Therefore, the overlapping region between DBD and FP, i.e. the Ftz-F1 box, is responsible for the in vitro contact to c-Jun. The BG/N-del mutant did not interact with c-Jun, further indicating amino acids 100–110 (KRDRALKQQKK) are critical for the binding (Fig. 5Go, A and B).



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Figure 5. Direct Interaction of SF-1 with c-Jun and TFIIB in Vitro

A, Structure of SF-1 domains used in the binding assay. The P box indicates the unique proline cluster. The results of the interaction assays are summarized at the right side. n.d., Not done; *, gel not shown. B, Binding of c-Jun to the Ftz-F1 box of SF-1. C, Binding of TFIIB to SF-1 derivatives. SF-1 and derivatives were cloned into T3 or T7 expression vectors for in vitro protein synthesis. The 35S-Met-labeled SF-1 and derivatives (1/10 input shown) were incubated with GST-c-Jun 1–169 (B) or GST-TFIIB (C) and subsequently analyzed by SDS-PAGE and autoradiography (left panels).

 
Figure 5Go showed that the Ftz-F1 box was required for interaction between SF-1 and c-Jun in vitro. However, it is not the only domain important for SF-1/c-Jun synergy. Our earlier transfection studies showed that truncation of the LBD or AF2 greatly diminished activity stimulation by AP1 (25). Taken together, our results suggest that the Ftz-F1 box and AF2 domain are both necessary for the SF-1/c-Jun transcriptional synergy. The transactivation strength of SF-1 itself is the prerequisite for the c-Jun/SF-1 synergy.

SF-1 Interacts Directly with Transcription Factor IIB (TFIIB)
Expression of SF-1 in JEG3 or COS-1 cells greatly increased CAT expression from SF-1-tk-CAT (Figs. 2BGo and 3AGo), but not from tk-CAT (data not shown). The binding of SF-1 to its cis-element apparently led to the transcriptional activation of SF-1-tk-CAT. Several nuclear hormone receptors, such as estrogen receptor (ER), progesterone receptor (PR), thyroid receptor (TR), and vitamin D3 receptor, directly target the TFIIB component of the transcription preinitiation complex (30, 31, 32, 33, 34, 35, 36, 37). To examine whether SF-1 physically interacts with TFIIB, we performed similar protein-protein interaction assays as described above.

The bead-bound GST-TFIIB interacted with radioactive SF-1 and derivatives N-tm, FH(L), FH(S), and FP (Fig. 5CGo), but did not bind C-tm (data not shown) and 9BD (Fig. 5CGo). Addition of 25-hydroxycholesterol did not affect the SF-1/TFIIB interaction (data not shown). The binding assay mapped the TFIIB-interacting domain of SF-1 to the FP region, which consists of the Ftz-F1 box and the proline-rich N-terminal half of the hinge domain (Fig. 5AGo).

TFIIB domains were examined similarly for interaction with SF-1. Deletion of amino acids 178–201 abolished the physical association of TFIIB with SF-1 (Fig. 6Go). The region of amino acids 178–201 is also involved in the interaction with thyroid receptor-{alpha} (TR{alpha}) (34), TRß (31), and acidic activator VP16 (38). The amphipathic {alpha}-helix contained in the region of amino acids 178–201 of TFIIB (39) likely plays an important role in the TFIIB-transcription factor interaction.



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Figure 6. Localization of the SF-1-Interacting Domain of TFIIB

A, Structure of TFIIB derivatives. The shaded box indicates a basic region and the open arrows mark imperfect repeats. Summary of SF-1 interaction is listed at the right side. B, Amino acid residues 178–201 are necessary for interaction with SF-1. The TFIIB deletion mutants listed in panel A were fused with GST for the in vitro interaction assay with 35S-incorporated SF-1.

 
SF-1 FP Actively Inhibits Endogenous SF-1 Activity
Since the FP region of SF-1 carries TFIIB- and c-Jun-interacting sites in vitro, we tested whether FP had a dominant negative function. The FP expression plasmid was cotransfected with SF-1-tk-CAT or tk-CAT into adrenocortical H295 cells. When the dosage of the FP expression plasmid was increased, the CAT activity derived from SF-1-tk-CAT dropped (Fig. 7Go). There was also repression of CAT expression from tkCAT with increasing amounts of FP, although this expression was low and therefore more difficult to see (Fig. 7Go). Apparently, excess FP peptides attenuated transcriptional activation of the reporter gene. This inhibition was mainly caused by FP, since cotransfection of increasing amounts of GAL4 DBD had less significant effects on SF-1-tk-CAT (data not shown). The inhibition of FP was probably due to squelching of limiting amounts of general transcription factors required for SF-1 transactivation through their interaction with FP. This squelching activity might also play a role in the dominant inhibition exerted by the mutants tested in Fig. 2Go.



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Figure 7. The FP Region Abolishes SF-1 Activation

The FP fragment of SF-1 was inserted into expression vector pcDNA3.1/HisB. Increasing amounts of the FP expression plasmid were cotransfected into H295 cells with 0.5 µg of SF-1-tk-CAT or tk-CAT. The DNA amount for each transfection was equalized by addition of the parental vector.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SF-1 is an important transcription factor controlling endocrine development (1, 2, 3). We have dissected the domains of SF-1 in an attempt to characterize their function. Different from most nuclear receptors, SF-1 lacks the N-terminal AF1 domain. Instead, it contains a unique basic Ftz-F1 box followed by a proline cluster. We find that this SF-1-specific region, designated FP, is involved in the physical interaction with TFIIB and c-Jun (Fig. 5Go). In addition, the Ftz-F1 box contains the NLS (Fig. 1Go) and controls the recognition of the SF-1-binding sequence TCAAGGTCA (13). Therefore, this FP motif exhibits multiple functions.

Many nuclear receptors stimulate transcription by recruiting TFIIB, which stabilizes the binding of TFIID to the TATA box and enables the incorporation of other general transcription factors and RNA polymerase II to form transcription preinitiation complex (30, 34, 36, 37, 40, 41, 42, 43). The FP region of SF-1 interacts with TFIIB in the absence of ligand (Fig. 5Go). This interaction requires amino acids 178–201 of TFIIB (Fig. 6Go), similar to the ligand-independent interaction between TFIIB and the N-terminal AF1 domain of TR{alpha} (34). Since SF-1 does not have the AF1 domain, the FP motif may replace the AF1 domain to recruit TFIIB. Our observation that increasing amounts of FP block the endogenous SF-1 activity in H295 cells (Fig. 7Go) is consistent with the notion that SF-1 activates transcription through TFIIB recruitment.

Certainly, the dominant negative effect of FP does not simply result from the direct interaction with TFIIB, since overexpression of TFIIB can not rescue this inhibition (data not shown). The FP region also interacts directly with c-Jun via the Ftz-F1 box (Fig. 5Go). This interaction greatly stimulates the transactivation activity of SF-1 in human placenta cell line JEG3 (Fig. 4Go, A and B). It is different from the negative interaction commonly observed between nuclear receptors and AP1. Glucocorticoid receptor (GR), thyroid receptors (TRs), and retinoid receptors (RARs and RXRs) all suppress AP1-mediated gene expression. AP1 also inhibits transcriptional induction by these nuclear receptors. All the mutual antagonism involves direct protein-protein interaction (reviewed in Ref. 44).

Indeed, SF-1 binds many proteins in addition to TFIIB and c-Jun (Fig. 8Go). Steroid receptor coactivator-1 (SRC-1) interacts with SF-1 at the AF2 domain and amino acids 187–245 (the C-terminal half of the hinge domain) (26, 27). General transcription coactivator CBP (CREB-binding protein)/p300 also interacts with SF-1 alone (45) or in synergy with SRC-1 (26). In addition, SF-1 synergizes with transcription factor WT1 (Wilms’ tumor 1) to up-regulate expression from the Mullerian-inhibiting substance gene. Transcription factor DAX-1 abolishes the SF-1/WT1 synergy either by directly antagonizing SF-1 or by recruiting N-CoR (nuclear receptor corepressor) to SF-1 through the SF-1/DAX-1 association (46, 47). Amino acids 226–230 and 437–447 of SF-1 are required for the negative interaction between SF-1 and DAX-1 (Fig. 8Go) (47). Taken together, SF-1 may confer a multitude of physiological functions via selective interaction with a variety of transcription factors and cofactors.



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Figure 8. Summary of SF-1 Domain Function

Functional regions are indicated in bars. F, Ftz-F1 box; P, proline cluster; AF2, activation function 2.

 
Furthermore, our experiments indicate that integrity of the AF2 domain is a prerequisite for SF-1-dependent gene activation. Disruption of the AF2 domain impairs the transactivation activity (Fig. 2Go) and the c-Jun synergy of SF-1 (25). Serine-430 of SF-1 can be phosphorylated by cAMP-dependent protein kinase A in vitro (12, 48). AF2 mutations attenuate the stimulation of SF-1 activity by protein kinase A (49). All these data manifest that the AF2 domain is necessary for transcriptional activation function of SF-1. Truncation of the AF2 domain eliminates the capacity to induce transcription. This explains why LBD Mut2 can not activate transcription either by itself (Fig. 2Go) or in synergy with c-Jun as wild-type SF-1 did (25).

In this report we show that the Ftz-F1 box situated downstream of the zinc finger motif directs the nuclear localization of SF-1. NLS2.2, which contains the bipartite basic motif located in the center of the Ftz-F1 box, is indispensable for nuclear transport (Fig. 1Go). Likewise, sequence after the second zinc finger of GR, PR, ER, androgen receptor, and vitamin D3 receptor contains putative bipartite basic motifs and possesses the nuclear targeting capacity (50, 51, 52, 53, 54, 55, 56). Based on the structural analysis of GR (57), Dingwall and Laskey (24) have proposed that the two basic clusters may be juxtaposed through looping of the spacer and function as a core basic motif, like the well studied NLS of SV40 large T antigen (PKKKRKKV). GR and PR have been demonstrated to possess an additional NLS (50, 52). Thus, it is possible that there is more than one NLS in SF-1.

In conclusion, our results show that the FP region C-terminal to the zinc finger motif of SF-1 is responsible for the direct interaction with TFIIB and c-Jun. The interaction is important for SF-1-mediated gene activation. SF-1 transactivation activity also requires an intact AF2 domain. In addition, the Ftz-F1 box contained within the FP region specifies nuclear targeting. The domain function of SF-1 is summarized in Fig. 8Go.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transfection and Cell Culture
Transfection was performed according to calcium phosphate precipitation procedures (Figs. 2BGo, 3BGo, and 4AGo) (58) or the lipofectamine-mediated method (Figs. 1BGo, 3AGo, 4BGo, and 7Go) (Life Technologies, Inc., Gaithersburg, MD) in human adrenocortical cell line H295, placental cell line JEG3, and monkey kidney cell line COS-1. While JEG3 cells were cultured in F-10 medium plus 2.5% FCS and 10% horse serum, H295 and COS-1 cells were grown in 10% FCS-containing DMEM-F12 and DMEM media, respectively. All the cells were cultured in 60 mm plates at 37 C in the presence of 5% CO2. The media and sera were purchased from Life Technologies, Inc.

Subcellular Localization
NLS1 and NLS2 sequences were amplified by PCR, while NLS2.1, -2.2, and -2.3 sequences were synthesized as oligodeoxynucleotides. Each sequence was cloned in frame behind the GFP of pEGFP-C1 (CLONTECH Laboratories, Inc., Palo Alto, CA) as an EcoRI-BamHI fragment. The pEGFP-NLS plasmids were transfected into cells using lipofectamine (Life Technologies, Inc.) following the supplemented protocol. The subcellular location of each fusion protein was detected using a model IX70 fluorescent microscope (Olympus Corp., Lake Success, NY) at 488 nm.

SF-1 Mutant Construction
The construction of several SF-1 mutants examined in this report has been described previously (25). The LBD Mut1 was generated by conversion of E455-M456 to G455-Y456 using site-directed mutagenesis. The Asp718I site created from this double-point mutation was then used to produce the LBD Mut2 and Mut3 by either deletion or insertion. BsrG-del, SalI-del, and Bpu-del mutants were generated by truncation digestion at the BsrGI, SalI, and Bpu1102I sites (Fig. 2AGo). The BsrGI, NcoI, and BssHI sites were used to yield BG/N-del, N/BH-del, N-tm, and C-tm. DBD, FH, FH(S), and FP were created by PCR (Fig. 5AGo). All wild-type SF-1 and mutants, except FP, were cloned into pCMV5 (59) for mammalian expression. The FP mutant was fused in frame to pcDNA3.1/HisB (Invitrogen, San Diego, CA) for proper translation, and the NLS in FP would lead the recombinant protein into nuclei. Vectors pBluescript KS(+) (Stratagene La Jolla, CA), pGEM7Z(f+) (Promega Corp., Madison, WI), and pET-29a (Novagen, Milwaukee, WI) were used for in vitro protein synthesis.

SF-1 Activity Assays
The activity of wild-type and mutant SF-1 was assayed by transient transfection experiments. SF-1-tk-CAT and SCC55 (29) were used as SF-1-responsive reporters, while RSV-CAT and tk-CAT were employed as nonspecific control reporters. Effects of c-Jun and c-Fos on SF-1 activity were tested by cotransfection of RSV-c-Jun and RSV-c-Fos with SF-1 expression plasmid. The total effector DNA amount was equalized by addition of parental expression vectors or OVEC (60) (Fig. 2BGo). Cytomegalovirus-ß-galactosidase was added as an internal control to equilibrate transfection efficiency (0.4 µg for calcium phosphate transfection and 0.2 µg for lipofectamine transfection). Reporter activity was measured usually 48 h after transfection, but was measured after 24 h in Fig. 3AGo. Production of antibody against full-length SF-1 and Western blot analysis was carried out following established procedures (61).

Protein-Protein Interactions
Protein-protein interactions were performed basically as described by Spencer et al. (62). SF-1 and its derivatives were synthesized using the TNT Lysate Coupled Transcription/Translation Kit (Promega Corp.) and appropriate phage RNA polymerase (T3 or T7) in the presence of [35S]methionine. GST and its fusion proteins were purified using glutathione sepharose 4B beads from lysates of E. coli harvesting pGEX-2T (Pharmacia Biotech, Piscataway, NJ) and pGEX recombinants after 2-h isopropyl-ß-D-thiogalactoside induction. GST-c-Jun169-331 was constructed by in-frame fusion of the PCR-generated c-Jun169-331 fragment into pGEX-2T, while GST-c-Jun1-169 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). GST-c-Jun1-79 and the GST-TFIIB series were gifts from Dr. H.-F. Yang-Yen and Dr. Danny Reinberg. For each interaction reaction, 35S-labeled protein from 1 µl of in vitro synthesis mixture was incubated with about 1 µg of bead-bound GST or GST fusion protein. After washing, bound 35S-labeled protein was detected by SDS-PAGE and autoradiography.


    ACKNOWLEDGMENTS
 
We thank Drs. Deepak Lala and Keith Parker for the SF-1 plasmids; Dr. Danny Reinberg for the TFIIB plasmids; and Drs. Jeffery Yen and Hsing-Fang Yang-Yen for the c-Jun and c-Fos plasmids.


    FOOTNOTES
 
Address requests for reprints to: Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China.

This work was supported by Grants NSC86–2313-B-001–011 and NSC87–2311-B-001–127 from the National Science Council, and by Academia Sinica, Republic of China.

1 Present Address: Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung, Taiwan, Republic of China. Back

Received for publication October 30, 1998. Revision received May 26, 1999. Accepted for publication June 18, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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