From the Department of Human Genetics and Cell and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618
Received for publication, September 22, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Gene regulation by steroid hormone receptors
depends on the particular character of the DNA response element, the
array of neighboring transcription factors, and recruitment of
coactivators that interface with the transcriptional machinery. We are
studying these complex interactions for the
androgen-dependent enhancer of the mouse sex-limited
protein (Slp) gene. This enhancer has, in addition to
multiple androgen receptor (AR)-binding sites, a central region (FPIV)
with a binding site for the ubiquitous transcription factor Oct-1 that
appears crucial for hormonal regulation in vivo. To examine
the role of Oct-1 in androgen-specific gene activation, we tested the
interaction of Oct-1 with AR versus glucocorticoid receptor
(GR) in vivo and in vitro. Oct-1
coimmunoprecipitated from cell lysates with both AR and GR, but
significant association with AR required both proteins to be DNA-bound.
This was confirmed by sensitivity of the protein association to
treatment with ethidium bromide or micrococcal nuclease. Addition of
DNA to micrococcal nuclease-treated samples restored interaction, even
when binding sites were on separate DNA molecules, suggesting
association was due to direct protein-protein interaction and not
indirect tethering via the DNA. AR/GR chimeras revealed that
interaction of the N and C termini of AR was required to communicate
the DNA-bound state that enhances interaction with Oct-1. Protease
digestion assays of hormone-bound receptors revealed further
conformational changes in the ligand binding domain of AR, but not GR,
upon DNA binding. Furthermore, these conformational changes led to
increased interaction with the coactivator SRC-1, via the NID 4 domain, suggesting DNA binding facilitates recruitment of SRC-1 by the AR-Oct-1
complex. Altogether, these results suggest that the precise arrangement
of binding sites in the Slp enhancer ensures proper hormonal response by imposing differential interactions between receptors and Oct-1, which in turn contributes to SRC-1 recruitment to
the promoter.
Androgen receptors (AR)1
and glucocorticoid receptors (GR) are members of the nuclear receptor
transcription factor superfamily (1). The steroid receptors share a
common structural arrangement and have been shown to recognize a
similar DNA-binding site, known as a hormone response element (HRE)
(2). Nevertheless, the hormones for each receptor elicit distinct
physiological functions. Multiple mechanisms have been demonstrated in
several cases to enforce steroid-specific gene activation, including
subtle preferences in DNA-binding site (3-5), domain interactions
within and between receptors that enhance cooperative function (6, 7),
and differential protein-protein interactions of the receptors with other regulatory factors (8, 9). The latter may include interactions
with other DNA-binding factors (e.g. AP-1, NFI, and Oct-1)
(10) or with transcriptional coactivators that do not themselves bind
DNA (e.g. SRC-1, GRIP1, and TIF2) (11). These interactions
sum to regulate the steroid response in a promoter- and cell
context-dependent manner.
To understand hormone-specific gene regulation, we study the mouse
sex-limited protein (Slp) gene, which expresses in adult male liver and kidney (12). Androgen dependence is mediated by a
120-base pair enhancer located 2 kb upstream of the gene (13). Although
both GR and AR are able to bind to HREs within the enhancer in
vitro, only androgens elicit a response in vivo. Previous studies have demonstrated that this androgen specificity is
determined by the interaction of AR with nonreceptor factors bound
adjacent to the HREs (14). One critical enhancer region, revealed
functionally in transfection assays and biochemically by in
vivo and in vitro protein binding studies, is called
FPIV (15, 16). This complex element includes an Oct-1-like recognition sequence and can bind several other proteins as well as Oct-1 in
vitro (17). Oct-1 is an intriguing candidate for interaction with
AR, in part because of its well established and diverse interactions with GR (18, 19). That is, Oct-1 can enhance or prevent glucocorticoid induction dependent on the precise sequence and arrangement of the
binding sites for these factors.
Oct-1 is a ubiquitous member of the POU-homeodomain family and is
involved in the regulation of a wide variety of genes (20). Previous
studies have demonstrated that Oct-1 can interact positively or
negatively with several nuclear receptors, in a promoter-specific manner. For example, with the mouse mammary tumor virus (MMTV) (21) and
gonadotropin-releasing hormone promoters (22), binding of both GR and
Oct-1 is required for transactivation. In contrast, GR repression of
the histone H2b promoter seems to involve sequestration of Oct-1 by GR
prior to DNA binding (23). These studies reveal alternative
actions of Oct-1 in nuclear receptor regulation, but the disparate
mechanisms responsible are not completely understood. Analysis has been
complicated in mammalian cells by pleiotropic effects of altering Oct-1
expression, making functional significance for steroid activation
difficult to determine. An alternative approach utilizing expression in
yeast cells has not yet overcome the transcriptional inactivity of
Oct-1 in that system (24).
To examine whether Oct-1 is involved in androgen specificity, we
compared physical interaction between Oct-1 and AR versus GR. Our data show that both AR and GR interact with Oct-1, but in
qualitatively different manners. First, in contrast to GR, AR interacts
well with Oct-1 only when both factors are bound to DNA. Second,
binding to the Slp enhancer induces selective changes in the
conformation of the ligand-binding domain of AR, but not GR, and leads
to increased interaction with Oct-1. Finally, the coactivator SRC-1
interacts more efficiently with the AR·Oct-1 complex when both
transcription factors are bound to DNA. These results suggest that
DNA-dependent protein-protein interactions, functionally
amplified by enhanced coactivator recruitment, may promote
receptor-selective activation. Thus differential interactions among
factors, rather than their stringent specificity, can confer precise
hormonal response.
Plasmids--
cDNAs for the mouse androgen receptor (mAR; D. Tindall) and the rat glucocorticoid receptor (rGR; K. Yamamoto)
contained in the eukaryotic expression vector, pCMV5, have been
described (16). Chimeras of AR and GR (AGA, GAG, GAA, and aGAA) have
also been described (7). For in vitro translation, the mouse
AR cDNA was excised from the expression vector and cloned into the
HincII site of pGEM4; rat GR cDNA was translated from
its original pC7 vector (pC7G). The pGEX3X vectors expressing
full-length Oct-1 and Brn-1 as GST-fused proteins were kindly provided
by N. Segil (25) and S. Young (26), respectively. To translate the AR DBD, the sequence encoding the first 37 amino acids of AR N terminus was fused via a SmaI site to the AR DBD and inserted into
pGem4. The chimeric receptors AGA, GAG, GAA, and aGAA were excised from pCMV5 at the KpnI/XbaI site and inserted into the
vector pGem3 for in vitro translation. For in
vitro translation, the AR dimerization mutant AR/R581D (gift of D. Pearce) (27) was subcloned from pCMV5 into pGem4. SRC-1 was in
vitro transcribed/translated from the pCR3.1 vector (gift from B. O'Malley). GST fusion proteins of SRC-1 NIDs were made by digesting
the pCR3.1 SRC-1 construct with XcmI/BamHI and
AvaI/XbaI. Fragments encoding NID 1-3 (amino acids 568-954; 1.1 kb) and NID 4 (amino acids 1139-1441; 1.2 kb) were
subcloned in the GST expression vector pGEX3.
Oligonucleotide Primers--
Fragments from the Slp
enhancer were generated by polymerase chain reaction, using the primer
sequences below. The oligo 1 (T) and (B) primers were used to produce a
70-bp DNA fragment containing the octamer-like binding site (FPIV) and
HRE-1 and -3 (15) (see Fig. 1C). Oligo 1 (T) and (B) are
complementary over 15 bases at their 3' ends; there is 16 bp
between HRE-1 and -3. Primers that introduced linker scanning mutations
into oligo 1 (oligos 5-8) have been described previously (3, 16). In oligo 5 the octamer-like binding site has been disrupted (italicized sequence), whereas in oligo 6 this site has been converted to the Oct-1
consensus sequence. Oligos 7 and 8 were used to mutate HRE-1 and HRE-3,
respectively. Oligo 2 and oligo 3 contain the binding sites for Oct-1
or receptor, respectively.
Cells and Transfection--
COS-7 cells were transiently
transfected with pCMV5 AR or GR DNA (10 µg/10 cm2 plate)
using the calcium phosphate method as previously described (28). The
DNA precipitates were left on the cells for 8 h, followed by a
glycerol shock with 15% glycerol in 1× HBS buffer (0.3 M NaCl, 50 mM HEPES, 1 mM
Na2HPO4, pH 7.05). After rinsing twice with
phosphate-buffered saline, cells were incubated in Dulbecco's modified
Eagle's medium, 8% charcoal-stripped fetal bovine serum containing
either 10 Whole Cell Extract Preparation--
Whole cell extracts were
prepared from transfected cells as described (29) with some
modifications. Cell pellets were resuspended in 1 ml of buffer A
(supplemented with 10 Western Blots--
For analysis of transfected cells, 5-10 µg
of whole cell extracts were resuspended in an equal volume of SDS
sample buffer and boiled for 5 min. Samples were electrophoresed on
7.5-8% SDS-PAGE gels. Proteins were transferred to a nitrocellulose
membrane using a semi-dry blotter (Buchler Instruments) at room
temperature (3 mA/cm2). Nonspecific sites were blocked for
30 min with 5% nonfat dry milk in Tween 20/Tris-buffered saline (TBS,
1% Tween 20, 10 mM Tris, pH 7, 5 mM NaCl).
Receptors and Oct-1 were revealed using antibodies specific for these
proteins (diluted in 5% nonfat dry milk, TBS), and a horseradish
peroxidase-conjugated secondary antibody (1:2000, ECL). The anti-AR
rabbit polyclonal antiserum used for Western blots was raised against a
GST fusion protein including amino acid residues 133-334 of mAR. GR
and Oct-1 were detected with mouse monoclonal antibodies (FIGR2 for GR,
a generous gift of W. Pratt, and 5G5 for Oct-1, kindly provided by N. Segil). The bands were visualized with the ECL chemiluminescence kit
(Amersham Pharmacia Biotech).
Coimmunoprecipitation Assays--
Coimmunoprecipitation
experiments were performed essentially as described by Kutoh et
al. (23). The lysates were precleared with protein A-Sepharose for
30 min and then incubated with either an anti-AR rabbit polyclonal
antibody directed against a peptide sequence between base 245 and 263 of mAR, or with anti-GR mouse monoclonal antibody FIGR2, for 1 h
at 4 °C. The antigen-antibody complexes were collected by the
addition of protein A-Sepharose, and pellets were washed in 20 mM sodium phosphate buffer and extracted with 25 µl of
SDS sample buffer. The eluted samples were analyzed on 7.5-8%
SDS-PAGE gels and subsequently blotted to nitrocellulose membranes.
Western blots were performed with anti-Oct-1 or receptor antibodies as
described above.
In some experiments the precipitated complexes were treated with
micrococcal nuclease (MNase) prior to elution (30). In assays where DNA
was added to the reaction, lysates were treated with 4 units of MNase
and 2 mM CaCl2 at 25 °C for 5 min (31), followed by centrifugation at 10,000 rpm for 1 min (4 °C). The supernatant was collected and 4-8 mM EGTA was added to
inhibit the nuclease activity. Before immunoprecipitation, these
MNase-treated extracts were incubated in the presence or absence of
30-100 ng of specific DNA or poly(dI/dC), for 15-30 min at
4 °C.
GST Pull-down Assays--
GST expression vectors were
transformed into Escherichia coli DH5 Limited Proteolytic Digestion--
Limited proteolytic digestion
of translated [35S]methionine-labeled receptors (AR and
GR) and chimeras (GAA and aGAA) with 20 µg/ml trypsin was carried out
essentially as described by Allan et al. (33). Before
proteolytic digestion, receptors were hormone-treated (100 nM DHT or Dex, as appropriate, for 10 min at room
temperature) and then incubated in the presence or absence of 100 ng of
oligo 1 (30 min on ice). 100 ng of nonspecific competitor, poly(dI/dC), was added to control samples. The difference in protease sensitivity among the receptors was quantified using a densitometer and scanning software (NIH Image 1.6) for which the results were plotted as previously described (34).
Binding to DNA Enhances Interaction of AR, but Not GR, with
Oct-1--
In vitro binding studies and analysis in
transfection of linker scan mutations of the Slp enhancer
support a role for Oct-1 in androgen-specific activation (17). To
confirm this, the ability of AR to associate with Oct-1 in
vivo was analyzed by coimmunopreciptitation with receptors
expressed in COS-7 cells by transient transfection (Fig.
1). AR or GR were precipitated from whole
cell lysates with receptor-specific antibodies, and immunoprecipitated
complexes were resolved by electrophoresis, and the presence of
associated Oct-1 was revealed by Western blot. The specificity of the
antibodies was demonstrated by immunoblotting the transfected COS-7
cell lysates (Fig. 1A). In the presence of AR or GR, Oct-1
coprecipitated with receptor antibodies but not with preimmune serum
(Fig. 1B). Interaction of GR with Oct-1 was readily detected
in the cell extract but was less evident for AR. Reprobing the Western
filters with receptor antisera suggested that the differences in
coprecipitated Oct-1 levels were not due to gross differences in levels
of receptor expression (Fig. 1B). Thus, Oct-1 detectably
interacts with both receptors in solution but apparently forms a more
stable or higher affinity complex with GR than with AR.
Since AR and Oct-1 presumably interact functionally when bound to DNA,
we asked whether adding their respective binding sites would allow
greater complex formation (Fig. 1C). Coimmunoprecipitation of Oct-1 with AR antibody was enhanced 2-3-fold by including a 70-bp
fragment from the Slp enhancer (oligo 1) containing binding sites for Oct-1 (within FPIV) and the receptors (HRE-1 and HRE-3). HRE-3 is a consensus binding site, whereas HRE-1 is a half-site which
AR, but not GR, appears to utilize in vivo (16). The
DNA-mediated increase in interaction with Oct-1 was not observed with
GR. Enhancement of the Oct-1 signal was not due to increased AR levels
in the immune complex (see Fig. 2). This
result suggested that binding to DNA enhanced the AR, but not GR,
interaction with Oct-1.
To distinguish whether binding to DNA promoted direct protein-protein
contact or simply tethered both factors via the nucleic acid,
coimmunoprecipitations were performed with separate oligonucleotides containing the binding site for Oct-1 (oligo 2) or receptor (oligo 3).
AR-Oct-1 interaction was strengthened even when both binding sites were
on separate molecules (Fig. 1C). Oligo 2 alone somewhat enhanced interaction, perhaps due to AR recognition of the partial HRE-1 included in its sequence (5 of 6 half-site bps). However, neither
binding site alone increased the association between AR and Oct-1 as
dramatically as the two together. Therefore, the DNA-dependent increase in AR-Oct-1 interaction involves
protein-protein contacts enhanced when both factors are DNA-bound.
AR Interaction with Oct-1 Depends on Sequence-specific DNA Binding
of Both Proteins--
To confirm the DNA dependence of AR-Oct-1
association, cell lysates were treated prior to immunoprecipitation
with reagents that disrupt DNA-protein interaction, by either
distorting DNA structure by intercalation (EtBr) or by degrading
nucleic acid (MNase), as described previously (30). Addition of 50 µg/ml EtBr reduced AR interaction with Oct-1 (Fig. 2) but did not
affect Oct-1 interaction with GR (see below). This suggested that the small amount of Oct-1 coprecipitated with AR from lysates without added
oligonucleotides (see Fig. 1B) might be due to residual cellular DNA. To eliminate this DNA, cell lysates were treated with
MNase, which was subsequently inactivated with EGTA. Similarly to EtBr,
MNase abrogated interaction of Oct-1 with AR but not GR (Fig. 2).
Furthermore, the association between AR and Oct-1 in MNase-treated
extracts could be restored by the addition of the enhancer DNA, oligo
1. In neither case was the amount of receptor precipitated affected.
DNA-dependent AR-Oct-1 interaction was also observed using
CV-1 (kidney) and LNCaP (prostate) cell lysates, confirming the
generality of the DNA
effect.2
To validate further the DNA-mediated increase in AR-Oct-1 interaction,
coimmunoprecipitations were performed with mutant enhancer fragments
(Fig. 3). Oligonucleotides 5, 7, and 8 contain mutations that disrupt binding of Oct-1 (oligo 5) or receptors
(oligo 7 and 8) to the enhancer. In oligo 6, the octamer-like site
within the FPIV region was converted to the consensus Oct-1 sequence. These mutations impair androgen response in transient transfection assays (16). Mutations that disrupt binding of either Oct-1 (oligo 5)
or AR (oligo 7 and 8) to the enhancer only weakly restored interaction
between AR and Oct-1 in MNase-treated COS-7 lysates (Fig. 3). However,
oligo 6, which increases Oct-1 binding to the enhancer (17), rescued
AR-Oct-1 interaction to nearly the same extent as the wild type
sequence. Thus, maximal DNA-dependent interaction of
AR-Oct-1 requires both AR and Oct-1 to be specifically bound to their
cognate elements.
Oct-1 Contacts the DBD of AR but Requires AR N/C Interaction for
DNA-dependent Enhancement--
To identify the receptor
domain(s) involved in the Oct-1 interaction and determine whether this
was sufficient to communicate DNA binding, GST pull-down assays were
performed with in vitro translated AR fragments (Fig.
4). Full-length receptors and fragments containing the LBD were treated following in vitro
translation with 100 nM of the appropriate hormone.
Interaction of full-length AR with GST-Oct-1 was specific since GST
alone did not retain the receptors nor did GST-Oct-1 retain luciferase
(Fig. 4A, upper panel). To confirm that the influence of DNA
binding could be detected in this approach, the pull-down experiment
was performed with full-length AR and GR in the presence and absence of
EtBr or MNase (Fig. 4A, lower panel). Interaction between AR
and GST-Oct-1 showed similar sensitivity to EtBr and MNase as in the
immunoprecipitation assays, and the interaction could be rescued after
MNase treatment by addition of enhancer DNA. Neither MNase nor EtBr
(not shown) treatments affected interaction of GR with Oct-1. In this
experiment, the source of "contaminating" DNA was apparently AR's
own cDNA used as the template for transcription/translation (data
not shown). HREs within the coding sequence of AR have been shown to be
involved in AR autoregulation (35). Thus, DNA binding stabilized the AR-Oct-1 interaction, regardless of the source of the receptor or the
influence of diverse cellular proteins.
To determine whether DNA dependence in AR-factor interactions was due
to an intrinsic instability of AR or to a particular association with
POU factors, we compared interaction with Brain-1 (Brn-1), a POU
protein expressed in kidney as well as neural tissue (36). In pull-down
assays, in vitro translated AR was retrieved with either
full-length Oct-1 or Brn-1 fused to GST (Fig. 4B). Whereas
both GST-Oct-1 and GST-Brn-1 interacted with AR, AR-Brn-1 association
did not require DNA binding (of either protein), as it was not
sensitive to EtBr. Thus DNA dependence is not a general feature of AR
interaction with other proteins.
Previous studies have shown that GR-Oct-1 interaction is mediated
through the receptor DNA binding domain (DBD) (21). For this reason,
the interaction of the AR DBD with GST-Oct-1, and its DNA dependence,
was examined in GST pull-down assays (Fig. 5). Interaction of the AR DBD with Oct-1
was insensitive to EtBr. Although MNase treatment reduced association
of Oct-1 and the AR DBD somewhat, addition of the enhancer fragment did
not increase interaction above that level. In general, protein-protein
interactions that were sensitive to EtBr and that could be rescued
after MNase treatment by the addition of DNA were considered
DNA-dependent. Thus, the AR DBD was sufficient for Oct-1
interaction but was not sufficient for enhanced interaction in the
presence of DNA.
To define the domains involved in communicating the DNA-bound state of
AR, AR and GR chimeras were tested for interaction with Oct-1 in the
presence and absence of DNA. These chimeras have been described
previously in functional studies of the Slp enhancer (7). In
the GAG and AGA chimeras, the AR and GR DBDs have been swapped. The
region of the DBD exchanged in the chimeras includes the residues
Cys-500 and Leu-501 of the GR DBD that are required for GR-Oct-1
interaction (21). Both GAG and AGA were retained by GST-Oct-1; however
AGA, but not GAG, was sensitive to MNase (Fig. 5B). This
confirmed that the DBD identity was insufficient for communicating the
effect of DNA binding. We reasoned that the N and C termini of AR might
be involved, as N/C interaction is fundamental to numerous AR functions
(37-39). To confirm this, receptor chimeras GAA and aGAA were tested
for DNA-dependent interaction with GST-Oct-1. In GAA, the N
terminus of GR is fused to the AR DBD and LBD. In aGAA, the first 37 amino acids of AR are fused to the translation start site of GAA. This
most N-terminal region of AR contains one of the two prominent sites
for interaction with the LBD of AR (40). In functional studies with the
Slp enhancer, aGAA conferred activation half that of AR
levels, whereas GAA behaved similar to GR and failed to transactivate
the androgen-specific target (7). In the GST pull-down assays,
interaction of aGAA, but not GAA, was sensitive to EtBr and MNase (Fig.
5B), suggesting that in fact AR N/C interaction was required
for DNA-dependence.
Since the interaction between the AR N terminus and LBD is required for
dimerization (39), we tested AR-Oct-1 DNA-dependent interaction with an AR mutant unable to form dimers (AR/R581D) (41).
Oct-1 interaction with AR monomers was weak and insensitive to EtBr
(Fig. 5C). Thus, DNA-dependent AR-Oct-1
interaction required both the N terminus and AR LBD and was influenced
by AR dimerization upon DNA binding.
AR, but Not GR, Is More Sensitive to Proteolysis When Bound to
DNA--
Enhancement of AR-Oct-1 interaction in the presence of DNA
suggested that binding to DNA induced a conformational change required for AR, but not GR, to interact with Oct-1. To test such an allosteric effect of DNA, limited proteolytic digestion of AR and GR was compared
in the presence and absence of oligo 1 (Fig.
6). Proteolytic analysis has been used to
contrast the conformation of unliganded receptor LBDs to LBDs occupied
by agonist or antagonist (33, 42). In vitro translated AR or
GR bound to hormone (100 nM DHT or Dex) was digested with
20 µg/ml trypsin for 1-10 min, with or without oligo 1. Trypsinization produced two major AR bands of 35 and 30 kDa and one
band of 35 kDa from GR (Fig. 6). Previous studies have shown that these
fragments derive from the LBDs of the receptors and are stabilized by
ligand (42, 43). The size of receptor fragments did not vary in the
presence of DNA (Fig. 6). However, the extent or rate of proteolytic
digestion differed between free versus DNA-bound receptors.
Autoradiograms from three independent experiments with each receptor
were scanned, and the density of the major LBD fragment at each time
point was determined relative to the density at 1 min without DNA;
these values are plotted in the graphs to the right in Fig.
6. Both the 35- and 30-kDa fragments of AR were more readily cleaved by
trypsin upon DNA binding. In contrast, the 35-kDa band of GR was, if
anything, more resistant to further digestion when GR was DNA-bound.
The effect of DNA binding on AR conformation was relatively subtle compared with that produced by ligand initially but was consistently demonstrable in these assays.
To examine the role of AR N/C interaction in the
DNA-dependent conformational change, trypsinization assays
were performed with the GAA and aGAA chimeras, as above (Fig.
7). The proteolytic pattern of the GAA
and aGAA chimeras was similar to the AR. However, the rate of digestion
of GAA was more similar to GR than to AR, although aGAA, like AR,
showed more sensitivity to trypsin when DNA-bound. Therefore, binding
to the Slp enhancer caused a detectable change in
conformation of the liganded AR, but not GR, LBD, which was in part
dependent on N/C interaction.
AR Interaction with the Coactivator SRC-1 Is Enhanced When Both AR
and Oct-1 Are DNA-bound--
That a conformational change in the
liganded AR LBD occurred upon DNA binding led us to conjecture that the
functional outcome might be to reposition the AF-2 surface in such a
way as to increase interaction with coactivators. We therefore tested
the influence of DNA on interaction of AR with steroid receptor
coactivator 1 (SRC-1), one of the p160 family whose members enhance
hormone response by direct interaction with ligand-occupied nuclear
receptors (11). The receptor LBDs interact with three nuclear receptor interaction domains (NID 1-3) of the p160s via LXXLL motifs
(44). SRC-1 is unusual in having a fourth NID (NID 4) close to the C terminus (see Fig. 8) that is involved in
facilitating AR N/C interaction and in enhancing AR activation (45,
46).
GST fusion proteins containing NID 1-3 or NID 4 of SRC-1 were
interacted with liganded receptors (Fig. 8). In vitro
translated AR association with GST-NID 4 was enhanced in MNase-treated
lysates by addition of oligo 1 (Fig. 8). However, association of AR
with the NID 1-3 region was not sensitive to such treatment. GR
interacted similarly with both NID 1-3 and NID 4 regardless of the
presence or absence of DNA.
Finally, we asked whether intact SRC-1 could be recruited to the
AR-Oct-1 complex in a DNA-dependent manner, by testing the ability of GST-Oct-1 to simultaneously retrieve AR and SRC-1 (Fig. 9). GST-Oct-1 was able to interact with
SRC-1 even in the absence of AR to some extent and appeared to enhance
interaction with AR when both were present (compare AR in
2nd and 4th lanes of top panel, Fig.
9). The SRC-1, AR, and Oct-1 ternary complex was sensitive to both EtBr
and MNase (Fig. 9). The multiprotein complex could be rescued after
MNase treatment, either by addition of the enhancer fragment or by
oligos 2 and 3 added together (Fig. 9, lower panel). Mutant
versions of oligo 1 incapable of binding AR or Oct-1 (oligos 5 and 7),
or oligo 2 or 3 individually, supported less interaction of SRC-1 and
AR with GST-Oct-1. Thus, recruitment of SRC-1 to the multiprotein
complex was more efficient when both AR and Oct-1 were DNA-bound.
Collectively, these results suggest that binding to the Slp
enhancer causes a conformational change in both AR and Oct-1 that
promotes protein-protein interaction and facilitates recruitment of the
coactivator SRC-1 to the ternary complex. In this manner, the
arrangement of the binding sites, rather than the specificity of the
individual factors, may contribute to differential hormonal
response.
We have contrasted the interaction of androgen and glucocorticoid
receptors with Oct-1 to determine whether the differential interplay of
generally versatile proteins can confer selective gene regulation.
Association of Oct-1 in cellular extracts with AR is weak relative to
GR but is markedly enhanced by inclusion of DNA fragments from the
androgen-specific Slp enhancer. This enhancer contains
intertwined binding sites for Oct-1 and receptor (16, 17). The
DNA-dependent AR-Oct-1 association requires that both
proteins are DNA-bound and that the N and C termini of AR are present
to communicate nucleic acid contact at the DBD. Both these features
suggest enhanced protein-protein interaction is due to conformational
changes imposed by the DNA rather than (or in addition to) DNA-directed
presentation of particular pre-existing protein facades. This is
supported by data from proteolytic digestions that reveal DNA-mediated
changes in the conformation of the liganded AR LBD, again dependent on
AR N/C interaction. A functional outcome of the altered conformation is
to enhance recruitment of the coactivator SRC-1, via its NID 4 domain.
Strikingly, SRC-1 detects the DNA-bound state of Oct-1 as well as AR.
Thus the coactivator may more efficiently integrate into a functional
transcription complex, directed by the precise array of enhancer
binding sites (see model in Fig. 10).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7 M dihydrotestosterone
(DHT) or 10
6 M dexamethasone
(Dex), as appropriate for 2 days prior to harvesting.
10 M DHT,
10
10 M Dex, and protease
inhibitors), homogenized by 20 strokes of a B pestle in a Dounce
homogenizer, and then centrifuged at 10,000 rpm for 10 min (4 °C).
The supernatant was collected, and the protein concentration was
determined by the Bradford method (Bio-Rad protein assay kit) using
bovine serum albumin as standard. Quality of extracts was assessed by
Western blot analysis for integrity of receptors.
cells and induced
with 0.2 mM
isopropyl-
-D-thiogalactopyranoside. The GST-fused
proteins were purified by glutathione affinity chromatography (32), and
the relative amount of bound fusion protein was determined by Coomassie
Blue staining of SDS-PAGE gels. The receptors were translated in
vitro using the coupled transcription-translation TNT reticulocyte
lysate system (Promega) in the presence of
[35S]methionine, using the manufacturer's protocol.
Following translation, appropriate hormone was added to each reaction
(100 nM DHT for AR, 100 nM Dex for GR). Binding
of in vitro translated receptors to isolated GST-fused
proteins was performed essentially as described by Préfontaine
et al. (21). Equal amounts (50 × 103 cpm)
of labeled proteins were incubated with 0.5 µg of immobilized GST
fusion proteins in 200 µl binding buffer (supplemented with protease
inhibitors) for 6-8 h at 4 °C. The precipitate was washed 5 times
with 1 ml of binding buffer. Retained proteins were eluted in SDS
sample buffer, electrophoresed, and analyzed by autoradiography (Autofluor; National Diagnostics). For GST pull downs of both SRC-1 and
AR simultaneously, equal amounts of labeled proteins (25 × 103 cpm) were incubated with 0.5 µg of immobilized
GST-Oct-1 as described above. Retained proteins were analyzed by
autoradiography as before.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA binding enhances interaction of Oct-1
with AR but not GR. A, AR and GR were transiently
expressed in COS-7 cells and detected by immunoblotting using 5 µg of
whole cell extracts to confirm efficient receptor expression.
Endogenous levels of Oct-1 (right panel of each pair) were
detected by reprobing the filter with Oct-1 antibody and appear
comparable in COS-7 and CV-1 cells, before or after transfection. LNCaP
and HTC cell extracts were used as positive controls for the expression
of AR and GR, respectively. Arrows show location of
full-length receptors and Oct-1. B, the ability of Oct-1 to
interact with receptors was tested by coimmunoprecipitation from
transfected COS-7 cell extracts with antisera
( -IP) specific for AR (ar), GR
(gr), or preimmune serum (pre). Mock-transfected
cell extracts (receptor
, last lane) were
immunoprecipitated with anti-AR to reveal specificity of complex
formation. Presence of Oct-1 in the complex was detected by Western
blot; Oct-1 coprecipitated efficiently with GR but much less so with
AR. Membranes were reprobed with anti-AR or anti-GR to confirm receptor
in the immunoprecipitation complex (lower panels).
C, coimmunoprecipitation of Oct-1 with receptor was tested
in the absence (
) or presence (+) of 30 ng of a fragment (oligo 1) of
the Slp enhancer (diagrammed above), containing receptor-
(HRE) and Oct-1 (FPIV)-binding sites, as shown. The sequence of the
fragment is given under "Materials and Methods." The 120-bp
androgen-specific Slp enhancer resides 2 kb upstream of the
start site of transcription; diamonds represent CBF
1
sites also critical for hormonal response. Reactions without specific
oligos contained 30 ng of poly(dI/dC). Oct-1 precipitation with AR was
markedly increased by the presence of specific DNA. In the lower
panel, oligos encompassing just the Oct-1- (oligo 2) or receptor
(oligo 3)-binding sites were added together or singly to
coimmunoprecipitation reactions, in comparison to the intact enhancer
fragment (oligo 1). Only when both DNA-binding sites were present,
whether in one or separate oligos, was protein interaction notably
enhanced. Specific DNAs (or poly(dI/dC) in the first sample) totaled 30 ng. Levels of binding were compared with 10% of the input lysate (5 µg) in both panels. All panels in this figure are representative of
at least six independent experiments.
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Fig. 2.
AR, but not GR, interaction with Oct-1 is
sensitive to treatments that disrupt DNA. In the upper
panel, coimmunoprecipitation of Oct-1 with AR from transfected
COS-7 cells was compared in the presence (+) or absence ( ) of oligo
1, as in Fig. 1, with the addition of 50 µg/ml EtBr where indicated
to test specific dependence on DNA structural integrity. This treatment
abolished the ability of Oct-1 to coprecipitate with AR. In the
lower panels, lysates were treated with 4 units of MNase
prior to immunoprecipitation with anti-AR (middle panels) or
anti-GR (lower panels), to degrade endogenous DNA. After
MNase treatment, lysates were incubated with 4 mM EGTA to
inactivate the enzyme, with or without oligo 1 as indicated. The
ability of Oct-1 to coprecipitate with AR was abrogated by MNase and
restored by subsequent addition of oligo 1, whereas coprecipitation of
Oct-1 with GR was unaffected by MNase and not enhanced by addition of
DNA. Uniform presence of receptors in the complexes was shown by
reprobing the membranes with anti-AR or anti-GR (lower panel
of pair). Panels are representative of at least eight
independent experiments.
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Fig. 3.
AR-Oct-1 interaction is most efficient when
both proteins are DNA-bound. Diagrammed relative to the enhancer
fragment (oligo 1) are mutant oligos designed to alter binding of Oct-1
or receptor. The octamer-like site within the FPIV region was mutated
to impair binding (oligo 5) or changed to match the Oct-1 consensus
element (oligo 6). Oligos 7 and 8 contain mutations in the AR-binding
sites of HRE-1 and -3. Oct-1 coimmunoprecipitation with AR was compared
in MNase-treated extract (except for untreated extract in the 1st
lane) as in Fig. 2, with addition of oligos as indicated
above each lane. Oligos 1 and 6, which have functional Oct-1
and HRE sites, led to efficient coprecipitation. Mutation of either HRE
(oligo 7 or 8) was sufficient to decrease AR-Oct-1 interaction. 20 µg
of lysate from COS-7 cells transfected with AR was used per sample.
This gel is representative of six independent experiments.
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Fig. 4.
Interaction of in vitro
translated AR with GST-Oct-1 is sensitive to EtBr and MNase
treatment. A, 50 × 103 cpm of
in vitro translated AR or luciferase were incubated with 0.5 µg of GST or GST-Oct-1 in the presence (+) or absence ( ) of 50 µg/ml EtBr. The labeled proteins were detected by autoradiography and
arrows indicate their expected migration. AR was retrieved
only by GST-Oct-1 in the absence of EtBr; nonspecific retrieval of
luciferase was not seen. In the lower panel, reticulocyte
lysates with in vitro translated AR or GR were treated with
4 units of MNase prior to incubation with GST (1st two
lanes) or GST-Oct-1 (next 6 lanes). After MNase
treatment, lysates were incubated with 4 mM EGTA in the
presence or absence of oligo 1 as indicated. AR, but not GR, binding to
GST-Oct-1 was severely decreased by MNase treatment but was restored by
addition of oligo 1. Levels of binding were compared with 10% of the
total input cpm. B, 50 × 103 cpm of
in vitro translated AR were incubated with 0.5 µg of GST,
GST-Brn-1 (Brn), or GST-Oct-1 (Oct) in the
presence (+) or absence (
) of 50 µg of EtBr. Brn-1, unlike Oct-1,
showed no dependence on DNA for interaction with AR. Levels of binding
were compared with 10% of the total input cpm. All experiments were
performed a minimum of three times.
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Fig. 5.
Interaction of AR N and C termini is required
for DNA-dependent AR-Oct-1 interaction. A,
50 × 103 cpm of in vitro translated AR DBD
(see "Materials and Methods") were incubated with 0.5 µg of
GST-Oct-1 (or GST alone in the 1st lane) in the presence (+)
or absence ( ) of 50 µg/ml EtBr or 4 units of MNase. After MNase
treatment and inactivation, oligo 1 was added as indicated. Input
lane contains 5% of the amount of in vitro translated
AR DBD used. The labeled proteins were detected by autoradiography.
Interaction of the AR DBD with GST-Oct-1 showed no effect of EtBr,
MNase treatment, or added DNA. B, 50 × 103
cpm of in vitro translated chimeric receptors AGA and GAG,
which are full-length AR and GR in which the DBDs have been swapped,
were tested for interaction with 0.5 µg of GST-Oct-1 in the presence
(+) or absence (
) of 4 units of MNase. After MNase treatment, oligo 1 was added as indicated. AGA but not GAG was sensitive to MNase in
interaction with Oct-1 and recovered upon addition of DNA. In the
lower panel, the chimeras GAA, which has the GR N terminus
fused to the AR DBD and LBD, and aGAA, which has the first 37 amino
acids of AR fused to GAA, were retrieved with GST-Oct-1 as described
above in the presence or absence of MNase or EtBr, and the labeled
proteins were detected by autoradiography. aGAA, but not GAA, was
sensitive to MNase in its interaction with Oct-1 but recovered upon
addition of DNA. C, the mutant AR R518D, which fails to
dimerize, was in vitro translated and retrieved with
GST-Oct-1 in the presence (+) or absence (
) of 50 µg/ml EtBr.
Compared with similar counts/min of AR in the 1st lane, AR
R518D interacts with Oct-1 less efficiently but is not dependent on
DNA. The input lane contains 10% of the amount of in
vitro translated AR R518D used. This experiment is representative
of three independent assays.
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Fig. 6.
Conformational change upon DNA binding is
apparent for AR but not GR. 50 × 103 cpm of
in vitro translated AR or GR were digested with 20 µg/ml
of trypsin for 0-10 min in the presence (+) or absence ( ) of 50 ng
of oligo 1. 100 nM DHT (AR) or dex (GR) was added to
receptors prior to protease digestion. Reactions without specific
oligos (
) contained 50 ng of poly(dI/dC). The trypsin-resistant
fragments of AR and GR were separated on 12.5% SDS-polyacrylamide gels
and detected by autoradiography. Arrows indicate receptor
fragments whose densities were quantified for the graphs to the
right by scanning with NIH image version 1.6. Points on the
graph represent the averaged density value from three independent
assays relative to the density of the AR 35-kDa protease fragment in
the absence of DNA at 1 min of digestion. Open circles are + DNA, black circles without DNA. * indicates
p < 0.05, by the Student's t test. AR but
not GR shows a statistically significant increased sensitivity to
protease in the presence of DNA.
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Fig. 7.
Conformational change of receptor upon DNA
binding involves N/C interaction. 50 × 103 cpm
of in vitro translated GAA, the chimeric receptor with the N
terminus of GR replacing that of AR, and aGAA, containing the first 37 amino acids of AR fused to the N terminus of GAA, were treated with 100 nM DHT and digested with 20 µg/ml trypsin in the presence
or absence of 50 ng of oligo 1 for the amount of time indicated, as in
Fig. 6. Resistant fragments of the receptor chimeras were detected by
autoradiography following PAGE; arrows indicate the
fragments whose densities were quantified for the graphs to the
right, as in Fig. 6. * indicates p < 0.05 by the Student's t test. aGAA, but not GAA, shows greater
sensitivity to trypsin in the presence of DNA.
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Fig. 8.
Interaction of NID 4, but not NID 1-3, of
SRC-1 with AR is increased in the presence of DNA. Shown at top is
a schematic representation of AR AF-1 and -2 regions and their
corresponding nuclear interaction domains (NID) in SRC-1.
In vitro translated AR or GR was retrieved with 0.5 µg of
GST-NID 4 (middle panel) or GST-NID 1-3 (lower
panel) in the presence (+) or absence ( ) of 4 units of MNase.
Oligo 1 was added after MNase treatment as indicated. Levels of binding
were compared with 10% of the total input counts/min. In all cases,
MNase treatment reduced interaction of receptors with SRC-1
subfragments; however, AR interaction with NID 4 was partially
recovered by addition of DNA.
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Fig. 9.
Recruitment of full-length SRC-1 to the
ternary complex is enhanced when both AR and Oct-1 are DNA-bound.
AR and SRC-1 were in vitro translated and tested singly and
together for interaction with 0.5 µg of GST or GST-Oct-1 in the
presence or absence of 50 µg/ml EtBr or 4 units of MNase (indicated
below the panel), as in Fig. 4. Levels of binding were
compared with 20% of the total input counts/min (50 × 103). The upper panel is without added DNA. In
the lower panel, MNase was inactivated with EGTA and the
oligos shown in Fig. 3 added as indicated below each
lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Model for DNA-dependent
protein-protein interactions. A, AR, Oct-1, and SRC-1
can interact in solution but with low affinity or stability.
B, binding to cognate sites within the enhancer induces
conformational changes in both AR and Oct-1 that favor their
interaction and facilitates recruitment of the coactivator SRC-1 to
promote transcription. One SRC-1 molecule is shown for simplicity
although the stoichiometry remains to be determined.
The interactions of AR and GR with Oct-1 differ qualitatively, in that association with GR, but not AR, is readily detectable in the absence of DNA. Previous studies demonstrated direct interaction between GR and Oct-1 via their DNA binding domains in the absence of DNA (21). This interaction in solution underlies glucocorticoid-induced repression of Oct-1 activation of the histone H2b promoter (23). Functional interaction between GR and Oct-1 also occurs on DNA, where the arrangement of the respective protein-binding sites dictates enhancement or repression of transcription. For example, on the MMTV promoter, hormonal induction is mediated by cooperative binding of GR and Oct-1 to adjacent sites (21). In contrast, repression of gonadotropin-releasing hormone transcription occurs by tethering of GR to DNA-bound Oct-1 (47). AR, like GR, cooperates with Oct-1 on the MMTV promoter, through multiple clustered HREs (19). AR-Oct-1 association on the Slp enhancer also requires both factors to be DNA-bound. The functional relevance of this is inferred since mutations that disrupt the AR-Oct-1 association in vitro also reduce androgen response of the enhancer in transfection. Synergism between transcription factors dictated by a precise array of binding sites has been shown for numerous promoters (9). In the case of FGF4, gene activation requires interaction between SOX 2 and Oct-3 that is dictated by the spatial arrangement of their cognate elements (48). In the case of the Slp enhancer, the DNA dependence of the AR-Oct-1 interaction, coupled with the particular array of consensus and nonconsensus binding sites, may accentuate the ability of AR, but not GR, to activate this element.
The DBD is a general prerequisite for DNA-dependent
effects, but it is interaction of the AR N and C termini that transmits the status of DNA binding to enhance association with Oct-1. This is
most apparent from experiments with AR/GR chimeric receptors, where
only those with both the N and C termini of AR (AGA and aGAA) show
sensitivity to EtBr or MNase, regardless of the identity of the DBD.
The N/C interaction promotes dimerization, ligand binding, and
functional synergism between AF-1 and AF-2 domains for PR and ER, as
well as AR (37, 39, 49). On the Slp enhancer, the N/C
interaction facilitates cooperative binding of AR, but not GR, thus
enhancing use of nonconsensus elements (7). Recently, N/C interaction
was shown to utilize two N-terminal sequences: 23FXXLF27, which contacts
primarily the AF-2 domain, and
433WXXLF437, which recognizes
a distinct region of the AR LBD (40). The FXXLF motif at
least appears critical for DNA-dependent association of AR
with Oct-1, since the first 37 amino acids of AR, including this
sequence, confer this ability on GAA when added to the N terminus
(aGAA). Conformational analysis of the liganded receptors by
proteolytic digestion reveals a subtle change in the AR, but not GR,
LBD in the presence of DNA. Previous studies of GR bound to HRE
sequences also reported absence of detectable changes in the GR LBD
(43). However, not all conformational changes are revealed by this
assay, since DNA-bound ER shows slower ligand dissociation but no
difference in sensitivity to trypsin (50). The greater sensitivity to
protease of aGAA compared with GAA supports the idea that
conformational differences of the AR LBD are due to N/C interaction.
Thus, DNA-dependent association of AR with Oct-1 is due to
DNA-mediated changes in the LBD that requires N/C interaction to
perceive modifications at the DBD surface. Conformational changes
induced upon DNA binding have also been reported for other members of
the nuclear receptor family. For example, binding to DR4 elements
induces conformational changes of retinoid X receptor in the
thyroid receptor/retinoid X receptor
heterodimer that modify
triiodothyronine-mediated transcription (34).
A functional outcome of the DNA-induced repositioning of the AR LBD appears to be increased recruitment of coactivators to the complex. SRC-1, a prototypic coactivator, interacts with the AF-2 region within the LBD by a central nuclear interaction domain that contains three LXXLL motifs (NID 1-3) or NR boxes (51). Unlike other p160s, SRC-1 contains an extra NR box (NID 4) that interacts with the AF-1 domain (52). The interaction of AR with the SRC-1, however, is unusual in comparison to that of other receptors, in that the AF-1 of the AR interacts with NID 1-3, whereas AF-2 contacts the NID 4 region (53, 54). Furthermore, SRC-1 enhances AR activation by facilitating N/C interaction. Additional reports indicate the SRC-1 interaction with AR is mediated by the N terminus and the DBD, whereas the AF-2 appears to be primarily involved in the N/C interaction (55, 56). Our results confirm that NID 4 interacts with the LBD of the AR. Furthermore, interaction with NID 4, but not with NID 1-3, is influenced by the DNA binding of the AR, since association is abrogated by EtBr treatment (not shown) and restored by addition of DNA following MNase treatment. Thus, the repositioning of either AF-2 or AF-1, or both, upon binding DNA seems to facilitate recruitment of SRC-1 in a manner somewhat particular to AR compared with other receptors. This unusual interaction may come into play in some cases of ligand-independent AR activity, such as in advanced prostate cancer.
In physical interaction assays with full-length SRC-1, we were
surprised to find that Oct-1 as well as AR associates directly with the
coactivator. Moreover, the formation of the multiprotein complex is
enhanced by the presence of DNA. This suggests that the functional
relevance of DNA dependence for both partners in the AR-Oct-1
interaction is to increase SRC-1 recruitment. Direct interaction
between SRC-1 and Oct-1 is not entirely unprecedented since AP-1 and
NF-B, which also functionally interact with nuclear receptors, also
recruit this coactivator (57, 58). In addition, the POU factor Pit-1
interacts with the coactivator CBP/p300, which is itself recruited by
SRC-1 (59). Furthermore, it was recently reported that a
DNA-dependent retinoic acid receptor/retinoid X
receptor interaction increases SRC-1 recruitment in a
ligand-dependent manner (60). This study also demonstrated
that retinoic acid receptor binding to a DR5 retinoic
acid response element facilitates recruitment of the corepressor N-CoR
in coimmunoprecipitation assays. The recently solved crystal structure
of the peroxisome proliferator receptor-
LBD shows a single
SRC-1 molecule can associate with a receptor dimer (61). The
stoichiometry of the multiprotein complex formed by AR, SRC-1, and
Oct-1 in the presence of DNA remains to be determined.
Similar to the interaction between AR and Oct-1, the association of
SRC-1 into the multiprotein complex relies on DNA elements and is
disrupted by the same mutations in these elements that disrupt AR or
Oct-1 binding. Furthermore, the binding sites for AR and Oct-1 need not
be contiguous for interaction with SRC-1, suggesting that the
coactivator is sensitive to contact between the transcription factors,
as well as their DNA-bound state (see Fig. 10). Direct contact between
Oct-1 and AR, together with the need for both factors to be DNA bound,
suggests SRC-1 is cooperatively recruited to the multiprotein complex.
Ultimately, it is the sequence and arrangement of binding sites in the
Slp enhancer that promotes a selective interaction between
Oct-1 and AR, rather than GR. That is, the nonconsensus Oct-1 binding
site overlaps a half-site HRE (HRE-1) shown in functional analysis to
be utilized by AR but not by GR (7). Binding of Oct-1 and AR to this
region induces a conformational change required for protein contacts.
This in turn facilitates cooperative recruitment of SRC-1 to a ternary complex that interfaces efficiently with the transcription machinery (see model in Fig. 10). Collectively, these results emphasize the context-dependent nature of transcriptional specificity,
where subtle preferences in DNA-binding sites, intrinsic differences in
the factors, and differential interactions with other nuclear proteins
sum to near-absolute selectivity in vivo.
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ACKNOWLEDGEMENTS |
---|
We thank our generous colleagues for the following reagents: Neil Segil for the Oct-1 antibody, 5G5, and the GST-Oct-1 plasmid; Bill Pratt for GR antibody, FIGR; Scott Young for the GST-Brn-1 plasmid; Dave Pearce for the AR mutant R581D; and Bert O'Malley for SRC-1 plasmid. We thank Alessandra Tovaglieri for help with the Brn-1 experiment. Michele Brogley and Elizabeth Hughes provided excellent technical assistance. Members of the laboratory, particularly Arno Scheller and Chris Krebs, provided invaluable advice throughout. We thank Ron Koenig for many helpful suggestions.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant DK-56356 (to D. M. R.).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.
Supported by training grants to the Reproductive Sciences Program
and the Cell and Molecular Biology Program.
§ To whom correspondence should be addressed. Tel.: 734-764-4563; Fax: 734-763-3784; E-mail: drobins@umich.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008689200
2 M. I. Gonzalez and D. M. Robins, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: AR, androgen receptor; GR, glucocorticoid receptor; GST, glutathione S-transferase; bp, base pair; kb, kilobase pair; HRE, hormone response element; MNase, micrococcal nuclease; MMTV, mouse mammary tumor virus; oligo, oligonucleotide; DHT, dihydrotestosterone; Dex, dexamethasone; PAGE, polyacrylamide gel electrophoresis; LBD, ligand binding domain; DBD, DNA binding domain.
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