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
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ABSTRACT
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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 178201 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.
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INTRODUCTION
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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
-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
-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
-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).
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RESULTS
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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. 1A
). 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.
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Two Lys/Arg-rich peptides, NLS1 and NLS2, were selected from the basic
region for the study of SF-1 nuclear localization (Fig. 1A
). 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. 1B
).
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. 1A
). 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. 1B
). 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. 2A
) 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. 2B
). 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 ( ) are shown. Restriction sites used for subcloning are
also shown.
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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. 2A
). All the mutations disrupt the AF2 domain (16).
Expression of the LBD Mut1, Mut2, and Mut3 in JEG3 cells yielded
1244% of wild-type SF-1 activity (Fig. 2B
). 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 6285% in H295
cells (Fig. 2B
). 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. 2B
). 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. 3A
) and H295 (Fig. 3B
) cell lines lacking
endogenous SF-1. Increasing amounts of the Bpu-del expression plasmid
diminished the CAT activity activated by SF-1 (Fig. 3
, 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. 3A
). Bpu-del, on the other hand, did not have
significant effects on Rous sarcoma virus (RSV)-CAT expression (Fig. 3B
). 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.
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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. 4
, 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 1169 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.
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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 1169 of c-Jun
(GST-c-Jun1-79 and GST-c-Jun170-331 did not
(Fig. 4C
). 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. 4C
).
The c-Jun-interacting domain of SF-1 was determined in a similar
experiment (Fig. 5
). The
GST-c-Jun1-169 protein interacted with the N-terminal
amino acids 1279 (SalI-del) but not the C-terminal amino
acids 259462 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 100110 (KRDRALKQQKK) are critical for the binding (Fig. 5
, 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 1169 (B) or GST-TFIIB (C)
and subsequently analyzed by SDS-PAGE and autoradiography (left
panels).
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Figure 5
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. 2B
and 3A
), 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. 5C
), but did not bind C-tm
(data not shown) and 9BD (Fig. 5C
). 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. 5A
).
TFIIB domains were examined similarly for interaction with SF-1.
Deletion of amino acids 178201 abolished the physical association of
TFIIB with SF-1 (Fig. 6
). The region of
amino acids 178201 is also involved in the interaction with thyroid
receptor-
(TR
) (34), TRß (31), and acidic activator VP16 (38).
The amphipathic
-helix contained in the region of amino acids
178201 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 178201 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.
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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. 7
). 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. 7
). 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. 2
.

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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.
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DISCUSSION
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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. 5
). In addition, the Ftz-F1 box
contains the NLS (Fig. 1
) 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. 5
). This interaction requires amino acids 178201 of
TFIIB (Fig. 6
), similar to the ligand-independent interaction between
TFIIB and the N-terminal AF1 domain of TR
(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. 7
) 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. 5
). This
interaction greatly stimulates the transactivation activity of SF-1 in
human placenta cell line JEG3 (Fig. 4
, 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. 8
). Steroid receptor coactivator-1
(SRC-1) interacts with SF-1 at the AF2 domain and amino acids 187245
(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 226230 and 437447 of SF-1 are required for the negative
interaction between SF-1 and DAX-1 (Fig. 8
) (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.
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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. 2
) 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. 2
)
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. 1
). 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. 8
.
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MATERIALS AND METHODS
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Transfection and Cell Culture
Transfection was performed according to calcium phosphate
precipitation procedures (Figs. 2B
, 3B
, and 4A
) (58) or the
lipofectamine-mediated method (Figs. 1B
, 3A
, 4B
, and 7
) (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. 2A
). 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. 5A
). 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. 2B
). 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. 3A
. 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 NSC862313-B-001011 and
NSC872311-B-001127 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. 
Received for publication October 30, 1998.
Revision received May 26, 1999.
Accepted for publication June 18, 1999.
 |
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