The Activation Function-2 Hexamer of Steroidogenic Factor-1 Is Required, but Not Sufficient for Potentiation by SRC-1
Peter A. Crawford,
Jeffrey A. Polish,
Gauri Ganpule and
Yoel Sadovsky
Department of Obstetrics and Gynecology (J.A.P., G.G., Y.S.) and
Pathology (P.A.C.), Washington University School of Medicine, St.
Louis, Missouri 63110
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ABSTRACT
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The orphan receptor steroidogenic factor 1 (SF-1)
plays a central role in development and differentiation of the adrenal
gland and gonads. It also regulates the expression of several pivotal
steroidogenic enzymes and other proteins that are essential for
reproductive function. Its mechanism of target gene activation that
directs these intricate processes has not been previously established.
We demonstrate here that the activation function-2 (AF-2) activation
hexamer (AF-2-AH) of SF-1, located within its carboxy-terminal region,
is required for reporter gene activation by SF-1, as well as for
SF-1-mediated induction of a steroidogenic phenotype in embryonic stem
cells. We further demonstrate that SF-1s AF-2-AH is not sufficient
for gene activation, requiring an additional, proximally located domain
of SF-1, positioned between residues 187245. Correspondingly, we show
that the coactivator SRC-1 potentiates the activity of SF-1 and that
the interaction between SF-1 and SRC-1 requires both AF-2-AH and the
proximal activation domain. We conclude that SF-1 harbors at least two
activation domains within its carboxy terminus and that both are
required for its transcriptional activation function and for direct
interaction with SRC-1. It is likely that SRC-1 plays a key role in
gene regulation by SF-1.
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INTRODUCTION
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Steroidogenic factor 1 (SF-1) is an orphan member of the steroid
receptor superfamily of proteins, which are essential for regulation of
embryonic development, differentiation, and cellular function (1, 2, 3, 4, 5, 6).
SF-1 is highly expressed in all three layers of the adrenal cortex, in
granulosa and theca cells in the ovary, and in Sertoli and Leydig cells
in the testis (7). Consistent with its anatomical distribution, SF-1
functions as a potent transcriptional activator of several
steroidogenic enzymes, including cytochrome P450scc, the
key rate-limiting enzyme in the steroid hormone biosynthetic pathway,
as well as P450c17, P450c21,
P450c11, P450arom, 3ß-hydroxysteroid
dehydrogenase, and steroidogenic acute regulatory protein (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19).
Significantly, SF-1 is also expressed in pituitary gonadotrophs, where
it is essential for production of the ß-subunit of LH and for
expression of the GnRH receptor (20, 21, 22, 23).
Developmentally, SF-1 is expressed as early as E9 in the mouse
urogenital ridge, before its morphological differentiation into adrenal
and gonadal tissues (24). In the brain, SF-1 expression is detected in
hypophyseal precursors, as well as in prosencephalic regions that give
rise to the hypothalamus (24). To study the developmental and
differentiated functions of SF-1 in vivo, Luo et
al (25) and our group (26) generated SF-1 -/- mice.
Intriguingly, these mice lacked gonads, resulting in the persistence of
Mullerian structures in genotypically male and female mice. They also
lacked adrenal glands, which led to their early neonatal death.
Furthermore, SF-1 -/- mice demonstrated a developmental defect in the
ventromedial hypothalamus, an area implicated in control of sexual
behavior (27, 28). Together, these results demonstrate that SF-1 is
expressed in tissues that are essential for reproductive and endocrine
homeostasis and that it is required for intact development of the
adrenal glands and gonads.
Members of the steroid receptor superfamily of proteins, like other
transcription factors, are composed of modular functional domains (6).
These include a DNA-binding domain, which is flanked by amino and
carboxy termini. A ligand-independent activation function (AF-1) is
commonly located at the amino terminus. The carboxy terminus harbors a
hinge region and a ligand-binding domain, which commonly includes a
second, ligand-dependent activation function (AF-2) domain (1, 29, 30, 31, 32).
Transcriptional activity by these domains is either constitutive or
ligand-dependent and determines the function of the nuclear receptors
in development and differentiation.
Transcriptional activation domains are presumed to mediate an
interaction with either components of the basal transcription machinery
(33, 34, 35) or with cellular proteins distinct from basal transcription
factors, termed coactivators. The interaction with coactivators may be
of paramount importance in regulating physiological functions (2, 3).
Several of these interacting proteins have been cloned recently and
found to interact with the conserved AF-2 domain at the carboxy
terminus of ligand-activated receptors. Several examples includes
receptor-interacting protein 140, a coactivator of estrogen receptor
[ER (36)], transcriptional intermediary factor 1, a regulator of ER,
progesterone receptor (PR), retinoid X receptor (RXR), retinoic acid
receptor (RAR), and vitamin D receptor (37), and cAMP response element
(CREB)-binding protein (CBP), a coregulator of RXR and thyroid hormone
receptor (TR) (38, 39). An additional coactivator, termed steroid
receptor coactivator (SRC)-1, functions as a coactivator for ER, PR,
glucocorticoid receptor (GR), TR, and RXR, as well as for several
nonsteroid receptor activators such as Sp1 (40).
While the role of SF-1 in development and differentiation has been
demonstrated, the mechanism of transcriptional activation by SF-1 has
not been previously analyzed. Despite its ability to be activated by
certain oxysterols (41), SF-1 differs from classic ligand-dependent
steroid receptors, which have known high-affinity ligands that are
essential for AF-2-mediated activation (42, 43, 44). Furthermore, SF-1 does
not harbor a transcriptionally active amino terminus and, unlike many
receptors that bind their response element as homo- or heterodimers,
SF-1 binds DNA half-site sequences as a monomer (45). To identify the
activation domains of SF-1, we initially characterized its AF-2
activation domain. While required for full SF-1 activity, this domain
is not sufficient. An additional domain, located carboxy-terminal to
the DNA-binding domain of SF-1, is also needed for gene activation.
Moreover, we demonstrate that SRC-1 potentiates SF-1 activity in
mammalian cells, and this interaction also requires both activation
domains of SF-1.
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RESULTS
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The AF-2 Activation Hexamer (AF-2-AH) Is Critical for
Transcriptional Activation by SF-1
The amino terminus of SF-1 contains only 12 nonconserved residues
amino-terminal to the first zinc module of the DNA-binding domain.
Therefore, to identify the activation domain of SF-1, we focused our
attention on the carboxy terminus, searching for domains that are
conserved between SF-1 and the carboxy-terminal region of other orphan
or ligand-dependent members of the steroid receptor superfamily. At the
carboxy-terminal end we found a region of six amino acids (AF-2
hexamer, see Fig. 1A
), previously
identified as a conserved activation domain in several steroid
receptors (42, 43, 44, 46, 47, 48). This motif is composed of a glutamate,
preceded by a variable amino acid, flanked by two pairs of hydrophobic
amino acids. To test whether or not this hexamer confers
transcriptional activity to SF-1, we initially deleted the entire
hexamer and compared the transcriptional activity of the truncated
protein (SF-1
AF-2) to the activity of wild type SF-1, using the S25
reporter gene. As shown in Fig. 1B
, removal of the 11 carboxy-terminal
amino acids of SF-1 diminished its activation capacity by 75% in both
JEG3 and CV-1 cells. Interestingly, removal of the terminal 183
residues (SF-1
279) did not cause further diminution of the
activation capacity of SF-1 (Fig. 1B
), suggesting that the AF-2 hexamer
is the most significant activation domain in this region of SF-1.

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Figure 1. AF-2-AH Is Required for the Transcriptional
Activation Function of SF-1
A, Mutations of the SF-1 AF-2-AH and corresponding mutagenic
oligonucleotides (see Materials and Methods). The
conserved hexamer is highlighted and
underlined. B, Transcriptional activation of an SF-1
reporter gene (S25, 0.5 µg) by transfected SF-1 constructs (0.1 µg)
that harbor a mutant AF-2-AH. Deleted AF-2-AH is labeled AF-2.
Results are expressed as fold luciferase activity over baseline
(mean ± SD) and represent four independent
experiments, each performed in duplicate. C, Mutations of AF-2-AH or a
deletion of this domain do not alter the DNA-binding capacity of SF-1.
Electromobility shift assay was performed as described in
Materials and Methods. Lysate is unprogrammed
reticulocyte lysate, and SF-1 WT AS is an SF-1 construct cloned into
pBS-KS in reverse orientation. Results represent two independent
experiments.
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Next, we determined the role of each pair of hydrophobic amino
acids (leucine-leucine or methionine-leucine), as well as the conserved
glutamate, in transcriptional activation by SF-1 (Fig. 1A
). As shown in
Fig. 1B
, we found that mutation of either pair of hydrophobic amino
acids (M1, M4) diminished the activation capacity of SF-1 to a level
similar to SF-1
AF-2, suggesting that each pair of hydrophobic
residues is essential for AF-2 function, as demonstrated for other
steroid receptors (42, 44, 46). In contrast, mutation of the conserved
glutamate (M3) had a weaker effect on the activation function of SF-1.
Not surprisingly, mutation of the nonconserved isoleucine (M2) had no
effect on activation by SF-1. A mutation of the glutamine at position
458 to either glutamate or alanine (M5) had no effect on activation by
SF-1. Similar results were obtained using a human P450scc
reporter construct (data not shown). These results indicate that,
unlike other steroid receptors such as ER, where mutation of either
pair of hydrophobic amino acids entirely abolish the activation
capacity of the protein, mutation of AF-2 activation hexamer (AF-2-AH)
or even a complete deletion of the hexamer renders SF-1 partly active,
albeit at a low level (2030% of wild type SF-1). This suggests the
presence of an additional activation domain in SF-1.
To confirm that mutations in AF-2-AH did not alter the ability of
SF-1 to bind its response element half-site, we tested the capacity of
wild type and mutant SF-1 constructs to bind a double-stranded
oligonucleotide probe that contains an SF-1 response element (45). As
shown in Fig. 1C
, we observed a comparable level of DNA binding by wild
type and mutant receptors in an electromobility shift assay. Taken
together, these results indicate that the AF-2-AH is an important
activation domain of SF-1.
The AF-2-AH Is Required for SF-1-Dependent Induction of
Steroidogenic Phenotype
Because SF-1 is required for development and differentiation
of steroidogenic tissues, we determined whether or not AF-2-AH is
important for this action of SF-1. For this purpose we used embryonic
stem (ES) cells, which differentiate into steroid-producing cells when
expressing SF-1 via stable transfection (49). Upon selection of
SF-1-transfected ES cells, we found that they expressed cytochrome
P450scc, a key enzyme in steroidogenic pathways (Fig. 2A
), and released progesterone into the
medium (Fig. 2B
). Remarkably, neither of these phenotypic changes was
observed when the SF-1 M4 mutant of AF-2-AH (Fig. 1A
) was introduced
(Fig. 2A
-B). These data suggest that SF-1s AF-2-AH is required for
its physiological function in induction of a steroidogenic
phenotype.

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Figure 2. SF-1 Requires AF-2-AH Activity for Induction of a
Steroidogenic Phenotype in ES Cells
A, Northern blot demonstrating basal as well as 8-bromo-cAMP-induced
expression of P450scc (SCC) in ES cells that stably express
either wild type SF-1, native ES cells, or ES cells that express an
AF-2-AH mutant SF-1 mAF-2 (M4, Fig. 1A ). The 18S ribosomal RNA band is
presented to illustrate equal loading. B, Progesterone production by ES
cells that express SF-1 constructs according to the paradigms described
in panel A. Results, expressed as mean ± SD,
represent four independent experiments, using three independent ES cell
clones.
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AF-2-AH Is Required, but not Sufficient for the Activation Function
of SF-1
The AF-2-AH bestows SF-1 with the ability to activate
transcription. To test whether AF-2AH is sufficient for this
activity, we tested for the independent activation capacity of the
carboxy-terminal region of SF-1, which contains the AF-2-AH. For this
purpose we fused the carboxy-terminal 35 amino acids, which include
AF-2-AH, to the DNA-binding domain of GAL4 (GAL41-147,
Fig. 3A
) and tested for its
transcriptional activation capacity in JEG3 cells using a reporter
(
GKI) that contains GAL4- binding sites. As shown in Fig. 3B
, the
activity of GAL4-SF-1428 was similar to that of GAL4 alone,
suggesting that the AF-2-AH containing SF-1428 was not
functional as an independent activation domain. We obtained a similar
result when longer fragments of SF-1 (residues 279462 or 245465)
were fused to GAL4. In contrast, we observed a strong activation of the
GAL4 reporter gene by GAL4-SF-1187 and
GAL4-SF-1119. As control, we identified a similar
activation by GAL4-ER282-595 (which contains the
carboxy-terminal region of ER) in the presence of E2. Thus,
in addition to AF-2-AH, there is a second critical component of SF-1
activation function, located between residues 187245. Next, to
determine whether AF-2-AH is required for the activity of this domain,
we tested for the activation capacity of
GAL4-SF-1119
AF-2, or GAL4-SF-1119mAF-2,
which contain a mutation in the terminal pair of hydrophobic residues
within AF-2-AH (M4, Fig. 1A
). The activation of the GAL4 reporter by
either mutant was diminished by 7080% (Fig. 3B
). Taken together,
these results indicate that AF-2AH is required for the activation
capacity of SF-1, but is not sufficient for this function, and requires
an additional activation domain, which is located further upstream in
the carboxy-terminal domain of SF-1 (between residues 187245).

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Figure 3. Transcriptional Activation by SF-1 Requires AF-2-AH
and an Additional Activation Domain
A, A schematic diagram depicting deletion fragments of the
carboxy-terminal domain of SF-1, fused to the DNA-binding domain of
GAL4 (residues 1147). Deleted AF-2-AH is labeled AF-2. B,
Activation of a GAL4 reporter construct ( GKI, 0.5 µg) in JEG3
cells by transfected GAL4-SF-1 fusion constructs, depicted in panel A.
Activation by GAL4-ER is used as a positive control. Results are
expressed as relative luciferase activity (mean ± SD)
and represent three independent experiments, each performed in
duplicate.
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SRC-1 Interacts with SF-1 and Potentiates Its Activity through
AF-2-AH and the Proximal Activation Domain
SRC-1 has been recently identified as a ligand-dependent
coactivator of several ligand-dependent steroid receptors [such as ER
and PR (40, 50)]. To determine whether or not SF-1 interacts with
SRC-1 in a manner dependent upon AF-2-AH, we used the two-hybrid system
in mammalian cells to identify the domains within SF-1 that are
required for interaction with SRC-1. For this purpose, we transiently
transfected CV-1 cells with GAL4-SF-1119, along with the
interacting domain of SRC-1 (residues 857-1061, 40 , fused to the
activation domain of VP16 (SRC-1-VP16). Using a coexpressed GAL4
reporter, we observed a dramatic enhancement of
GAL4-SF-1119-dependent activity, as the concentration of
SRC-1-VP16 was increased (Fig. 4A
). This
increase was similar to that seen with a fusion of GAL4 and the
carboxy-terminal region (residues 282595) of ER (not shown). This
result indicates that SRC-1 interacts with the carboxy-terminal,
autonomously active region of SF-1.

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Figure 4. The Interaction between SF-1 and SRC-1 Requires
Both Activation Domains of SF-1
A, The concentration-dependent interaction between SF-1 and SRC-1
(residues 857-1016, fused to the activation domain of VP16) is detected
using a two-hybrid system in CV-1 cells. The GAL4 reporter gene
( GKI, 0.5 µg) is activated by transfected GAL4-SF-1119
(0.2 µg). B, The interaction between SF-1 and SRC-1-VP16 (0.7 µg)
requires SF-1s AF-2-AH. C, The interaction between SF-1 and
SRC-1-VP16 in a two-hybrid system requires the proximal activation
domain of SF-1, located between residues 187245. Results are
expressed as relative luciferase activity (mean ± SD)
and represent three independent experiments, each performed in
duplicate. D, Schematic diagram depicting currently identified domains
within SF-1.
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Because we previously determined that AF-2-AH is required for
activation by SF-1, we tested whether or not AF-2-AH is required for
the interaction between SF-1 and SRC-1. We transfected CV-1 cells with
either GAL4-SF-1119 or a similar construct that contains a
mutated AF-2-AH (M4, Fig. 1A
), along with the SRC-1-VP16 vector, and
found that the interaction between SRC-1 and SF-1 absolutely requires
an intact AF-2-AH (Fig. 4B
). Similarly, to test whether the proximal
activation domain is required for the interaction between SF-1 and
SRC-1, we transfected CV-1 cells with SRC-1-VP16, along with a series
of SF-1 fragments, fused to GAL4. We observed that the interaction of
SF-1 with SRC-1 in the two-hybrid system was abrogated when residues
between 187245 were deleted from the SF-1 construct (Fig. 4C
).
Together, these results indicate that both AF-2-AH and the proximal
activation domain (between residues 187245) are required for the
activation capacity of SF-1, as well as for interaction with the
coactivator SRC-1. The relative positions of these domains are
indicated in Fig. 4D
.
To determine whether these activation domains of SF-1 are required for
potentiation by SRC-1, we transiently transfected CV-1 cells with SF-1,
in the presence or absence of human SRC-1. For a positive control we
used ER (and its cognate reporter, Vit2P36-LUC), as SRC-1
has been previously shown to synergize with ER in regulation of
estrogen-responsive reporter genes (50). As shown in Fig. 5A
, the addition of SRC-1 potentiated the
transcriptional activity of holo-SF-1 on its reporter construct S25,
while SRC-1 alone had no effect on promoter activity. Therefore, SRC-1
is a coactivator for SF-1. Next, we determined whether AF-2-AH and the
proximal activation domain are both required for potentiation of SF-1
activity by SRC-1. Using GAL41-147 fusions of the SF-1
carboxy terminus we found that the same SF-1 domains required for
autonomous activation capacity, and for two-hybrid interaction with
SRC-1-VP16, were also required for potentiation by intact SRC-1 (Fig. 5B
). Therefore, the transcriptional activity of SF-1 depends on both
the proximal activation domain and AF-2-AH. The interaction of these
domains with SRC-1 may provide the mechanism for regulation of SF-1
signaling by SRC-1 in vivo.

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Figure 5. SRC-1 Potentiates Transcriptional Activation by
Wild Type SF-1
A, CV-1 cells were transfected with CMV-SF-1 expression vector (0.1
µg), in the presence or absence of a human SRC-1, cloned in a pCR
vector (1 µg), and S25 reporter (0.5 µg). ER, in the presence of
E2 (10-8 M), was used as a
positive control (using the ER reporter Vit2P36-LUC). B,
Potentiation of SF-1 activity by SRC-1 requires AF-2-AH and the
proximal activation domain. CV-1 cells were transfected with GAL4
reporter gene and GAL41-147 fusions to SF-1 carboxy
terminus as above. SRC-1 expression vector (1 µg) was cotransfected
where indicated. Results are expressed as relative luciferase activity
(mean ± SD) and represent two independent
experiments, each performed in duplicate.
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DISCUSSION
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In this study we have dissected the transcriptional activation
function of SF-1. Like other members of the steroid receptor
superfamily, SF-1 is composed of modular functional domains. The
DNA-binding domain is highly homologous to other steroid receptors, and
its interaction with half-site sequences has been previously described
(45). In the absence of a functional AF-1 region in the amino terminus,
our analysis of the activation function of SF-1 focused on its carboxy
terminus, which harbors a conserved AF-2-AH sequence of six amino
acids. We have clearly shown that this AF-2 hexamer is required for
transcriptional activation by SF-1. This was demonstrated using a
synthetic SF-1 reporter gene, P450scc promoter, as well as
induction of differentiation in ES cells (49), where SF-1 requires
AF-2-AH to promote the expression of P450scc and
progesterone production.
Although SF-1s AF-2-AH is conserved among other steroid receptors
(42, 43, 44, 46, 47, 48), there are two features that distinguish the AF-2-AH
in SF-1: 1) Unlike the AF-2 of other ligand-dependent steroid receptors
(such as ER) where mutations of either pair of hydrophobic amino acids
within AF-2-AH entirely abolished the transcriptional activity of the
protein, mutations in SF-1s AF-2-AH only diminished its activity to
2530% of wild-type SF-1. 2) A replacement of the conserved glutamate
with glutamine completely disrupts the activity of the chick
T3R
(43), yet a similar replacement in SF-1 only
diminished its activity by 50%. Interestingly, a replacement of this
pivotal residue with alanine had no effect on the activation capacity
of ER (42). As shown for other steroid receptors, none of these
mutations altered the DNA- binding capacity of SF-1. These differences
between SF-1 and ligand-dependent steroid receptors may be related to
the ligand independence of SF-1. Alternatively, these differences may
specify the interaction of AF-2-AH with other domains within SF-1 or
with a distinct repertoire of coactivators.
While essential for transcriptional regulation by SF-1, the AF-2-AH of
SF-1 does not exhibit an independent activation domain when fused to a
GAL4 DNA-binding domain. In contrast, a region of 35 amino acids that
span the AF-2 hexamer of T3R
exhibits a strong
transcriptional activity when fused to GAL4 (43). Our results establish
that the AF-2-AH of SF-1 is necessary, but insufficient, for
transcriptional activation, and domains located further upstream
(residues 187245) are essential for the transcriptional activity of
SF-1. Several steroid receptors contain additional activation domains
amino-terminal to the ligand-binding domain. For example, two proximal
activation domains (
2,
3) were identified in TR
, and
homologous sequences have been found in RAR and RXR (46). Similarly, a
proximal transcriptional activation function resides between residues
181310 in the SF-1 homolog xFF1rA (51). These activation domains are
capable of independent transcriptional activation when fused to
GAL41-147. The proximal activation domain of SF-1 is also
capable of activating a reporter gene even in the absence of the
AF-2-AH, albeit at a markedly reduced potency. These results suggest
that AF-2-AH and the proximal activation domains synergize in their
activation function. This synergy may be achieved through
intramolecular interaction between complementary domains or may furnish
a platform for complex interaction with coactivators. Importantly, the
proximal activation domain of SF-1 (amino acids 187245) does not
appear to share significant homology with any protein other than the
closest relatives of SF-1, such as LRH-1 (FTF, PHR) and xFF1rA.
It has been proposed that coactivators are needed for bridging between
the steroid receptor and components of the basal transcriptional
machinery (52). This has been shown for RAR, which requires a E1A-like
factor to interact with transcription factor IID (53). Similarly, the
SWI/SNF family of yeast proteins are components of the basal
transcription machinery that enhance transcription by the steroid
receptors (54). SRC-1 is a recently identified coactivator for several
steroid receptors that can associate with nuclear receptor-CBP
complexes (see below), but the exact mechanism by which it potentiates
transcriptional activation by steroid receptors is presently unknown.
SRC-1 interacts with PR and ER and enhances the transcriptional
activation of PR, ER, TR, GR, and RXR in the presence of their
respective agonists (40, 52). It forms a family of proteins with
ER-associated protein 160, which interacts with ER, RARß, and RXR
(55), as well as GR-interacting protein 1 (transcriptional intermediary
factor 2), a protein that interacts with GR, ER, AR, RAR, and RXR in a
ligand-dependent fashion in yeast and mammalian cells (56, 57). Unlike
the SRC-1 association with most of these receptors, the interaction
between SF-1 and SRC-1 may be ligand-independent, as seen with some
steroid receptor coactivators (58). Our results indicate that SRC-1
potentiates the activation function of SF-1 in mammalian cells. The
AF-2-AH of SF-1 is required for SF-1 interaction with SRC-1, as the
interaction is abolished in the presence of a mutated AF-2-AH. However,
AF-2-AH is not sufficient for this interaction and requires an
additional, amino-terminal region of SF-1, located between residues
187245. These data suggest that SRC-1 requires the correct
conformation of the two activation domains to interact with SF-1,
allowing signaling to the basal machinery or to additional
coactivators. Interestingly, a GAL4-SF-1119 construct that
is devoid of functional AF-2-AH does not interact with SRC-1, yet it
retains some transcriptional activity (Fig. 3B
). This suggests that
additional coactivators may interact with proximal regions of SF-1.
The two-hybrid data presented herein demonstrate a functional
interaction between SF-1 and SRC-1. However, our data do not prove that
the interaction of the two proteins is direct, and it is possible that
a complex of coactivating proteins is required for the functional
interaction between SF-1 and SRC-1 to occur. Indeed, the coactivator
CBP/p300 has been shown to participate in nuclear receptor-SRC-1
complexes and, in this case, could bridge SF-1 to SRC-1 (39, 59, 60, 61).
Nevertheless, two lines of evidence argue for the direct interaction
between SF-1 and SRC-1 in our two-hybrid assays. First, the interactive
fragment of SRC-1 we employed for our two-hybrid assays contains none
of the CBP/p300 interactive region (60). Second, using residues 1450
of CBP in an analogous two-hybrid assay, we found that SF-1 and CBP do
not interact in CV-1 cells (data not shown). Thus, because CBP/p300
does not appear to directly interact with SF-1, endogenous CBP probably
does not bridge SF-1 to SRC-1 in the CV-1 two-hybrid assay. While CBP
and SF-1 do not directly interact in CV-1 cells, it is still possible
that an SF-1-SRC-1 interaction permits a ternary complex among SF-1,
SRC-1, and CBP in vivo.
That SRC-1 plays a role in SF-1-mediated differentiation or function of
steroidogenic cells remains to be determined. This is plausible, as a
naturally occurring mutation within the AF-2-AH of human TRß disrupts
interaction with SRC-1, implying that SRC-1 transduces the biological
activity of TR (62). The results presented here demonstrate a clear
correlation between the activation function of SF-1 and its interaction
with SRC-1, implying that SRC-1 may be a primary determinant of SF-1
activity.
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MATERIALS AND METHODS
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Plasmids
The SF-1 expression vector [cytomegalovirus(CMV)-SF-1] was
generated by sequential subcloning of the SF-1 cDNA (a gift from K.
Parker, Duke University, Durham NC) into the EcoRI sites of
pBS-KS (generating pBS-SF-1 and pBS-revSF-1, cloned in reverse
orientation), then using the KpnI and XbaI sites
from the BS polylinker to clone into pCMV-Neo. Using PCR, we generated
SF-1
AF-2 by placing a stop codon flanked by a 3'-EcoRI
site downstream from residue 451, thereby terminating SF-1 immediately
upstream from the AF-2-AH. The amplified EcoRI fragment was
cloned into pBS-KS and pCMV-Neo as described above for full-length
SF-1. CMV-SF-1
279 was generated by SalI digestion of
pBS-revSF-1 and subcloning the EcoRI fragment of the
truncated SF-1 cDNA back into pBS, then using the KpnI and
XbaI sites as detailed above to clone into pCMV-Neo.
For mutagenesis of the SF-1s AF-2-AH we used an inverse PCR-based
site-directed mutagenesis, using pBS-SF-1 as a template. The forward
primer harbored the mutations depicted in Fig. 1A
and abutted a reverse
primer that encoded a wild type sequence. Using T4 kinase we
phosphorylated the reverse primer, then used KlenTaq (63) in ten cycles
of amplification with the following parameters: 94 C for 0.5 min, 56 C
for 2 min, 72 C for 5 min. The product was treated with 200 µg/ml
proteinase K for 30 min at 56 C, then phenol-chloroform-extracted,
ethanol-precipitated, treated with Pfu polymerase (Stratagene, La
Jolla, CA) at 37 C for 30 min, then ligated overnight. To remove the
original template, the ligation mix was treated with DpnI
for 1 h and transformed into XL-1 blue bacteria. The authenticity
of the mutant constructs was confirmed using the dideoxynucleotide
sequencing method on an Applied Biosystems (Foster City, CA) model 373A
DNA sequencer. All SF-1 constructs used for transfection of ES cells
were cloned as an EcoRI fragment (from pBS) into the vector
pCAGGS, which uses the cytomegalovirus immediate early enhancer and the
chicken ß-actin promoter and first intron enhancer, as described
elsewhere (49).
We used PCR to generate fusion proteins between the DNA-binding domain
of GAL4 (1147) and carboxy-terminal fragments of SF-1. Utilizing a
BamHI-linked forward primer, a T3 reverse primer, and
pBS-revSF-1 as a template, we amplified the desired fragments of SF-1
(residues 428462, 279462, 245462, 187462, 119462, and
119452), digested with BamHI and HindIII, and
cloned downstream from GAL4 in pM2 vector (64). The correct frame of
each chimeric protein was verified by sequencing.
GAL4-ER282-595 was cloned in a similar fashion, using
pBS-ER (a gift from Stuart Adler, Washington University) as a
template.
A human SRC-1 expression vector (pBK-SRC1) was kindly provided by
M. J. Tsai and B. W. OMalley (Baylor College of Medicine,
Houston, TX). To generate the SRC-1-VP16 fusion we amplified the
receptor-interacting region of SRC-1 [corresponding to amino-acids
857-1061 (see Refs. 40 and 57); amino acids 1243-carboxy-terminus (see
60 ], and cloned it in frame upstream of the activation domain of
VP16 (amino acids 413440).
The SF-1 reporter construct (S25) contains the PRL minimal promoter
downstream from two SF-1-binding elements (TCAAGGTCA) separated by five
nucleotides, upstream from the luciferase gene. The
hP450scc-luciferase construct, which includes 2327 bp of
the human cytochrome P450scc promoter (65), was kindly
provided by W. L. Miller (University of California, San
Francisco). The GAL4 reporter gene (
GKI), which contains five GAL4-
binding sites upstream of an E1B-TATA box, linked to luciferase,
was kindly provided by P. Webb and P. J. Kushner
(University of California, San Francisco). The ER reporter
construct (Vit2P36-LUC) was previously described (66).
Cell Culture and Transfection
CV-1 cells are maintained in DMEM that contains 10% FBS and
antibiotics at 37 C and 10% CO2. JEG3 cells are maintained
in MEM that contains 10% FBS and antibiotics at 37 C and 5%
CO2. All tissue culture reagents were obtained from the
Tissue Culture Support Facility, Washington University School of
Medicine (St. Louis, MO). RW4 mouse strain 129/SvJ embryonic stem (ES)
cells are maintained on a feeder layer of murine embryonic fibroblasts,
as described elsewhere (49).
One day before transfection, CV-1 or JEG3 cells were plated in either
six- well plates at a density of 125,000 cells per well, or in 12-well
plates at a density of 60,000 cells per well. Four hours before
transfection the standard growth medium was replaced by fresh DMEM that
contained additives as above, and the cells were incubated in 10%
CO2 at 37 C. Transfection was performed using the
standard calcium-phosphate precipitation method previously described
(67) in six- and 12-well plates using 2.5 and 0.6 µg total DNA,
respectively, which included 0.1 µg CMV ß-gal plasmid (to normalize
for cell viability and transfection efficiency). Luciferase assay was
performed 4048 h after transfection. Cells were lysed in a lysis
buffer that contains 50 mM Tris-2-(4-morpholino)-ethane
sulfonic acid (pH 7.8), 1 mM dithiothreitol, and 1% Triton
X-100. Lysates were assayed for luciferase using a luminometer
(Monolight 2010 Analytical Luminescence Laboratory, San Diego, CA), and
for ß-GAL using a 96-well plate reader (anthos htIII, Anthos Labtec,
Salzburg, Austria). All experiments were performed in duplicate and
repeated at least three times. Results (mean ±SD),
normalized to ß-GAL activity, were expressed as relative luciferase
units. In experiments in which ER was used we used phenol-red free
medium with serum that contains negligible levels of
E2.
For stable transfection of ES cells, 5 x 106 cells
were electroporated with 25 µg plasmid DNA as described elsewhere
(49). Transfected cells were plated on 100-mm tissue culture dishes
coated with a confluent layer of murine embryonic fibroblasts, and 300
µg/ml G418 or 1 µg/ml puromycin were administered the following day
for 5 days. On the sixth day, colonies were picked and expanded. For
stimulation experiments, ES cells were plated at 5 x
104 cells per gelatinized well of 24-well plates in 0.5 ml
ES media. After 1 day, cells were given 5 µg/ml
20
-hydroxycholesterol in the presence or absence of 1 mM
8-bromo-cAMP.
Electromobility Shift Assay
Wild type or mutant SF-1 cDNA was transcribed and translated
from a pBSKS expression vector, using a TnT reticulocyte lysate system
(Promega, Madison, WI). A double-stranded oligonucleotide (100 ng) that
contained the SF-1 response element [TCAAGGTCA in tandem (45)], was
end-labeled by 20 µCi [
-32P]ATP, using
polynucleotide kinase. For each binding reaction 4 µl of the
translation mixture were mixed with 1 ng labeled probe in a binding
buffer (68) and incubated for 30 min at 25 C. Each binding reaction was
loaded onto a 5% polyacrylamide gel, run in 0.5x TBE buffer at 150 V
for 3 h. The gel was then dried and exposed to a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) screen or film.
RNA Analysis and Progesterone Measurement
RNA was extracted by the guanidinium thiocyanate-acid phenol
method (69). Total RNA (15 µg) was electrophoresed on a denaturing
gel of 1% agarose and 1.5% formaldehyde and blotted onto Zeta-Probe
GT membranes (Bio-Rad, Hercules, CA). The blot was probed with a
P450scc probe (a PCR product that spans the first 300 bp of
the mouse coding sequence), which was labeled with
[
-32P]-dCTP, as previously described (70). Membranes
were hybridized for 16 h at 45 C, then washed with 0.2x SSC
(1xSSC is 0.15 M NaCl, and 0.075 M sodium
citrate) and 0.1% SDS for 20 min at 68 C, and analyzed by
PhosphorImager.
For progesterone assay, media were harvested 24 h after the
addition of hormones, and progesterone was determined by RIA using the
Coat-a-Count system (Diagnostic Products Corporation, Los Angeles, CA).
Inter- and intraassay coefficients of variation were 5.1% and 2.6%,
respectively.
 |
ACKNOWLEDGMENTS
|
---|
We thank K. L. Parker (Duke University, Durham, NC),
M. J. Tsai, and B. W. OMalley (Baylor University, Houston,
TX), P. Webb, P. J. Kushner, W. L. Miller (University of
California, San Francisco, CA), and S. Adler (Washington University,
St. Louis, MO) for plasmids, and E. Sadovsky and J. Willand (Washington
University) for technical assistance. We also thank J. Milbrandt for
insightful discussions and suggestions in preparation of the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Yoel Sadovsky, M.D., Department of Obstetrics and Gynecology, Washington University School of Medicine, Box 8064, St. Louis, Missouri 63110.
This work was supported, in part, by NIH Grant HD-34110 and the Berlex
Scholar Award (both to Y.S.).
Received for publication May 7, 1997.
Revision received July 7, 1997.
Accepted for publication July 17, 1997.
 |
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