The DEAD Box Protein DP103 Is a Regulator of Steroidogenic Factor-1
Qinglin Ou,
Jean-François Mouillet,
Xiaomei Yan,
Christoph Dorn,
Peter A. Crawford and
Yoel Sadovsky
Department of Obstetrics and Gynecology and Department of Cell
Biology and Physiology Washington University School of Medicine
St. Louis, Missouri 63110
Department of Obstetrics and
Gynecology (C.D.) University of Bonn Bonn 53105, Germany
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ABSTRACT
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The nuclear receptor steroidogenic
factor-1 (SF-1) is essential for development of the gonads, adrenal
gland, and the ventromedial hypothalamic nucleus. It also regulates the
expression of pivotal steroidogenic enzymes and other important
proteins in the reproductive system. We sought to elucidate the
mechanisms that govern the transcriptional activity of SF-1. We
demonstrate here that a previously uncharacterized domain, located
C-terminal to the DNA binding domain of SF-1, exhibits transcriptional
repression function. Point mutations in this domain markedly potentiate
the transcriptional activity of native SF-1. Using an SF-1 region that
spans this proximal repression domain as bait in a yeast two-hybrid
system, we cloned an SF-1 interacting protein that is homologous to
human DP103, a member of the DEAD box family of putative RNA helicases.
DP103 directly interacts with the proximal repression domain of SF-1,
and mutations in this domain abrogate its interaction with DP103. DP103
is expressed predominantly in the testis and is also expressed at a
lower level in other steroidogenic and nonsteroidogenic tissues.
Functionally, DP103 exhibits a native transcriptional repression
function that localizes to the C-terminal region of the protein and
represses the activity of wild-type, but not mutant, SF-1. Together,
the physical and functional interaction of DP103 with a previously
unrecognized repression domain within SF-1 represents a novel mechanism
for regulation of SF-1 activity.
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INTRODUCTION
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Normal development and differentiation depend on intricate
regulatory loops through which hypothalamic and pituitary hormones
regulate the production and release of steroid hormones. Steroidogenic
factor-1 (SF-1), a member of the nuclear receptor superfamily of
transcription factors, is a major regulator of endocrine and
reproductive function through its influence on development and
differentiation of hormone-producing tissues (1, 2). Developmentally,
SF-1 is essential for formation of female and male gonads, the adrenal
gland, and the ventromedial hypothalamus (1). Absent development of
these tissues in SF-1 null mice is associated with abnormal gonadotrope
differentiation, phenotypic male-to-female sex reversal, low serum
levels of corticosteroids, and, consequently, early neonatal death
(3, 4, 5). In addition, SF-1 synergizes with SOX9 in regulation of
Mullerian Inhibitory Substance expression in Sertoli cells (6, 7). In
adult tissues SF-1 is highly expressed in steroid-producing cells
within the adrenal glands, testis, and ovary, where it regulates the
expression of diverse genes that are essential for steroid hormone
biosynthesis, such as cytochrome P450 hydroxylases, ACTH receptor, and
steroidogenic acute regulatory protein (1). SF-1 is also expressed in
pituitary gonadotropes, where it activates the promoter for
-gonadotropin, LH-ß, and GnRH receptor (2, 8). Together, these
data indicate that SF-1 activity is imperative for intact embryonic
development, sex determination, and endocrine differentiation and is a
central regulator of the hypothalamic-pituitary-adrenal/gonadal
axis.
SF-1 is defined as a nuclear receptor based on structural and
functional homology with members of this family of proteins (9, 10).
The action of many receptors from this family is modulated by an
activating ligand and a homo- or heterodimeric partner. Furthermore,
several regulatory domains within these proteins interact with
coactivators or corepressors, which influence the activity of the
nuclear receptor through protein-protein interaction (11, 12). While
SF-1 regulates the expression of proteins that exhibit variable
expression during endocrine and reproductive function, the mechanisms
that modulate the activity of SF-1 are poorly understood. Unlike many
other nuclear receptors, SF-1 is defined as an orphan receptor because
a direct ligand, which is essential for its activity, has not yet been
identified. Whereas 25-OH cholesterol enhances the transcriptional
activity of SF-1, its role as a physiological ligand of SF-1 is
uncertain (13, 14). Furthermore, SF-1 binds to its DNA response element
as a monomer and thus is not a target for modulation by a DNA binding
heterodimerizing partner (15). Lastly, unlike many steroid receptors
that harbor an N-terminal ligand-independent activation domain (AF-1)
and a C-terminal, ligand-dependent activation domain (AF-2), SF-1 lacks
a functional AF-1 domain and depends entirely on domains C-terminal to
the DNA binding domain (DBD) for its activity (2, 16). We and others
have previously dissected the interaction of SF-1 with the coactivator
SRC-1, which potentiates the activity of SF-1 utilizing the
highly-conserved AF-2 hexamer at the C terminus of the protein and the
proximal interaction domain at residues 226230 (17, 18). Similarly,
we have determined that SF-1 utilizes a distal repression domain to
interact with DAX-1, which, like SF-1, is germane for reproductive
development and represses the activity of SF-1 in vitro (19, 20). Additional proteins, including early growth response protein 1
(Egr-1), Wilms tumor-1 (WT-1), pituitary homeobox 1 (Ptx1),
glucocorticoid receptor-interacting protein 1 (GRIP1), and multiprotein
bridging factor 1 (MBF1) modulate the activity of SF-1, but the
mechanism of their influence on SF-1 is unclear (21, 22, 23, 24, 25).
In our pursuit of mechanisms that modulate the transcriptional
activity of SF-1, we report here the identification of a previously
unknown repression domain within SF-1. Using this repression domain in
a yeast two-hybrid system we cloned a novel regulator of SF-1, which
represses the transcriptional activation function of SF-1 by direct
interaction. Using a BLAST database search we determined that this
regulator of SF-1 is a mouse homolog of DP103 (also known as Gemin3), a
recently cloned member of DEAD box-containing RNA helicases (26).
Although DP103 directly interacts with the survival motor neuron (SMN)
protein, as well as with the Epstein Barr Virus proteins EBNA2 and
EBNA3C, its function is unknown (26, 27, 28). We found that DP103 is
expressed predominantly in the testis and is also expressed in other
tissues as well as in cell lines that express SF-1. DP103 interacts
with the proximal repression domain (PRD) of SF-1 and represses its
transcriptional activity.
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RESULTS
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Identification of the PRD within SF-1
The transcriptional regulatory domains of SF-1 reside C-terminal
to its DBD (Fig. 1A
). To further explore
the function domains within SF-1, we generated a series of N-terminal
truncations of SF-1, fused to GAL4 DBD (Fig. 1B
), and tested their
transcriptional activity in JEG3 cells. Using the GAL4 reporter plasmid
GKI, we found that the transcriptional activity of
GAL4-SF-1120-462 was
5-fold higher when compared with GAL4 alone, as expected (Fig. 1C
). A
similar activity was observed using N-terminal truncations down to
residue 192. Surprisingly, the activity of
GAL4-SF-1202-462 was
10-fold higher than the activity of
GAL4-SF-1120-462 (Fig. 1C
). Further truncations that deleted the proximal interaction domain
at residue 226 (17) markedly diminished the activity of SF-1. These
data pointed to the presence of a repression domain located between aa
193201 of SF-1, which we termed PRD. We generated several point
mutations within PRD to identify the residues that are required for
transcriptional repression. As shown in Fig. 1C
, mutations in residues
194197 (KSEY) exhibited the most dramatic enhancement of
transactivation, resembling the transcriptional activity of
GAL4-SF-1202-462. We
therefore used mSF-1-AAEY in subsequent analyses. To confirm the
repressive function of PRD in the context of native SF-1, we mutated
KSEY residues to AAEY in CMV-SF-1 vector. Using a Western analysis we
confirmed that the expression of SF-1 was unchanged by the PRD mutation
(not shown). As shown in Fig. 1D
, the transcriptional activity of
mutated native SF-1 was higher than the activity of wild type SF-1 in
either JEG3 or CV-1 cells. Together, these data indicate that PRD
exhibits a repression function within SF-1.

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Figure 1. SF-1 Harbors a Repression Domain at aa 193201
A, A diagram depicting key regulatory domains of SF-1, including the
PRD. B, A series of N-terminal truncations of SF-1 (the number denotes
the aa residues), fused to GAL4 DBD. Five different mutations
(underlined) within the PRD in GAL4-SF-1 are shown. C,
The transcriptional activity of chimeric proteins (2 ng) composed of
either truncated or PRD-mutated SF-1, fused to GAL4 DBD, transfected
into JEG3 cells along with 0.5 µg of the reporter plasmid GKI. D,
The transcriptional activity of wild-type or AAEY-mutant SF-1 (0.1
µg), transfected into either JEG3 or CV-1 cells, along with 0.5 µg
of the SF-1-responsive reporter plasmid S25. Results are expressed as
RLU, normalized to ß-galactosidase activity, and represent three
independent experiments performed in duplicate.
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We next examined the autonomous transcriptional capacity of PRD,
independent of SF-1 context. Using a GAL4x5-tkLuc reporter, we found
that fusion of SF-1 residues 169220 to GAL4 DBD markedly reduced
(56%) the transcriptional activity of GAL4 in JEG3 cells (Fig. 2A
). Similar data were obtained with the
reporter
GKI (not shown). Importantly, either AAEY or KSAA mutations
of PRD abrogated this repression (Fig. 2A
). We further assessed whether
or not PRD can confer a repression function to a different nuclear
protein, estrogen receptor (ER). For this purpose, we created a
chimeric protein of SF-1s PRD fused to the AF-2 domain of ER,
downstream of GAL4
(GAL41-147-SF-1169-220-ER283-595).
Whereas estradiol stimulated the transcriptional activity of the
chimeric protein, the activity was further potentiated (7.6-fold) when
the SF-1 169220 segment harbored the AAEY mutation (G4-mSF-1-PRD/ER,
Fig. 2B
). These results confirm the transcriptional repression function
of SF-1s PRD and indicate that this repression function is
independent of SF-1 context. Thus, it is likely that PRD acquires its
repression function via interaction with cellular coregulatory
proteins, and not via intramolecular interactions within SF-1.

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Figure 2. SF-1s PRD Exhibits Intrinsic Transcriptional
Repression Function
A, PRD containing GAL4-SF-1169-219 (0.2 µg)
was cotransfected into JEG3 cells along with 0.5 µg of the reporter
vector GAL4x5-tkLuc. Wild-type denotes aa KSEY, whereas AAEY or KSAA
denote mutations in aa 194197 of SF-1. B, The transcriptional
activity of a chimeric protein (0.1 µg) composed of SF-1s PRD (aa
169219) fused to the AF-2 domain of ER (aa 283595) downstream from
GAL4 DBD, cotransfected into JEG3 cells along with 0.5 µg of the
reporter vector GKI. Estradiol (E2, 10-8
M) was added 24 h after transfection, and ethanol
(0.2%) was used as control. Results are expressed as RLU normalized to
ß-galactosidase activity and represent three independent experiments,
performed in duplicate.
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Cloning and Expression of SF-1 Regulatory Protein
To identify a putative corepressor of SF-1, we used a PRD-spanning
region of SF-1 (residues 109280) as a bait in a two-hybrid screen for
an interacting protein within a rat ovary cDNA expression library. Of
16 positive clones, we identified one candidate that interacted with
wild-type PRD, but not with an AAEY-mutant PRD. This clone encoded 406
aa upstream of a stop codon and a poly-A tail. Using this clone as a
probe in a Northern analysis, we detected an approximately 3-kb
transcript in mouse testis or ovary. To obtain a full-length cDNA, we
used this clone to screen a mouse testis cDNA library and identified a
2,910-bp clone that encodes an open reading frame of 825 aa. The
identified amino acid sequence exhibits high homology at its N terminus
region with proteins from the DEAD-box family of putative ATP-dependent
RNA helicases (reviewed in Ref. 29). These proteins are conserved at
their N-terminal region, which harbors eight discrete domains,
including a DEAD (Asp-Glu-Ala-Asp) box element (Fig. 3A
). Recently, a new member of this
family termed DP103 and a human ortholog of our newly identified SF-1
regulator was cloned by Grundhoff et al. (26) from a
human B cell lymphocyte line (BJAB) (26). The function of DP103 in
lymphocytes is presently unknown. DP103 is identical to Gemin3, a
protein of unknown function that interacts with SMN protein (27, 28).
The function of DP103/Gemin3 in the nervous system remains unknown. A
Western analysis of JEG3 cell lysate, transfected with mouse DP-103
fused to a FLAG-tag and detected using an anti-FLAG antibody, revealed
a 98-kDa protein (Fig. 3B
), which was consistent with the size of human
DP103, and was detected using immunocytochemistry in both the nucleus
and cytoplasm (not shown).

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Figure 3. The Structure and Size of DP103
A, A diagram of DP103, denoting domains in the N-terminal region that
are conserved in the DEAD-box containing RNA helicase eIF4A-II, as well
as other members of putative ATP-dependent RNA helicases (for details,
see Ref. 29). The location of the first aa in each domain is shown.
Sequences in the C-terminal region, which interact with SF-1, are not
conserved among members of this family of proteins. B, Immunoblot of
JEG3 cells, which were transiently transfected with a FLAG-DP103
expression vector (2 µg) and detected in cell extract by an antibody
against FLAG.
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Using Northern analysis of mouse tissues, we identified the
highest expression of DP103 in the testis (Fig. 4A
). In
situ hybridization demonstrated highest expression in the
periphery of the seminiferous tubules as well as in intertubular
regions, which contain Leydig cells, but not in mature sperm. This
pattern parallels SF-1 expression (Fig. 4C
). DP103 is also expressed,
albeit at a lower level, in the steroid-producing ovary and adrenal, as
well as in the placenta, brain, and kidney (Fig. 4A
). Interestingly,
DP103 expression is dramatically higher in the adult testis when
compared with P10 testis, suggesting a role in adult testicular
function. While DP103 is expressed in the adult ovary, its level does
not seem to fluctuate during the estrous cycle. The expression of DP103
in cell lines correlates with SF-1 expression (Fig. 4B
), exhibiting a
strong signal in the adrenocortical cells Y1, Leydig MA-10, and
gonadotrope LßT2. DP103 is also expressed in trophoblast Rcho cells,
and at a weak level in JEG3, Hela, DC3, and NIH3T3 lines.

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Figure 4. Expression of DP103 in Tissues and Cell Lines
Total RNA (25 µg), isolated from either tissues or cell lines, was
hybridized with a probe for either DP103 or SF-1, as described in
Materials and Methods. 18S subunit was used to control
the quantity of loaded RNA. A, DP103 expression in mouse tissues. B,
DP103 expression in cell lines, including CV-1 (monkey kidney
fibroblasts), Y1 (mouse adrenocortical), JEG3 (human choriocarcinoma),
Hela (human cervical adenocarcinoma), MA-10 (mouse Leydig), LßT2
(mouse gonadotropes), DC3 (rat ovarian granulosa), NIH3T3 (mouse
embryonic fibroblasts), Rcho (rat choriocarcinoma), JAR, and Bewo
(both human choriocarcinoma cells). To
ensure that the detection level in human cell lines does not reflect
species specificity of the murine probe, we hybridized the membrane to
a probe derived from a human DP103 ortholog (26 ) and obtained an
identical result (not shown). C, In situ hybridization
of SF-1 and DP103 expression in adult mouse testis, performed as
described in Materials and Methods (magnification
x400). No signal was detected using respective sense probes (not
shown). S denotes seminiferous tubules, and black arrows
demarcate the tubular margin. White arrows point to
Leydig cells containing intertubular region (bar =
50 µm).
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DP103 Interacts with SF-1 and Regulates Its Activity
To test for physical interaction between DP103 and PRD domain of
SF-1, we performed an immunoprecipitation experiment using a
FLAG-DP103-transfected JEG3 extract and in vitro expressed
full-length 35S-labeled SF-1. Precipitation by
anti-FLAG affinity gel revealed a strong SF-1 signal with wild-type PRD
(Fig. 5A
). A 4.3-fold weaker signal was
detected when SF-1 was mutated (AAEY at PRD), supporting the role of
PRD in the physical interaction between SF-1 and DP103 in
vitro. This interaction was recapitulated in yeast, where
SF-1109-280, which
contains the PRD, interacted with DP103, yet a similar AAEY-mutated
fragment failed to interact (Fig. 5B
). Finally, we confirmed the
interaction of DP103 and the PRD domain in mammalian cells, using
GAL4-SF-1120-462 and
DP103-VP16 hybrids cotransfected into CV-1 cells along with the
reporter plasmid
GKI. We found a strong interaction (6-fold) between
GAL4-SF-1120-462 and DP103
(Fig. 5C
). In contrast, this two-hybrid interaction was diminished when
SF-1 was either mutated at PRD (AAEY) or harbored an internal PRD
deletion (
187220), thus supporting a physical interaction of
SF-1s PRD and DP103.

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Figure 5. DP103 Physically Interacts with the PRD Domain of
SF-1
A, Immunoprecipitation of wild-type or AAEY-mutant
35S-labeled SF-1 by FLAG-DP103. FLAG-DP103 was generated by
a nuclear extract from JEG3 cells transiently transfected with
pCMV2-FLAG-DP103 (15 µg). SF-1 was transcribed, translated, and
35S-labeled using TnT. 35S-SF-1 and nuclear
extract containing transfected FLAG-DP103 were incubated and
precipitated by anti-FLAG affinity gel, and SF-1 detected by
autoradiography. Input contained 20% of 35S-labeled SF-1.
When corrected to input expression, the binding of mSF-1AAEY to DP103
was 23% of SF-1wt binding. B, The interaction of DP103 with either
wild-type or AAEY-mutated SF-1109280 fragment in yeast,
as described in Materials and Methods. Results represent
two independent experiments. C, The interaction of the two hybrids
GAL4-SF-1 (1 ng) and DP103-VP16 (0.4 µg), transiently transfected
into CV-1 cells along with the reporter plasmid GKI (0.5 µg).
Results, which represent three independent experiments performed in
duplicate, are expressed as fold activation over control in which the
empty pVP16 plasmid was used and normalized to ß-galactosidase
activity.
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To determine the transcriptional function of DP103, we initially
assessed its activity when tethered to DNA. As shown in Fig. 6A
, expression of GAL4 fused to
full-length DP103 in CV-1 cells repressed the activity of GAL4 reporter
(GAL4x5-tkLuc) in a concentration-dependent manner. Because DP103
directly interacts with SF-1 through the PRD, we tested for the ability
of DP103 to repress the activation function of SF-1. Using a GAL4
reporter and
GAL4-SF-1120-462 in CV-1
cells, we found that cotransfection of DP103 resulted in a 60%
reduction in the transcriptional activity of GAL4-SF-1 (Fig. 6B
). As
expected, the activity of GAL4-SF-1 mutated at residues AAEY of the PRD
was markedly higher. Importantly, the activity of the mutated SF-1 was
not repressed but slightly enhanced after cotransfection of DP103 (Fig. 6B
). Similarly, DP103 did not repress the activity of
GAL4-ER283-595. Together,
these results indicate that the repression effect of DP103 is abrogated
when the PRD is mutated or absent. Interestingly, because of the
proximity of PRD to serine-203 of SF-1, which was shown important for
SF-1 activity (25), we tested whether or not a mutation of serine-203
to alanine alters the influence of DP103 on SF-1. As expected, the
activity of SF-1S203A was markedly lower than the
activity of wild-type SF-1, yet the repression effect of DP103 on SF-1
was unchanged (not shown). We further confirmed the effect of DP103 on
native, full-length SF-1 using the wild-type or AAEY-mutant SF-1 (both
expressed using pCMV-SF-1), in the presence or absence of DP103. As
expected, DP103 diminished the activity of wild-type, but not
AAEY-mutated SF-1 (Fig. 6C
). Next, we analyzed the effect of DP103 on
the transcriptional activity of SF-1 using the SF-1-responsive
P450scc-luciferase reporter plasmid, which
harbors SF-1 binding elements (30, 31). When transfected into Y1
adrenocortical cells, which express SF-1 endogenously, we found that
DP103 repressed the activity of the P450scc
promoter in a concentration-dependent fashion (Fig. 6D
). Importantly,
DP103 had no effect on the P450scc-luciferase
reporter when transfected into JEG3 cells, which do not express SF-1
(Fig. 6E
). However, DP103 diminished the activity of
P450scc-luciferase when enhanced by cotransfected
SF-1. This effect was abolished when the
P450scc-luciferase reporter was mutated at the
two SF-1 binding sites (30, 31). Taken together, we conclude that DP103
exhibits a native transcriptional repression function, and that its
interaction with SF-1 in a PRD-dependent manner represses the
transcriptional activity of SF-1.

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Figure 6. DP103 Represses the Transcriptional Activity of
Wild-Type SF-1
A, The transcriptional activity of GAL4-DP103 transfected into CV-1
cells along with 0.5 µg of the reporter plasmid GAL4x5-tkLuc. Maximal
transcriptional repression was 87%. B, The effect of DP103 on the
transcriptional activity of wild-type
GAL4-SF-1120-462,
GAL4-SF-1120-462mAAEY, or
GAL4-ER283-595 (in the presence of
10-8 M estradiol). Twenty nanograms of GAL4
fusion proteins and 2 µg of pcDNA3-DP103 (or pcDNA3 empty vector)
were cotransfected into CV-1 cells, along with 0.5 µg of the reporter
plasmid GKI. C, The effect of DP103 on the transcriptional activity
of native, wild-type, or AAEY-mutant SF-1. Either SF-1 construct,
expressed from CMV-SF-1 (1 ng), was transfected into CV-1 cells along
with 3 µg of pcDNA3-DP103 and 0.5 µg of the reporter plasmid S25.
The empty expression vectors CMV-Neo or pcDNA3 were used as control for
SF-1 or DP103, respectively. D, The effect of DP103 on the activity of
the SF-1-responsive P450scc-luciferase reporter. A total of
3 µg of pcDNA3-DP103 and pcDNA3 empty vector were cotransfected into
Y1 cells, along with 0.5 µg of the reporter plasmid
P450scc-luciferase. E, The effect of DP103 on the activity
of the P450scc-luciferase reporter in JEG3 cells. A total
of 3 µg of pcDNA3-DP103 and pcDNA3 empty vector were cotransfected
along with 0.1 µg of SF-1 where indicated, as well as 0.5 µg of
P450scc-luciferase reporter plasmid, which contained two
wild-type or mutated SF-1 binding elements, described previously (31 ).
Results are expressed as RLU, normalized to ß-galactosidase activity
and represent at least two independent experiments, performed in
duplicate.
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DP103 interacts with EBNA2, EBNA3C, or SMN via its nonconserved C
terminus. We therefore fused either the N-terminal (aa 1410) or the
C-terminal fragments of DP103 to the activation domain of VP-16 and
determined their interaction with wild-type or AAEY-mutated G4-SF-1 in
a mammalian two-hybrid experiment. As shown in Fig. 7A
, the interaction of
VP16-DP103410-825 with
SF-1wt was similar to that seen with full-length DP103. In contrast,
VP16-DP1031-410 did not
interact with SF-1, and both DP103 fragments did not interact with SF-1
AAEY mutant. Moreover, when we fused these DP-103 fragments to GAL4 and
tested for their transcriptional function, we found that the C-terminal
fragment of DP103 repressed the activity of the GAL4 reporter gene,
whereas the N-terminal fragment had no effect (Fig. 7B
). This
difference did not reflect an altered expression level, as both
fragments were expressed at a level similar to the expression of
full-length GAL4-DP103 (Fig. 7C
). Together, these findings provide
further support to the specific effect of DP103 and indicate that DP103
interacts with SF-1 and represses its activity utilizing domains
located at its nonconserved C-terminus.

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Figure 7. The C-Terminal Region of DP103 Interacts with SF-1
and Harbors a Repression Domain
A, The interaction of either wild-type or mutant GAL4-SF-1 (1 ng) with
DP103 full-length, N terminus (aa 1410), or C terminus (aa 411825)
fused to VP16, transiently transfected (0.4 µg) into CV-1 cells along
with the reporter plasmid GKI (0.5 µg). Results, which represent
two independent experiments performed in duplicate, are expressed as
fold activation over control in which the empty pVP16 plasmid was used
and normalized to ß-galactosidase activity. B, The
transcriptional activity of GAL4 fused to either DP103-VP16 full-length
N terminus (aa 1410) or C terminus (411825), transfected into CV-1
cells along with 0.5 µg of the reporter plasmid GAL4x5-tkLuc. C,
Immunoblotting of the GAL4-DP103 fusion proteins analyzed in Fig. 7B , detected using an anti-GAL4 antibody as described in Materials
and Methods.
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DISCUSSION
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SF-1 plays a pivotal role in reproductive development and function
(1, 2, 16). Dynamic regulation of SF-1s activity seems essential to
account for the diverse spatial and temporal influences of SF-1 on a
wide array of target genes, and protein coregulators are likely to play
a central role in modulating SF-1 activity during that process (2, 16).
In this manuscript we examined a previously unidentified domain within
SF-1, located at residues 193201. Upon deletion or point mutation of
key residues within this domain, the transcriptional activity of SF-1
is markedly increased, compared with that of wild-type SF-1. The
repression function of the PRD is retained when it is transferred to
other transcriptional factors, indicating that the PRDs function is
independent of SF-1 context. Interestingly, sequence analyses revealed
that the nine-residue PRD is conserved among nuclear receptors that are
closely related to SF-1, such as LRH-1 (FTF) and xFF1R (32, 33). The
function of PRD in these proteins has not been examined.
Using the region of SF-1 that spans PRD as bait in a yeast two-hybrid
system, we cloned a protein of 825 aa from an ovarian cDNA library.
This protein is a mouse ortholog of human DP103, a member of DEAD box
family proteins (29, 34). Members of this family are defined by at
least eight evolutionary conserved motifs (one of which is the
Asp-Glu-Ala-Asp, or DEAD motif) that are necessary for RNA helicase
activity. DEAD box proteins play an important role in processes related
to RNA metabolism, including pre-mRNA splicing, mRNA transport, and
ribosome biogenesis and translation initiation (29, 34). Thus, they are
germane for cell development, differentiation, and proliferation.
Although it is likely that RNA helicase is a key function of DEAD box
proteins, it is noted that members of this family contain regions that
share little or no sequence homology with other members in the family.
Human DP103 was recently cloned by Grundhoff et al. (26)
based on its interaction with Epstein-Barr virus nuclear antigens EBNA2
and EBNA3C. In addition, DP103 (also termed Gemin3), directly interacts
with SMN protein (27, 28). Mutations in SMN are responsible for
different forms of the neurodegenerative disease, spinal muscular
atrophy. The function of DP103 in regulation of these proteins is
unknown. In contrast, we demonstrated that DP103 regulates the
transcriptional activity of SF-1. Interestingly, the relevance of a
different RNA helicase to a steroid receptor was demonstrated by Endoh
et al. (35), who identified an RNA helicase (p68) as a
transactivator for human ER
, acting through the AF-1 domain. DP103,
on the other hand, is a repressor of wild-type SF-1, as shown using
either a GAL4 fusion of SF-1 or native SF-1. DP103 also represses a
reporter plasmid when tethered to DNA through fusion with GAL4 DBD.
Furthermore, when overexpressed in Y1 adrenocortical cells, DP103
represses the activity of the SF-1-dependent
P450scc promoter, which regulates the
transcription of a rate-limiting enzyme in corticosteroid biosynthesis.
The direct interaction of SF-1 with the nonconserved C-terminal region
of DP103 resembles the interaction of DP103 with either EBNA proteins
or SMN. This suggests that the C-terminal region of DP103 is capable of
forming a protein complex, which exhibits transcriptional functions
distinct from the conserved RNA-regulatory domain in the N terminus of
DP103 (29).
Although DP103 is expressed at a low level in diverse tissues, highest
expression is found in the testis. Additionally, DP103 is expressed in
the ovary and adrenal gland, which also depend on SF-1 expression for
morphogenesis and differentiation (1, 16). Correspondingly, the level
of DP103 is high in steroidogenic cell lines Y1 and MA-10, as well as
in LßT2 gonadotropes, all of which express functional SF-1. Whereas
the level of DP103 in the ovary was unchanged during the estrous cycle,
its expression in the testis was considerably enhanced in the adult,
when compared with P10. Taken together, the expression pattern of DP103
and its interaction with SF-1 suggest that DP103 may play a role in
differentiation of steroid-producing tissues. DP103-dependent
repression of SF-1 activity may play a role in down-regulation of SF-1
target genes during development and function of reproductive and
steroid-producing tissues. Studies are currently underway to assess
these possibilities.
Intriguingly, we noted that DP103 somewhat enhanced transcription
when coexpressed with mutant SF-1 (see Fig. 6
, B, C, and E). These data
suggest that DP103 diminishes the activity of wild-type SF-1 through
interaction with PRD, yet an additional interaction with a different
domain of SF-1, which may result in enhanced transcriptional
activation, cannot be excluded. Recent evidence indicates that other
coregulators of nuclear receptors, such as NSD1 and mZAC1b, can act as
coactivators or corepressors in a promoter and cell type-dependent
manner (36, 37). The mechanism underlying this bipolar effect is
presently unknown. Similarly, the mechanism used by DP103 to influence
gene activity remains to be determined and may involve modulation of
RNA processing. It is noted that RNA helicase activity is dispensable
for the transcriptional influence of the DEAD box protein p68 on ER.
Moreover, p68 recruits the coactivator CREB-binding protein (CBP) to
its complex with ER (35). These findings support a function of DEAD box
proteins, which is independent of RNA helicase activity.
We and others have previously identified several SF-1 domains
that modulate its activity through interaction with coregulators. These
include the C-terminal AF-2 hexamer, interacting with SRC-1 (17) and in
turn, CBP (18), the distal repression domain, interacting with DAX-1
that binds nuclear receptor corepressor (N-Cor) (19, 20); and the
proximal interaction domain that interacts with both SRC-1 and DAX-1
(17, 19). Additional segments of SF-1, including
serine203 (interacting with GRIP1) and the
proline-rich region near the DBD play an important role in regulating
SF-1 activity (25, 38). Whereas additional proteins, such as Egr-1 and
Sp1 (21, 39, 40), synergistically interact with SF-1, the mechanism of
this interaction is currently unknown. Unlike these proteins, DP103 is
a regulator of SF-1 and interacts with SF-1 through a previously
unidentified repression domain. Although our results do not elucidate
the specific function of DP103 in development and differentiation of
steroidogenic tissues, the identification of a repression domain within
SF-1, which interacts with DP103, suggests the involvement of this
protein in modulating SF-1 function. Further dissection of DP103
function is likely to shed light on the mechanisms that modulate the
transcriptional activity of the orphan receptor SF-1, as well as on the
role of DP103 in other tissues.
 |
MATERIALS AND METHODS
|
---|
Plasmids and Cloning of Mouse DP103
We used PCR to generate fusion proteins of GAL4 DBD (aa 1147)
in pM2 vector and carboxy-terminal fragments of SF-1 (residues
120462, 187462, 192462, 202462, 220462, and 230462) and
169219, as previously described (17). The plasmid pBS-SF-1 was used
as a template for PCR-based site-directed mutagenesis within the PRD
(IKSEYPEPY) of SF-1, performed as previously described (21). Each
mutant and chimeric protein was sequenced using the dideoxynucleotide
sequencing method on a model 373A DNA sequencer (PE Applied Biosystems, Norwalk, CT). Mutated SF-1 fragments were fused to
GAL41-147, and mutated
SF-1 clones were cloned back into pCMV-Neo (17). GAL4-SF-1
187220
was generated by reverse PCR amplification of GAL4-SF-1 using forward
and reverse primers that correspond to nucleotides at residue 220 and
187, respectively, followed by ligation. The chimeric protein
GAL4-SF-1169-219-ER283-595
was generated by amplification of
SF-1169-219 with flanking
BamHI/SpeI sites and
ER283-595 with flanking
SpeI/SalI sites, and subsequent three-way cloning
downstream from GAL41-147.
The vector pBS-ER (a gift from S. Adler, Washington University, St.
Louis, MO) was used as a template. We used two GAL4 reporter genes:
GKI, composed of five GAL4 binding sites upstream of an E1B promoter
(a gift from P. Webb and P. Kushner, University of California, San
Francisco), or GAL4x5-tkLuc, composed of five GAL4 binding sites
upstream of a thymidine kinase promoter (a gift from J. Milbrandt,
Washington University). The rat
P450scc-luciferase reporter construct was
subcloned from the rat P450scc -894/+37-GH
reporter construct (a gift from J. S. Richards, Baylor College of
Medicine, Houston, TX), as previously described (31). The SF-1 reporter
S25 was previously described (17).
The cloning of DP103 was performed according to the Matchmaker yeast
2-hybrid protocol (CLONTECH Laboratories, Inc., Palo Alto,
CA). A murine SF-1 bait spanning aa 109280 was cloned downstream of
GAL4 DBD in a pAS1 vector [a gift from J. Milbrandt (41)]. A similar
fragment harboring the AAEY-mutation was used as a negative control.
The bait, along with a rat ovary cDNA library (CLONTECH Laboratories, Inc.), was sequentially transformed into HF7C
yeast, and 16 positive colonies were selected by growth in HIS-minus
medium and ß-galactosidase assay. To eliminate false positive clones,
we transformed either the bait or the mutant bait into yeast strain
HF7C (MATa), and positive clones into yeast strain Y187 (MAT
).
Mating was performed according to the CLONTECH manual, and one clone
(ARO19), which interacted with wild-type but not AAEY mutant, was
identified. To clone full-length DP103, a 400-bp fragment between
PstI and ScaI of ARO19 was used to probe a
-phage mouse-testis cDNA library (CLONTECH Laboratories, Inc.). Five independent clones were obtained, two of which were
sequenced and identified as ARO19 matches with an open reading frame of
825 aa and a poly-A tail, designated as full-length DP103.
To generate a DP103 expression vector, we subcloned the full-length
DP103 from pTripIEX into the BamHI/XbaI sites in
pcDNA3 vector (Invitrogen, San Diego, CA). The fusion
protein FLAG-DP103 was generated by cloning full-length DP103
downstream from FLAG at KpnI/XbaI sites of
pFLAG-CMV2 vector (Sigma, St. Louis, MO). DP103-VP16,
DP103411825-VP16 (N-terminal), and
DP103411825-VP16 (C-terminal) were generated with
DP103 fragments downstream from the VP16 activation domain at
BamHI/XbaI sites in a pVP16 vector
(CLONTECH Laboratories, Inc.). GAL4-DP103,
GAL4-DP1031410, and GAL4-DP103411825 were
generated by cloning DP103 fragments between
BamHI/XbaI sites downstream from GAL4 DBD.
Cell Culture and Transfection
JEG3 and CV-1 cells were maintained in culture as previously
described (17). Y1 cells were cultured in DMEM, supplemented with 10%
FBS and antibiotics. Cells were transfected with a total of 1 µg or 5
µg plasmid DNA per plate in either 12- or 6-well plates,
respectively, using the calcium phosphate coprecipitation technique as
described previously (17). Transfections were performed in duplicate
and repeated at least three times. Results were expressed as relative
luciferase units (RLU), normalized to ß-galactosidase
activity. In experiments in which estradiol was used, the cells were
cultured in phenol-red free medium with serum that contains negligible
levels of 17ß-estradiol.
Expression
Using TRI-REAGENT (Molecular Research Center, Inc.,
Cincinnati, OH) we isolated total cellular RNA from either mouse (Bl/6
or Bl6/129 strains) tissues or diverse murine or human cell lines. RNA
samples (25 µg) were separated by electrophoresis. A mouse DP103
probe, which corresponds to aa 216516, was generated using 900 bp
HindIII fragment from pTripIEX-DP103. The SF-1 probe, its
labeling, RNA transfer, hybridization, and detection were previously
described (42).
For immunoblotting of FLAG-DP103 we transfected 2 µg of
pFLAG-CMV2-DP103 into JEG3 cells plated on a 100-mm plate. After
48 h the nuclear extract was isolated and mixed with anti-FLAG-M2
affinity gel suspension (Sigma) followed by TBS (25
mM Tris, pH 7.4, 150 mM NaCl) washings
(Sigma). The protein was separated on an 8%
polyacrylamide gel, and then transferred to an Immobilon-P
polyvinylidene fluoride membrane (Millipore Corp.,
Bedford, MA) at 150 mA overnight. The membrane was probed with a mouse
anti-FLAG antibody (1:360 dilution, Sigma) followed by
peroxidase-labeled antimouse secondary antibody (1:1,000 dilution,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody
staining was visualized with an enhanced chemiluminescence system
(Amersham Pharmacia Biotech, Arlington heights, IL). For
immunoblotting of GAL4 fusion proteins, we transfected 3 µg of G4
alone, G4-DP103, G4-DP103 N, and G4-DP103 C into CV-1 cells plated on a
six-well plate. After 48 h the nuclear extract was isolated with
sample buffer (12.5 mM Tris-HCl, pH 6.8, 8% vol/vol
glycerol, 0.4% SDS, 1% vol/vol 2-mercaptoethanol, and 0.004%
bromophenol blue). The protein was separated as described above, and
the membrane was probed with mouse anti-GAL4 DBD antibody (1:1,000
dilution, Santa Cruz Biotechnology, Inc.) followed by
peroxidase-labeled antimouse secondary antibody (1:1,000 dilution,
Santa Cruz Biotechnology, Inc., Torrance, CA), and
developed as described above.
For in situ hybridization we collected mouse tissues, and
embedded them in OCT compound (Sakura, Torrance, CA).
Cryostat sections (10 µm) were postfixed in 4% paraformaldehyde
followed by two PBS washes. Fragments of DP103 (which corresponds to aa
515618) or SF-1 (which corresponds to aa 1205) were cloned into
pBSK plasmid. Sense and antisense digoxigenin-labeled riboprobes were
synthesized using DIG RNA labeling mix (Roche,
Indianapolis, IN). Slides were prehybridized for 2 h and
hybridized overnight at 58 C in 5x sodium chloride-sodium citrate
buffer with 50% formamide and 40 µg/ml heat-denatured ssDNA,
followed by stringency washes. Levamisole solution (Zymed Laboratories, Inc., South San Francisco, CA) was used to inhibit
endogenous alkaline phosphatase according to the manufacturers
protocol. Signals were detected using DIG Nucleic Acid Detection Kit
(Roche).
Immunoprecipitation
To generate FLAG-DP103 protein, we transfected JEG3 cells with
15 µg of pFLAG-CMV2-DP103 in 100-mm plates and isolated nuclear
extracts 48 h later. SF-1 and mutSF1AAEY were
35S-Met labeled in vitro using a TNT
kit (Promega Corp., Madison, WI). We mixed 10 µl of
labeled wild-type or mutant SF-1 with the FLAG-DP103-containing
extracts and then added to 20 µl anti-FLAG-M2 affinity gel suspension
(Sigma). After washes the proteins were separated using
8% polyacrylamide gel and exposed to Kodak X-OMAT film at
-80 C overnight.
Data Deposition
The sequence of mouse DP103 reported in this paper has been
deposited in the GenBank database under Accession no. AF220454, and a
partial sequence of rat DP103 under Accession no. AF220455.
 |
ACKNOWLEDGMENTS
|
---|
We thank S. Adler, P. Webb, P. Kushner, K. Parker, and J.
S. Richards for plasmids, and E. Sadovsky and L. Rideout for technical
assistance. We also thank J. Milbrandt for plasmids and for insightful
discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Yoel Sadovsky, M.D., Department of Obstetrics and Gynecology, Washington University School of Medicine, 4566 Scott Avenue, Campus Box 8064, St. Louis, MO 63110. E-mail: sadovskyy{at}msnotes.wustl.edu
This work was supported by NIH Grants HD-34110 and HD-37571 and Howard
Hughes Medical Institute Pilot Research Projects Award (to Y.S.), and
DFG DO 653/1 (to C.D.).
Received for publication May 17, 2000.
Revision received September 8, 2000.
Accepted for publication October 2, 2000.
 |
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