From the Laboratory of Molecular Endocrinology, CHUL Research Centre and Laval University, Sainte-Foy, Québec, G1V 4G2, Canada
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ABSTRACT |
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Regulation of the human CYP11A gene
encoding cytochrome P450scc, which catalyzes the first step of steroid
synthesis, is regulated by many trans-acting transcription
factors including steroidogenic factor 1 (SF-1). Transfection
experiments in human adrenal NCI-H295 cells demonstrate
regulation of the P450scc gene promoter region that contains several
putative SF-1 binding sites. Cotransfection of SF-1 with a
luciferase reporter construct containing the P450scc gene 5'-flanking
region from nucleotides 1676 to +49 increased promoter activity, and
deletion of the nucleotide sequence from position
1676 to
1620,
which removes a putative cAMP response element (CRE), did not affect
the stimulatory response to SF-1. As well, further deletion of the
promoter region to nucleotide
110, which contains only one SF-1
binding site, still retained the ability to respond to exogenous SF-1.
However, mutation of the remaining site which abolished SF-1
protein/DNA interaction also abrogated any functional response to the
factor. All the P450scc reporter constructs which responded to SF-1
were further stimulated by exogenous p300 and CREB-binding protein
(CBP), suggesting interaction between SF-1 and p300/CBP. As well,
mutation of the binding site that abrogated the response to SF-1 also
abolished the response to p300 and CBP. Cotransfection of the
adenovirus E1A oncoprotein, which has been shown to interact with
p300/CBP and interfere with its function, decreased the stimulatory
effect of SF-1 and p300/CBP. Cotransfection of a mutated E1A protein, RG2, which does not interact with p300/CBP, did not alter the stimulatory effect of SF-1 and p300/CBP on the P450scc promoter. Deletion of the region from amino acid residues 2-67 in E1A, which has
been postulated to interact with p300/CBP, also abolished the
inhibitory effect of E1A, whereas deletion of the region from residues
120 to 140 had no effect. Two regions of CBP from amino acids 1 to 451 and from 1460 to 1891 were demonstrated to interact with SF-1 in
vitro. Coexpression of fragments of the p300 protein fused to the
VP16 protein in the presence of SF-1 and the
110 P450scc reporter
construct indicated in vivo the interaction of two regions
of p300 with SF-1, thus confirming the in vitro results. Taken together these results indicate that regulation of the human P450scc gene by SF-1 is mediated by p300/CBP. Due to the many putative
roles of SF-1 to regulate many genes, its interaction with p300/CBP is
potentially a key component effecting important physiological
processes.
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INTRODUCTION |
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Cytochrome P450scc is a mitochondrial enzyme that catalyzes the conversion of cholesterol to pregnenolone and is the first step in the synthesis of all steroid hormones (reviewed in Miller (1)). The CYP11A1 gene that encodes P450scc is expressed in steroidogenic tissues such as the adrenal, ovary, testis, and placenta, and some expression has also been detected in the brain (2). The hormonal regulation and developmental pattern of expression of P450scc are specific to each steroidogenic tissue where adrenocorticotropin increases steroidogenesis and accumulation of P450scc transcript in the human adrenal cortex, and similar effects are seen with luteinizing hormone and follicle stimulating hormone in human ovarian granulosa cells, and with luteinizing hormone and human chorionic gonadotropin in human testicular Leydig cells (3-5). In each case, interaction of the tropic hormone with the cell surface receptor activates a G protein (Gs) that increases intracellular cAMP, which acts as the second messenger in a cAMP-dependent pathway to increase P450scc gene transcription (6).
The human P450scc cDNA has been cloned and the gene mapped to chromosome 15q23-24 (7). Preliminary studies with the 5'-flanking region of the gene demonstrated the ability of a 2.5-kilobase pair DNA fragment to confer basal and cAMP-responsive activity when transiently transfected into mouse adrenal Y1 tumor cells (8-10). Subsequent studies in mouse Leydig MA-10 (11), I-10 (12), human placenta JEG-3 (13), and adrenal NCI-H295 (14) cells identified regions of the 5'-flanking DNA that conferred basal promoter activity and a response to cAMP. However, it is apparent that the cAMP-responsive element identified in human JEG-3 cells is different from the element identified when studies were performed in mouse Y1, MA-10, and I-10 cells.
The orphan nuclear receptor steroidogenic factor 1 (SF-1)1 also known as adrenal
4-binding protein (Ad4BP) has been demonstrated to promote
cell-specific expression of the human P450scc gene promoter in Y1 cells
(15-17). The human P450scc gene promoter is inactive in
nonsteroidogenic CV-1 cells, but could be activated by expression of
exogenous SF-1 (15, 18). In addition to P450scc (15-17, 19), the SF-1
protein is involved in the regulation of several other steroidogenic
genes including P450c17 (20, 21), P450c11 (15, 16, 22) and P450
aromatase (23-25), where it has been postulated to play a role in
constitutive and cAMP-regulated expression.
In addition to its function as regulator of steroidogenic genes, SF-1 is implicated in endocrine differentiation and sexual development (26, 27). The observation of embryonic SF-1 expression early in the adrenal primordium before the acquisition of steroidogenic competence, expression of SF-1 in the urogenital ridge, differential regulation of SF-1 expression in the male versus the female gonads at the onset of morphological sexual differentiation (28-31), regulation of the Müllerian inhibiting substance gene by SF-1 (32, 33), and expression of SF-1 in the embryonic diencephalon and the anterior pituitary all indicate that SF-1 plays important roles in endocrine and sexual differentiation in addition to regulating the endocrine system. These putative roles of SF-1 is further supported by gene disruption studies where knock-out mice displayed female external genitalia irrespective of genetic sex, and the absence of adrenal glands, gonads, and the ventromedial hypothalamic nucleus (34-36). Based on the many potentially important roles of SF-1, it is clear that other target genes of SF-1 remain unidentified, and the mechanism of SF-1 function remain to be understood. As seen with other nuclear receptors, it is reasonable to speculate that SF-1 is a component of a multiprotein complex required to become transcriptionally active. Identifying and understanding how individual components of this complex function will be important to understand how SF-1 modulates transcription.
CREB-binding protein (CBP) and p300 are closely related ubiquitous coactivators that have been postulated to play key roles in modulating initiation of transcription by RNA polymerase II (37-39). Although the mechanism of CBP/p300 action is unresolved it is apparent that these coactivators can bridge transcriptional activators and the components of the basal transcriptional apparatus; CBP/p300 bind TFIIB, which in turn contacts the TATA box-binding protein in the TFIID complex (40-43). In addition, CBP/p300 as well as CBP/p300-associated factor have intrinsic histone acetyltransferase activity that may play a role in initiating transcription (44, 45).
This present study addresses the role of SF-1/Ad4BP in regulation of
the human P450scc gene in human adrenal carcinoma NCI-H295 cells.
NCI-H295 cells express the steroidogenic enzymes P450scc, P450c17,
P450c21, P450c11, P450c11AS, aromatase, adrenodoxin, adrenodoxin
reductase, and 3
-HSD type II (46, 47). To date, these cells
represent the only model of the human adrenal that has retained the
ability to express steroidogenic genes regulated in a hormonally
responsive fashion and to secrete a wide panel of different steroids.
Reporter constructs under the control of the 5'-flanking DNA of the
human P450scc gene, cotransfected with an SF-1 expression vector, show
the ability of the nuclear receptor to regulate P450scc promoter
activity in NCI-H295 cells. 5' deletions of the P450scc gene promoter
region demonstrate that the putative SF-1 binding site at position
46
to
38 is sufficient to confer responsiveness, and mutation of this
site abolishes both protein binding and SF-1 response. Coexpression of
exogenous SF-1 and p300 shows the ability of p300 to potentiate SF-1 to
increase P450scc promoter activity. Incubation of partially purified
CBP with SF-1 indicate in vitro the interaction between the
two proteins. Protein-protein interaction between the carboxyl- and
amino-terminal region of p300 with SF-1 is shown in vivo by
the ability of p300/VP16 fusion proteins to activate the P450scc
promoter in the presence of SF-1. Recruitment of p300/CBP coactivators
to form a multiprotein complex with SF-1 may be a key step by which
this nuclear receptor modulates the expression of steroidogenic
genes.
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EXPERIMENTAL PROCEDURES |
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Tissue Culture-- Human adrenal NCI-H295 cells obtained from the American Type Culture Collection were cultured in monolayer as described previously (14) in RPMI 1640 medium (Life Technologies, Inc.) supplemented with penicillin (50 mg/liter) streptomycin (105 units/liter), and 10% fetal calf serum (Hyclone, Logan, UT).
Plasmids--
The P450scc luciferase reporter constructs contain
fragments of the human P450scc gene which span from nucleotide 1676,
1620, and
110 at the 5'-end to nucleotide +49 at the 3'-end. The
DNA fragments were amplified by PCR from a P450scc genomic clone kindly provided by Dr. Bon-chu Chung (Academia Sinica, Nankang, Taipei) using
oligonucleotides which introduced a KpnI site and
BglII site at the 5'- and 3'-end, respectively. Following
amplification, PCR products were digested with KpnI and
BglII, subcloned into the pGL3 reporter plasmid
(Promega, Madison, WI), and verified by dideoxynucleotide
sequencing.
Transfections and Luciferase Assay--
NCI-H295 cells were
plated at a density of 3 × 105 cells per well in
6-well plates (35 mm per well). Twenty-four hours later, cells were
transfected with plasmid constructs using Lipofectin as instructed by
the manufacturer (Life Technologies, Inc.) and incubated for 48 h.
Following transfection, the medium was removed, and cells were lysed by
addition of 500 µl of lysis buffer (1% Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM
MgSO4, 2 mM EGTA, pH 8.0) for 15 min. Twenty
µl of the cell lysate were assayed for luciferase activity with a
luciferase assay system (Promega, Madison, WI) in a Berthold LUMAT
LB9501 luminometer. All experiments were normalized by cotransfection
of a CMV--galactosidase expression vector, and 10 µl of the cell
lysate were assayed for
-galactosidase activity with Galacto-Light
Plus (Tropix, Bedford, MA).
Western Blot Analyses-- NCI-H295 cells were transiently transfected as described above for luciferase assays and were harvested in SDS-sample buffer (0.2% SDS, 12.5 mM Tris-Cl, pH 6.8, 30 mM 2-mercaptoethanol, 2.5% glycerol). 20 µg of total protein were separated by 10% SDS-PAGE and electrophoretically transferred to nitrocellulose (53). The blot was probed with an anti-HA antibody (Babco, Richmond, CA) to detect SF-1-HA, and the signal was visualized by chemiluminescence using the Renaissance system as described by the manufacturer (DuPont NEN).
EMSA--
EMSAs were performed using double-stranded
oligonucleotides containing the human P450scc gene sequence from
nucleotides 52 to
31, which contains the putative wild type SF-1
binding site TCAAGGCCA in the noncoding strand. The mutant probe
contains the identical sequence but has two nucleotide changes in the
SF-1 binding site (see Fig. 3A for the sequence). The probes
were end-labeled using [
-32P]ATP and T4 polynucleotide
kinase (New England Biolabs, Beverly, MA). For all experiments, 2 µg of total protein extract from BL21 Escherichia coli
cells expressing GST-SF-1 fusion protein was incubated with 100 fmol
(150,000 cpm) of DNA probe in the presence of 2 µg of poly(dI·dC)
in 15 mM Hepes, pH 7.9, 50 mM KCl, 42 mM NaCl, 0.15 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 2.5% glycerol, and 4% Ficoll in a final reaction volume of 20 µl for 30 min at room
temperature. To ascertain the presence of SF-1 in the observed DNA-protein complex, the protein extract was preincubated with a
polyclonal anti-SF-1 antiserum (Upstate Biotechnology, Lake Placid, NY)
for 10 min prior to the addition of the DNA probe. In the competition
experiments, reactions contained a 500-fold molar excess of unlabeled
double-stranded wild type or mutant SF-1 oligonucleotides. DNA-protein
complexes were resolved by native 5% PAGE in 0.5 × Tris
borate-EDTA for 2 h at 150 V. Gels were dried and exposed to film
for 6 h at
80 °C.
In Vitro Protein Binding Assay-- To ascertain the interaction between CBP/p300 and SF-1 in vitro, fragments of CBP polypeptides from different regions of the protein were expressed as GST fusion proteins and were immmobilized on glutathione-coupled Sepharose as described by Frangioni and Neel (54) prior to incubation with radiolabeled SF-1 protein. 35S-Labeled SF-1 protein was produced from an SF-1 expression construct provided by Dr. Keith Parker (University of Texas Southwestern) using rabbit reticulocyte lysate and T7 RNA polymerase (Promega). GST-CBP fusion proteins were produced from cDNA constructs in E. coli BL21 following induction with 0.1 mM isopropythiogalactopyranoside. Equal quantities of GST-CBP fusion proteins were incubated with glutathione-coupled Sepharose for 30 min at 4 °C in binding buffer (100 mM NaCl, 1 mM EDTA, pH 8, 20 mM Tris, pH 8.0, 0.5% Nonidet P-40). The beads were then washed three times with phosphate-buffered saline, once with binding buffer, and then incubated with radiolabeled SF-1 for 2 h at 4 °C in the same buffer. After incubation, the beads were washed five times with washing buffer (20 mM Tris-Cl, pH 8, 500 mM NaCl, 5 mM EDTA, pH 8.0, and 0.1% Triton X-100). Bound proteins were released from the Sepharose by boiling in SDS sample buffer and were analyzed by SDS-PAGE (55). The gels were stained with Coomassie Blue to ascertain that equal amounts of GST proteins were loaded, after which the gels were incubated for 30 min with Amplify (Amersham Corp., Oakville, Ontario, Canada), dried, and exposed to film.
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RESULTS |
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Effect of SF-1 and CBP/p300 on the Human P450scc
Promoter--
Previous gene transfer experiments have demonstrated the
ability of the 5'-flanking region of the human P450scc gene to confer significant promoter activity in NCI-H295 cells (14). To determine the
ability of CBP/p300 to mediate SF-1 regulation of the human P450scc
gene, luciferase reporter constructs (1676Luc,
1620Luc, and
110Luc) which contain 5' deletions of the 5'-flanking region of the
gene, were cotransfected with SF-1 and p300 or CBP in human adrenal
NCI-H295 cells (Fig. 1). In transient
transfection experiments, expression of exogenous SF-1 protein
increased luciferase activity of the three constructs by 3-fold,
indicating that the putative SF-1 binding site at position
46 is
sufficient to confer a response. Transfection of p300 alone activated
transcription above basal levels; however, cotransfection of SF-1 and
p300 further increased expression of the three reporter constructs.
Similar results were obtained with the CBP protein transfected in place
of p300 (Fig. 1). To determine if the putative SF-1 binding site
TCAAGGCCA, which is found in the inverted position at nucleotides
38
to
46, can confer a response to SF-1, p300, and CBP, the site in the
110 construct was mutated to TCAATTCCA. Mutation of the putative SF-1
binding site abolished the response to SF-1, SF-1 cotransfected with
p300, and SF-1 cotransfected with CBP (Fig. 1). Since it is possible
that the response of promoter activity to SF-1 is concentration-dependent, Western blot analysis was performed to measure the level of exogenous SF-1 (Fig.
2). Coexpression of p300 and the
HA-tagged SF-1 fusion protein (SF-1-HA) led to a 3-fold increase in the
level of exogenous SF-1 protein over the transfection of SF-1-HA alone.
However, transfection of an 8-fold increase of the SF-1-HA expression
plasmid alone, which led to a 8-fold increase in the level of expressed
protein, conferred a significantly lower level of P450scc promoter
activity than the combined effect of p300 and SF-1. Thus, activation of
the P450scc promoter is at least partially dependent on p300 and
indicates a functional interaction between CBP/p300 and SF-1.
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Inhibition of SF-1 and p300 Activity by E1A-- Based on previous studies it is apparent that gene regulation mediated by CBP and p300 is repressed by the adenovirus 12 S E1A oncoprotein (56). Cotransfection of an E1A expression construct had a inhibitory effect on the activity of SF-1, and it also significantly inhibited the stimulatory activity mediated by p300 (Fig. 4). It has been shown that the positive arginine residue at position 2 of E1A is important for stable p300 interaction where the RG2 mutation abrogates p300 binding selectively (49). To ascertain the specificity of the inhibitory effect of E1A on p300 and SF-1, transfection of RG2 in place of E1A was found to abolish the inhibitory effect and did not decrease the stimulatory activities of SF-1 and p300. In addition, the region between residues 37 and 80 (region 1) of E1A has been suggested to interact with p300 (49, 57). As shown in Fig. 5, the deletion mutants E1A del2-36 and E1A del37-68 abolished the inhibitory activity and had no effect on SF-1- and p300-mediated regulation, whereas deletion of residues 120-140 was able to inhibit SF-1 and p300 function.
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Interaction between CBP/p300 and SF-1--
To determine if SF-1
interacts with CBP/p300, GST fusion proteins that contain different
regions of CBP were bound to glutathione-Sepharose and incubated with
35S-labeled SF-1 protein produced from rabbit reticulocyte
lysate. SF-1 interacted with the region between residues 1-451 and
1460-1891 of CBP, but no interaction was observed with the regions
between residues 451-721, 721-1100, 1100-1460, and 1892-2441 (Fig.
6). Similar experiments performed with
fragments of the p300 protein demonstrate interaction of SF-1 with both
the carboxyl- and amino-terminal region of the protein (data not
shown). To ascertain if SF-1 can interact with p300 in vivo,
two-hybrid assays were carried out in NCI-H295 cells. The amino region
of p300 (Np300) from residues 1 to 1275 and the carboxyl region from
residues 1257 to 2378 (Cp300) were fused to VP16 and cotransfected with
SF-1 and the 110Luc reporter plasmid (Fig.
7). The coexpression of SF-1 with
Np300/VP16 or Cp300/VP16 yielded a higher activity than cotransfection
of SF-1 with VP16, which indicates the interaction of both the amino- and carboxyl-terminal region of p300 with SF-1. These in
vivo results also indicate a stronger interaction between the
carboxyl region of p300 and SF-1, which concurs with the interaction
experiments performed in vitro.
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DISCUSSION |
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Regulation of the human P450scc gene has been examined in several
studies in which the promoter region was transfected into different
cell lines. However, depending on the cells used, different putative
cis-acting elements have been implicated to confer
regulation. The first studies in mouse adrenal Y1 cells (8-10)
identified an upstream region in the 5'-flanking DNA, which confers
responsiveness to cAMP and implicates a putative element TGATGTCA
homologous to the concensus CRE. These results were later confirmed in
mouse leydig MA-10 (11) and I-10 cells (12); however, experiments in
human JEG-3 cells localized a cAMP response element to a different region further downstream and implicate an Sp1 site (13, 58). Interestingly, recent studies in human adrenal NCI-H295 cells were
unable to localize a cAMP-responsive element that correlates with the
elements found in the other cells (14). The substantial differences
seen between the cell lines were generally thought to reflect
tissue-specific differences, since trans-acting nuclear factors are believed not to vary much among mammalian species. However,
the recent results in human adrenal NCI-H295 cells when compared with
results in mouse adrenal Y1 cells further raise the possibility of
species-specific differences. The putative CRE at nucleotide 1633 of
the human P450scc gene 5'-flanking region, which confers cAMP
responsiveness in Y1, MA-10, and I-10 cells, is found 11 nucleotides
upstream of an SF-1 binding site TCAAGGTCA, and it has been proposed
that both elements are required to confer a full cAMP response (17).
However, neither of these elements can confer a response to cAMP in
NCI-H295 cells as demonstrated in transient gene transfer experiments
(14). As seen in this study, the addition of 5'-flanking sequences from
nucleotides
110 to
1676, which introduced several putative SF-1
binding sites, conferred only a slight increase of SF-1 response beyond the activity obtained with the
110 construct. These results suggest that the SF-1 site TCAAGGCCA found between nucleotides
38 and
46
confers a response to SF-1. The relevance of this site to regulate the
P450scc gene in NCI-H295 cells is further demonstrated by the mutation
of two nucleotides which abrogates the SF-1/DNA complex and abolishes
any functional response to SF-1.
It has been demonstrated that steroidogenic gene promoters that are
inactive when transfected into nonsteroidogenic cell lines such as CV-1
(15, 18) and Hela (59) can be induced by expression of exogenous SF-1.
Considering the essential role of this factor to regulate steroidogenic
gene expression including P450scc, relatively little is known about the
mechanism of SF-1 action. This present study clearly demonstrates the
ability of p300 to mediate SF-1 function by increasing P450scc promoter
activity. The cotransfection of SF-1 with p300 or CBP regulated the
shortest promoter construct that contains only one SF-1 binding site at
position 46; however, the presence of additional sites in the longer
constructs conferred slightly higher responses to p300 and CBP in
NCI-H295 cells. Recent studies with the rat aromatase CYP19
gene (25) in rat granulosa and R2C Leydig cells indicate the
interaction of phosphorylated SF-1 and CREB, which bind to two adjacent
sites, similarly to the sites found in the upstream region of the human
P450scc gene 5'-flanking region. As stated by Carlone and Richards
(25), it is probable that CBP is involved in mediating the action of SF-1 and CREB on the rat aromatase gene; however, the role of the
analogous sites in the human P450scc gene remains to be determined in
human cells. In human placenta JEG-3 cells, endogenous SF-1 expression
has not been detected (60), and interestingly, the putative CREB
binding site at position
1633 does not confer a cAMP response (13,
58). In human adrenal NCI-H295 cells, the putative CREB binding site
also does not confer a cAMP response (14), despite the expression of
endogenous SF-1 (59).2
CBP/p300 proteins have also been speculated to play a role in conferring a basal and cAMP response of the bovine P450scc gene by Sp1
and SF-1 (61). Although the human P450scc promoter region also contains
a putative Sp1 binding site at position
117 found in close proximity
to the SF-1 binding site at
46, the addition of an Sp1 sequence
upstream of the
110 construct did not confer greater response to cAMP
(14) and had a minimal effect on the activity conferred by exogenous
SF-1. However, the longer constructs did confer a greater response to
the coexpression of SF-1 and p300 or CBP.
It is becoming clear that CBP and p300 are general coactivator
proteins, which can interact specifically with a wide array of
transcription factors and may serve as integrators of multiple signal
transduction pathways (62). CBP/p300 have been demonstrated to mediate
the activity of several factors such as MyoD (63), AP1 (64), SRC-1 (65,
66), p65 (67) including the nuclear receptors ER (68), RAR, RXR, and TR
(62, 69). It has been postulated that the negative cross talk between
nuclear hormones receptors and AP1, which all interact with CBP/p300,
may be the result of competition for interaction with limiting amounts
of coactivator proteins (64). Recently, Cheng et al. (70)
reported the positive cross-talk between nuclear hormone receptors and p45/NF-E2 mediated by CBP. Similarly, the interaction of SF-1 with
CBP/p300 may be a mechanism by which SF-1 can exert its effect on
P450scc gene expression by influencing the interaction of p300 with
other factors to trigger RNA polymerase II-dependent
transcription. In addition to the results shown in this study with the
human P450scc gene promoter, activation of the human P450c17 gene
promoter by SF-1 is also mediated by CBP/p300 in NCI-H295
cells.2 Similar to other nuclear receptors, SF-1 interacts
with the amino-terminal region of p300; however, SF-1 also binds p300
at the carboxyl-terminal region, which have been shown to interact with
several transcription factors including SRC-1 (50). The association of
SF-1 with both the amino- and carboxyl-terminal regions of CBP/p300 is
similar to their interaction with p65 of the NF-B family of
transcriptional activators (67). One possible explanation for
association with CBP/p300 at two independent sites is the simultaneous
interaction of two or more molecules of SF-1. Although it was
demonstrated that the SF-1 site at position
46 of the P450scc gene is
functional, the addition of putative sites further upstream led to a
higher response to SF-1 and p300. Binding of SF-1 to one site of the coactivator may facilitate occupancy of the other SF-1 site, leading to
recruitment of other factors required for transcriptional activity. Interaction with nuclear hormone receptors and receptor-mediated transactivation by CBP/p300 have been shown to be
ligand-dependent. However, the ligand for the orphan
nuclear receptor SF-1 has not been determined, and our results
demonstrate interaction between SF-1 and p300 without the addition of
exogenous ligand. Recently, it has been demonstrated that oxysterols
can enhance SF-1-dependent transcriptional activity,
however, it is unclear if these compounds serve as a ligand (71).
Human p300 was identified initially by its ability to bind the adenoviral E1A oncoprotein (72), which can transform primary cells, block cellular differentiation, and inhibit certain transcriptional enhancer elements. Binding of E1A to CBP/p300 has been shown to abolish or down-regulate the stimulatory effects of c-Fos (45), cJun, JunB (52), MyoD (63), and CREB (39, 56). In this study, the positive effect of SF-1 on P450scc gene promoter activity was also inhibited by E1A, which most likely is mediated by binding to endogenous p300. In agreement with previous studies, which show that mutation of E1A at position 2 can abolish interaction with p300 and alleviate its inhibitory effect, we show that RG2 is unable to inhibit the stimulatory effect of SF-1 and p300 on the P450scc gene promoter. As well, the deletion of residues 2-36 and 37-68 in E1A, which are regions postulated to interact with p300, also abolished its effect on SF-1 and p300.
Analogous to the action of E1A that can alter cellular differentiation,
SF-1 plays an important role in endocrine differentiation as well as
regulating sex determination and P450 steroid hydroxylase gene
expression. In addition to the many identified genes including P450c21,
P450scc, P450c17, P450 aromatase, 3-HSD, and Müllerian inhibiting substance, which are regulated by SF-1, it is likely that
other novel targets of SF-1 will be identified. Regulation of the
numerous steroidogenic genes by SF-1 most likely involves CBP/p300 to
integrate the effects of such factors as Sp1 and CREB. The interaction
of SF-1 with p300 is potentially a key step in the mechanism of SF-1
action; however, further studies will be required to identify the other
factors involved in the SF-1/p300 protein complex.
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ACKNOWLEDGEMENTS |
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We thank Dr. Bon-chu Chung (Academia Sinica, Nankang, Taipei), Dr. Richard H. Goodman (Oregon Health Sciences University), Dr. Ralf Janknecht (Hannover Medical School), Dr. Keith L. Parker (University of Texas Southwestern), Dr. Claude Labrie (Laval University), Dr. Elizabeth Moran (Cold Spring Harbor Laboratory), and Dr. Yang Shi (Harvard Medical School) for supplying plasmid constructs.
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FOOTNOTES |
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* This work is supported by the Medical Research Council of Canada no. MT12901, and the Fonds de la Recherche en Santé du Québec no. 950031-103 (to D. W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Holder of a postdoctoral fellowship from the Medical Research Council of Canada.
¶ To whom correspondence should be addressed: Laboratory of Molecular Endocrinology, Centre Hospitalier de l'Université Laval, 2705, Boul. Laurier, Sainte-Foy, Québec, G1V 4G2 Canada. Tel.: 418-654-2296; Fax: 418-654-2761; E-mail: Dean.Hum{at}crchul.ulaval.ca.
1 The abbreviations used are: SF-1, steroidogenic factor 1; Ad4BP, adrenal 4-binding protein; CRE, cAMP response element; CBP, CREB-binding protein; PCR, polymerase chain reaction; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin, CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis.
2 F. DeWitte and D. W. Hum, unpublished data.
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REFERENCES |
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