From the Departments of Biological Sciences,
¶ Neuroscience, and
Pharmacology, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260 and the § Laboratory of
Receptor Biology and Gene Expression, NCI, National Institutes of
Health, Bethesda, Maryland 20892-5055
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ABSTRACT |
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An element required for glucocorticoid repression
of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the
distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1
present in GT1-7 cell nuclear extracts or by Oct-1 purified from HeLa
cells. Furthermore, purified full-length GRs interact directly with
purified Oct-1 bound to the distal nGRE. Increasing the extent of
distal nGRE match to an Oct-1 consensus site not only increases the
affinity of Oct-1 binding, but also alters the conformation of
DNA-bound Oct-1 and the pattern of protein DNA complexes formed
in vitro with GT1-7 cell nuclear extracts. In addition,
the interaction of purified GR with DNA-bound Oct-1 is altered when
Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE
to a consensus Oct-1 site is also associated with reduced
glucocorticoid repression in transfected GT1-7 cells. Furthermore,
repression of GnRH gene transcription by
12-O-tetradecanoylphorbol-13-acetate, which utilizes
sequences that overlap with the nGRE, is reversed by this distal nGRE
mutation leading to activation of GnRH gene transcription. Thus,
changes in the assembly of multi-protein complexes at the distal nGRE
can influence the regulation of GnRH gene transcription.
Gonadotropin-releasing hormone
(GnRH)1 is secreted by
neurons in the hypothalamus and is at the top of the endocrine axis
that controls reproductive function. In recent years, molecular studies of GnRH gene regulation have been facilitated by the development of the
immortalized GnRH-secreting GT1 cell lines (1). In these cell lines,
GnRH expression is regulated by various neurotransmitters (2-5),
second messengers, and other signal transduction pathways (6-14).
Our laboratory has utilized the GT1-7 cell line to investigate the
molecular mechanism responsible for glucocorticoid regulation of GnRH
gene expression. Glucocorticoids have been implicated in physiological
regulation of GnRH, as stress-related reproductive disorders and high
cortisol levels in women have been associated with reductions in
circulating luteinizing hormone (15-17). Glucocorticoids can directly
suppress gonadotropin secretion from the pituitary (18), but the
possibility that they also act at a hypothalamic level, i.e.
directly on GnRH neurons, has been suggested in a few physiological
studies (19, 20). The detection of glucocorticoid receptors (GRs), a
member of the steroid/thyroid hormone family of nuclear receptors
(21-23), within a subset of GnRH neurons (24), provides additional
support for the notion that glucocorticoids exert direct effects on
GnRH gene expression. The GT1-7 cell line also contains functional GRs
that, in the presence of glucocorticoid agonist dexamethasone, repress
GnRH promoter activity (7). Two regions of the mouse GnRH promoter, the
distal and proximal glucocorticoid response elements (nGREs), mediate
glucocorticoid repression of transcription (25). GRs do not bind
directly to either of these nGREs, but at the distal nGRE they are part
of a multi-protein complex that also includes Oct-1 (25), a member of
the POU domain family of transcription factors (26-30).
The fact that GR and Oct-1 co-occupy the distal nGRE (25) of the mouse
GnRH promoter suggests that functionally relevant interactions between
GR and Oct-1 may not be limited to solution as observed in other
studies. We therefore have examined whether GR interacts directly with
DNA-bound Oct-1. We show here that purified GR indeed can associate
with Oct-1 bound to the GnRH distal nGRE. This provides the first
evidence of glucocorticoid repression of transcription that is mediated
by the tethering of GR to a DNA-bound transcription factor. We
furthermore show that the nature of Oct-1 interactions at the distal
nGRE influences transcriptional repression, not only by
glucocorticoids, but also by the tumor-promoting phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA). Thus,
alternative conformational states of Oct-1, which are dictated by
precise DNA contacts made at different binding sites, may influence the
assembly of multi-protein complexes that impart unique regulatory
properties upon linked promoters.
In Vitro Mutagenesis--
DNA fragments containing 3446 base
pairs of the mouse GnRH promoter (25) were mutagenized to create a
consensus Oct-1 binding site at the distal nGRE using the
protocol supplied in the in vitro mutagenesis kit
(CLONTECH). The primer used for the mutagenesis, 5'-GCTAAGATTTGCATGACCAGG-3', contained nucleotide changes at base pairs
The mouse GnRH promoter containing either the wild type GnRH distal
nGRE or the consensus Oct-1 binding site linked to the luciferase
reporter gene (25) was digested with HindIII and BstXI to generate two fragments, one from base pairs Cell Culture and Transfections--
GT1-7 cells were grown as
described previously (25). 5 µg of plasmid DNA along with 5 µg of
herring sperm DNA carrier were transfected into GT1-7 cells on 60-mm
dishes using the calcium phosphate precipitation method as described
previously (25). The precipitate was allowed to sit on the cells for
12-16 h, after which the cells were washed with Tris-buffered saline
and refed with fresh medium. The cells were then treated with either
10 Statistical Analyses--
The mean luciferase activity for each
construct was determined from at least five independent experiments.
The luciferase activity between controls and after dexamethasone or TPA
treatment was compared using the Wilcoxon signed rank test. The percent change in luciferase activity generated by wild type and mutant reporters was compared using the Mann-Whitney rank-sum test.
Antibodies--
Either commercially available BuGR2 monoclonal
antibody or clone 57 polyclonal antibody (Affinity BioReagents,
Neshanic Station, NJ) was used to detect GR in protein-DNA complexes.
The antibody against Oct-1 was obtained from Santa Cruz Biologicals
(Santa Cruz, CA).
Electrophoretic Mobility Shift Assays (EMSAs)--
The sequences
of L7, L73M, and L7M oligonucleotides are shown in Fig. 1.
Single-stranded oligonucleotides were 32P-labeled with
polynucleotide kinase (31) and then hybridized to their complementary
strands to generate double-stranded probes. Nuclear extracts from
GT1-7 cells were prepared as described previously (25). 0.05 µM 32P-labeled double-stranded
oligonucleotide (L7 or L7M) was incubated with 1× gel shift buffer
(100 mM Tris-HCl, pH 8.0, 50% glycerol, 10 mM
EDTA, 10 mM dithiothreitol) and 1 µg/ml poly(dI-dC) in a 15-µl reaction volume with 5 µg of GT1-7 cell nuclear extract for
15 min at room temperature. To resolve protein-DNA complexes, the
reaction mix was run on native 10% polyacrylamide (75:1) gels in 1×
TBE (Tris borate, EDTA) buffer. Gels were electrophoresed in 1× TBE at
180 V for approximately 30 min. For competition assays, unlabeled
double-stranded competitor DNA was incubated with GT1-7 nuclear
extract for 10 min prior to the addition of radiolabeled probe. For
supershift assays with BuGR2 monoclonal GR antibody, 1 µl of BuGR2
was first incubated with GT1-7 extract for 10 min at room temperature,
after which the radiolabeled probe was added. The incubation was then
continued for another 15 min. For supershift assays with the Oct-1
polyclonal antibody, probe and nuclear extract were incubated for 15 min at room temperature, after which the antibody was added. The
incubation at room temperature was continued for an additional 30 min.
For EMSAs with purified Oct-1, 0.05 µM
32P-labeled double-stranded oligonucleotide was incubated
with 1× Oct-1 gel shift buffer (4% glycerol, 20 mM
Hepes-KOH 7.9, 60 mM KCl, 2 mM dithiothreitol, and 1 mg/ml BSA), 1 µg of poly(dI-dC), 1 µl of 10 mg/ml BSA (New England Biolabs, Beverly, MA). and 2 µl of purified Oct-1 for 15 min
at room temperature. For supershift assays. 2 µl of Oct-1 antibody
was added and the incubation continued for an additional 15 min.
Protein-DNA complexes were resolved as described above.
For EMSAs with purified Oct-1 and purified rat GR (32), 0.05 µM 32P-labeled double-stranded
oligonucleotide was incubated in 1× Oct-1 gel shift buffer (see
above), 1 µg of poly(dI-dC), 1 µl of 10 mg/ml BSA, 2 µl of
purified Oct-1, and 0.5 µl of purified GR. The reaction was incubated
for 15 min at room temperature and then 5 min on ice. Protein-DNA
complexes were resolved as described above.
Protease Sensitivity Assay--
0.05 µM
32P-labeled double-stranded oligonucleotide (L7 or L7M) was
incubated with 1× Oct-1 gel shift buffer in a 15 µl reaction volume
with 5 µg of GT1-7 cell nuclear extract for 15 min at room temperature. Sequencing grade trypsin (Promega, Madison, WI) was then
added at increasing concentrations (1.25-5 ng/ml) and the reaction
incubated for another 10 min on ice. To resolve protein-DNA complexes,
the reaction mix was run on native 10% polyacrylamide (75:1) gels in
1× TBE buffer. Gels were electrophoresed in 1× TBE at 180 V for
approximately 30 min.
Oct-1 Purification--
Oct-1 was either purified from HeLa
cells using established methods (33, 34) or from a
Drosophila cell (S2) expression system (Invitrogen Corp.,
Carlsbad, CA). In the latter case, FLAG epitope-tagged full-length
human Oct-1 was purified as described (35). Briefly, cells were lysed
by sonication, and the supernatant from a 11000 × g
centrifugation incubated with anti-FLAG M2 affinity gel (Sigma) at
4 °C overnight. The M2 agarose was washed five times and the bound
protein eluted by incubation with the FLAG peptide (Sigma) for 1 h
at 4 °C. The eluate was concentrated on a Centricon-30 centrifugal
concentrator (Amicon Inc., Beverly, MA), and aliquots were frozen at
Purified Oct-1 Binds to the GnRH nGRE--
Previous results from
our laboratory showed that Oct-1 present in GT1-7 cell nuclear
extracts binds to the mouse GnRH distal nGRE in vitro (25).
As the Oct-1-containing complex formed on this nGRE appeared to contain
multiple proteins, we wished to determine whether Oct-1 binding to this
site required GT1-7 cell-specific nuclear factors. Oct-1 was purified
from HeLa cell nuclear extract by a combination of wheat germ
agglutinin chromatography and DNA affinity chromatography as described
previously (33, 34). The purity of the Oct-1 preparation was confirmed
by silver staining, which showed a single protein band (data not
shown). The DNA binding activity of the purified Oct-1 protein was
tested using a consensus Oct-1 binding site (29) oligonucleotide
(i.e. L73M) that was derived from the GnRH distal nGRE (Fig.
1). The 23-base pair L73M and 55-base
pair L7M oligonucleotides (hereafter designated "consensus Oct-1
site") contain two nucleotide substitutions at the Oct-1 homology
sequence in the GnRH distal nGRE. As shown in Fig.
2A (lane
2), purified Oct-1 binds strongly to the consensus Oct-1 site. Oct-1 binding to this sequence is not competed effectively by the
distal GnRH nGRE (Fig. 2A, lane 3),
suggesting that Oct-1 binds to the consensus Oct-1 site more strongly
than to the GnRH distal nGRE. Oct-1 binding to the consensus site is
competed by excess cold consensus site oligonucleotide (data not shown)
and is completely inhibited by the inclusion of an Oct-1 antibody in
the binding reaction (Fig. 2A, lane
4).
The inability of the GnRH distal nGRE to compete with the consensus
Oct-1 site for Oct-1 binding was not surprising, given the limited
homology of the Oct-1 sequence in this nGRE to a consensus Oct-1 site
(Fig. 1). Specifically, the Oct-1 binding site in this element has only
a 5/8 match on the bottom strand and 6/8 match on the top strand to a
consensus Oct-1 binding site. Nonetheless, as shown in Fig.
2B (lane 2), purified Oct-1 also binds
the distal GnRH nGRE oligonucleotide L7, although with lower affinity
than to the nGRE mutant containing a consensus Oct-1 site. A 250-fold molar excess of the consensus Oct-1 site effectively competes for
binding with the distal nGRE (Fig. 2B, lane
3). As shown previously, a 250-fold molar excess of the
distal nGRE is not able to compete for binding with the consensus Oct-1
site (Fig. 2A, lane 3). The fact that
the protein-DNA complex formed on the distal nGRE contains Oct-1 and
not a contaminant in our Oct-1 preparation was confirmed by the ability
of an Oct-1 antibody to prevent the formation of an Oct-1·nGRE
complex (Fig. 2B, lane 4). Thus, Oct-1
has the capacity to interact directly with the GnRH distal nGRE
in vitro, i.e. in the absence of other GT1-7
nuclear proteins.
The Nature of the Oct-1 Binding Sequence within the Distal GnRH
nGRE Influences the Binding of GT1-7 Nuclear Factors--
The binding
of purified Oct-1 to the GnRH distal nGRE generates a single
protein-DNA complex, which is indistinguishable in its electrophoretic
mobility from the complex formed on the consensus Oct-1 site. When
crude GT1-7 cell nuclear extract was used, multiple distinct
protein-DNA complexes formed on the distal nGRE (Fig. 3A, lane
3). Previously, we showed that one multi-protein DNA complex
formed on this nGRE (i.e. complex C1-L7) contains Oct-1 and
GR (25). Interestingly, when the consensus Oct-1 site oligonucleotide was incubated with GT1-7 cell nuclear extract, only a single
protein-DNA complex was formed (Fig. 3A, lane
4). As expected from our previous studies with purified
Oct-1 (see Fig. 2), the distal nGRE did not effectively compete with
the consensus Oct-1 containing oligonucleotide for binding of Oct-1
present in the GT1-7 cell nuclear extract (data not shown). A
comparison of the competitive strength of the wild type nGRE
versus the consensus Oct-1 sequence for GT1-7 nuclear
extract binding revealed that the apparent affinity of GT1-7 nuclear
extract for the consensus Oct-1 sequence is approximately 10-fold
higher than for the GnRH distal nGRE (data not shown). Furthermore, the
Oct-1 containing C1-L7M protein-DNA complex, formed on the consensus
Oct-1 site had a slightly increased electrophoretic mobility as
compared with the Oct-1-containing complex, C1-L7, formed on the distal
nGRE (Fig. 3A, compare lanes 4 and
3). This apparent migration difference, although small, was
noted in multiple EMSAs performed with these oligonucleotides using
different GT1-7 cell nuclear extract preparations (data not shown).
These large complexes migrate slowly through the 10% polyacrylamide
gels and are difficult to resolve. However, we were able to resolve the distal nGRE-protein complex, C1-L7, and the consensus Oct-1
site-protein complex, C1-L7M, on a lower percentage gel (Fig. 3B,
lanes 1 and 2). As will be shown below, this
differential migration most likely reflects the fact that GR is present
within the multi-protein C1-L7 complex, but not the C1-L7M complex (see
Fig. 3C).
In addition to Oct-1, one multi-protein complex formed on the distal
nGRE when using GT1-7 cell nuclear extracts also includes GR (25). To
determine whether GR is also present in the complex formed at the
mutant nGRE possessing a consensus Oct-1 binding site, we added the
BuGR2 anti-GR antibody to a binding reaction containing the mutant nGRE
and GT1-7 cell nuclear extract. As shown in Fig. 3C
(lanes 1 and 2), BuGR2 does not
supershift the complex formed on the consensus Oct-1 binding site.
Thus, increasing the affinity of Oct-1 binding site by mutation of the
GnRH distal nGRE alters the recruitment of GR into a multi-protein
complex formed on this site with GT1-7 cell nuclear proteins.
GR Interacts with Oct-1 at the GnRH Distal nGRE--
GRs contained
within a multi-protein complex on the distal nGRE do not bind DNA
directly at this site (25). The only GT1-7 cell nuclear protein that
we have established is directly bound to the distal nGRE is Oct-1 (25).
Does GR bind Oct-1 in this complex and does GR distinguish between
Oct-1 bound at either a consensus site Oct-1 site or the distal nGRE?
GR and Oct-1 have been shown to interact in vitro in the
absence of DNA. In fact, it has been postulated that repression of
histone H2b promoter activity by glucocorticoids is brought about by
the sequestration of Oct-1 by GR in solution (29). Furthermore,
although GR and Oct-1 can bind simultaneously in vitro to
two distinct sites on the mouse mammary tumor virus long terminal
repeat (MMTV LTR; Ref. 23), GR and Oct-1 have never definitively been
shown to interact with each other while DNA-bound. In order to reveal
whether GR and Oct-1 can co-occupy the GnRH distal nGRE in
vitro, we performed EMSAs with this nGRE using purified
preparations of rat GR (32) and Oct-1. As shown in Fig.
4 (lanes 1 and
2), an Oct-1·nGRE complex (i.e. complex C1-L7)
formed in vitro was supershifted (i.e. complex S1-L7) by the addition of purified GR to the binding reaction. The
supershift occurs only when purified GR is added to Oct-1 and not when
another protein fraction from the GR purification procedure is
incubated with Oct-1 (data not shown). We also tested the ability of
purified GR and Oct-1 to bind to the mutant nGRE possessing a consensus
Oct-1 binding site. The binding of Oct-1 to the consensus Oct-1 binding
site oligonucleotide (complex C1-L7M) is also supershifted by the
addition of GR (Fig. 4, lanes 3 and 4), but this supershift (complex S1-L7M) is much less
pronounced than the GR supershift of the Oct-1·wild type nGRE complex
(Fig. 4, compare lanes 2 and 4).
Therefore, GR appears to interact differently with Oct-1 depending upon
the context of the Oct-1 binding site. It is surprising that purified
GR interacts with Oct-1 at the consensus site since GR was not part of
the protein complex formed at the consensus Oct-1 binding site in EMSAs
with GT1-7 nuclear extract (see Fig. 3C). This may be due
to differences in the relative ratios of GR and Oct-1 in EMSAs with
purified proteins versus GT1-7 nuclear extract. Thus, GR
can interact with DNA-bound Oct-1 in vitro, particularly at
the wild type GnRH distal nGRE. Altering the sequence of the Oct-1
recognition site within the nGRE may affect GR interactions with
DNA-bound Oct-1, but additional characterization of these ternary
Oct-1·GR·DNA complexes is required to fully address this issue.
Oct-1 Adopts a Different Conformation When Bound to the Distal GnRH
nGRE Versus a Consensus Oct-1 Site--
POU domain proteins such as
Oct-1 exhibit some flexibility in their interaction with target DNA
sites (36, 37). Such diversity in sequence recognition allows for the
recruitment by Oct-1 of unique transcriptional coactivators (37). This
selectivity is dictated, in part, by the ability of Oct-1 to adopt
different conformations at different binding sites (37). Since GR
interactions with Oct-1 in vitro were altered upon
conversion of the distal GnRH nGRE to a consensus Oct-1 site, we set
out to examine whether Oct-1 conformation was indeed distinguishable at
these two sites.
Purified Oct-1 bound to either an nGRE or a consensus Oct-1 site was
subjected to a limited trypsin digestion (38, 39) and resulting
protein-DNA complexes examined by EMSAs. As shown in Fig.
5A, treatment with increasing
amounts of trypsin resulted in the appearance of trypsin-resistant
fragments on both the L7 and L7M probes. However, Oct-1 bound at these
different DNA elements exhibited a differential sensitivity to trypsin.
Complexes labeled 1 and 2 represent intact Oct-1
bound to the L7 and L7M probes (Fig. 5A, lanes
2 and 8). When bound to the consensus Oct-1 site, predominant trypsin-resistant Oct-1-DNA complexes, i.e.
designated a-e (Fig. 5A, lanes
9-12) were observed, while at the nGRE, a different set of
trypsin-resistant Oct-1·DNA complexes, i.e. A and B (Fig. 5A, lanes 5 and
6), were noted. Complexes A and B on
the distal nGRE exhibit different electrophoretic mobilities than
complexes d and e on the consensus Oct-1
oligonucleotide. The difference in mobility is particularly evident
when lanes 5 and 11 are placed next to
each other (Fig. 5B, lanes 1 and
2).
The Nature of the Oct-1 Recognition Sequence within the Distal GnRH
nGRE Influences Glucocorticoid and TPA Effects on GnRH Promoter
Activity--
The GnRH distal nGRE contributes to glucocorticoid
repression of GnRH promoter activity (25). As this repression is
mediated by GR recruitment to this site, we set out to examine whether subtle differences in GR interaction with the nGRE generated by altering Oct-1 binding affinity and conformation affected
glucocorticoid regulation of GnRH promoter activity in vivo.
A 5'-deletion of the GnRH promoter to
Transcription from the mouse and rat GnRH promoters is also repressed
by TPA (a tumor-promoting phorbol ester; Refs. 8-10). The elements
responsible for this repression (i.e. the negative TPA-responsive elements or nTREs) have been mapped to a region of the
promoter that coincides with the nGREs (40). The mechanism of this
repression has not been definitively established, although in one model
down-regulation of protein kinase C has been implicated as being
responsible for TPA effects (40). Given the effect of the Oct-1
mutation in the distal nGRE on glucocorticoid repression, we examined
whether effects of TPA on the promoter would also be altered by
conversion of the Oct-1 binding site at the distal nGRE to a consensus
Oct-1 binding site. As shown in Fig. 6, mutation of the Oct-1 site
within the distal nGRE to a consensus Oct-1 binding site reversed TPA
effects on promoter activity, i.e. TPA treatment led to
induction of GnRH promoter activity. Specifically, TPA treatment
significantly decreased luciferase activity generated from the wild
type Our results provide the first demonstration of GR co-occupancy on
an nGRE that does not involve direct DNA binding by GR (41), but rather
protein-protein interactions between GR and a DNA-bound transcription
factor. Furthermore, the mechanism of glucocorticoid repression of GnRH
that we have elaborated is distinct from other examples of repression
that are mediated by either direct DNA binding of GR (42) or the
association of GR with other transcription factors or coactivators in
solution (29, 43, 44). Glucocorticoid repression of GnRH gene
transcription in GT1-7 cells is dictated by two distinct nGREs that
lie close to the GnRH promoter (25). Although GRs do not bind directly
to these nGREs, they appear to be recruited to these elements by virtue
of their interaction with POU domain transcription factors (25). We had
previously suggested that Oct-1 could be responsible for directing GR
to the distal GnRH nGRE since both GR and Oct-1 in GT1-7 nuclear extracts were components of a unique protein-DNA complex formed in vitro on this nGRE (25). In this report, we demonstrate
that the in vitro binding of purified Oct-1 to the distal
nGRE can occur in the absence of any other GT1-7 cell-specific nuclear factors. Furthermore, purified GR can bind directly to nGRE-bound Oct-1
in vitro, also in the absence of any GT1-7 cell specific nuclear factors.
Oct-1 and GR have previously been shown to participate in both
transcriptional activation (21-23, 33, 45-51) and repression (29,
41-43, 52-54). Glucocorticoid activation of transcription from the
MMTV LTR promoter requires the binding of GR and Oct-1 to separate
sites (23). The simultaneous direct binding of GR and Oct-1 has been
observed on the relevant sites within MMTV LTR DNA in vitro
(55), but there is no evidence for direct interaction between these two
DNA-bound factors. Glucocorticoid repression of histone H2b gene
transcription is apparently brought about by the association of GR with
Oct-1 in solution, which eliminates Oct-1 binding to the histone H2b
promoter (29). It is important to note that the Oct-1 binding site in
this case does not appear to be co-occupied by GR and Oct-1, as we have
shown for the GnRH distal nGRE. The interaction between Oct-1 and GR in
solution requires the receptor DNA-binding domain (29). The
identification of GR domains involved in interactions with DNA-bound
Oct-1 at the GnRH distal nGRE will be the subject of future studies.
How do we account for the clear distinctions between GR repression of
transcription of histone H2b that involves GR·Oct-1 interactions in
solution (29) versus repression of GnRH gene transcription
where GR associates with DNA-bound Oct-1? As we have demonstrated in
this report, Oct-1 binds with relatively low affinity to the GnRH
distal nGRE, which was expected given the limited degree of homology
between this nGRE and an Oct-1 consensus sequence (25). However, the
relatively weak binding of Oct-1 to the distal nGRE appears to be an
essential feature of the mechanism of glucocorticoid repression. When
the distal nGRE is mutated to increase Oct-1 binding capacity,
glucocorticoid repression in transfected GT1-7 cells is affected.
Furthermore, GR does not associate as effectively in vitro
with tightly bound Oct-1 at the Oct-1 consensus site nGRE. This was
shown in examinations of GR·Oct-1 interactions on this consensus site
nGRE with both purified GR and Oct-1 and GT1-7 cell nuclear extracts.
As discussed by others (for review, see Ref. 27), we hypothesize that
DNA-bound Oct-1 may adopt different conformations depending upon the
precise nature of its recognition sequence. In fact, using protease
sensitivity of Oct-1·DNA complexes as an assay, we provide direct
evidence for alternative Oct-1 conformations at the distal GnRH nGRE
versus a consensus Oct-1 site. As diagrammed in Fig.
7A, the conformation adopted
by Oct-1 upon its binding to the GnRH distal nGRE may allow efficient
tethering of GR (or other factors) and lead to
glucocorticoid-dependent repression of transcription. Altering
the Oct-1 recognition sequence within the nGRE may still allow Oct-1
binding, but generate a different Oct-1 conformation that recruits a
distinct set of co-factors to this element (Fig. 7B).
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
206 and
207 of the mouse GnRH promoter, to generate a consensus
Oct-1 binding site at the mouse GnRH distal nGRE. The GnRH nGRE
mutation was confirmed by dideoxy sequencing (31).
3446
to
471, and the other containing the GnRH promoter up to base pair
471 linked to the luciferase reporter gene. The GnRH promoter deletion to base pair
471 was then blunt-ended and ligated to generate the
471mGnRH-Luc.
6 M dexamethasone or 100 ng/ml TPA (Sigma)
where indicated. Following a 24-h incubation, cells were harvested and
assayed for luciferase activity (Luciferase kit, Promega, Madison, WI)
using the same amount of total protein from each of the plates.
140 °C.
RESULTS
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Fig. 1.
Sequence of the L7, L7M, and L73M
oligonucleotides. The sequences of the L7 (distal GnRH nGRE, 238
to
201), L73M, and L7M oligonucleotides are shown. The region of the
L7 oligonucleotide that is homologous to the Oct-1 binding site is
overlined and underlined, and the mismatches to
the perfect Oct-1 site are shown by asterisks. The L7
exhibits a 5/8 match to a consensus Oct-1 binding sequence
(5'-ATGCAAAT-3') on the top strand and a 6/8 match on the bottom
strand. Both L7M and L73M contain a consensus Oct-1 binding site
(underlined), which was derived by mutating the two
nucleotides at positions
206 and
207 of the mouse GnRH promoter.
These nucleotides are shown by asterisks in the L7 sequence.
L7M contains base pairs
237 to
201 of the GnRH distal nGRE, and
L73M contains base pairs
218 to
201 of the GnRH distal nGRE.
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Fig. 2.
Pure Oct-1 binds to the consensus Oct-1
binding site and to the GnRH distal nGRE. A, 2 µl of
pure Oct-1 was incubated with 0.05 pmol of 32P-labeled L73M
oligonucleotide for 15 min at room temperature (lane
2) as described under "Experimental Procedures."
Protein-DNA complexes were resolved on a 10% polyacrylamide gel
(75:1). For the competition analysis, 250-fold molar excess of
unlabeled L7 oligonucleotide (GnRH distal nGRE) was also included in
the reaction mixture (lane 3). For EMSAs in the
presence of Oct-1 antibody (lane 4), 2 µl of
Oct-1 was first incubated with 0.05 pmol of 32P-labeled L7M
oligonucleotide for 15 min at room temperature, after which 2 µl of
Oct-1 antibody was added and reaction continued for another 15 min at
room temperature. The Oct-1-DNA complex is shown by an arrow.
B, 2 µl of pure Oct-1 was incubated with 0.05 pmol of
32P labeled L7 oligonucleotide for 15 min at room
temperature (lane 2) as described under
"Experimental Procedures." The protein-DNA complexes were resolved
on a 10% polyacrylamide gel (75:1). For competition analysis, 250-fold
molar excess of unlabeled L7M oligonucleotide was included in the
reaction mixture (lane 3). For EMSAs in the
presence of Oct-1 antibody, after the incubation of pure Oct-1 with the
32P-labeled L7 oligonucleotide, 2 µl of an Oct-1 antibody
was added and the reaction incubated for another 30 min at room
temperature (lane 4). The complex that represents the Oct-1
DNA complex is shown by an arrowhead.
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Fig. 3.
In vitro binding of GT1-7
nuclear proteins to the consensus Oct-1 binding site
oligonucleotide. A, 5 µg of GT1-7 nuclear extract
was incubated with 0.05 pmol of 32P- labeled L7 or L7M
oligonucleotides (lanes 3 and 4,
respectively) for 15 min at room temperature as described under
"Experimental Procedures." The protein DNA complexes were resolved
on a 10% polyacrylamide (75:1) gel. The protein-DNA complex
(i.e. C1-L7), which was previously found to include GR and
Oct-1 (25), is shown by an arrowhead, and the protein-DNA
complex, C1-L7M, formed by consensus Oct-1 site oligonucleotide is
shown by an arrow. Lanes 1 and 2 contain L7 and L7M probes, respectively, incubated in the absence of
GT1-7 nuclear extract. B, 3 µl of GT1-7 nuclear extract
was incubated with 0.5 pmol of 32P-labeled L7 (lane
1) or L7M (lane 2) for 15 min at room
temperature. The complexes were resolved on a 8% (75:1) polacrylamide
gel. C, 5 µg of GT1-7 nuclear extract was incubated with
0.05 pmol of 32P-labeled L7M oligonucleotide for 15 min at
room temperature (lane 1). For EMSAs in the
presence of the anti-GR antibody BuGR2, 2 µl of the antibody was
first incubated with 5 µg of GT1-7 nuclear extract for 15 min at
room temperature. 0.05 pmol of 32P-labeled oligonucleotide
was then added and the reaction incubated for another 15 min at room
temperature (lane 2). L7M oligonucleotide
incubated with GT1-7 nuclear extract in the absence of BuGR2 antibody
is shown in lane 1. The protein-DNA complexes
were resolved on a 10% polyacrylamide gel (75:1). The major
protein-DNA complex C1-L7M formed by GT1-7 nuclear extract with the
L7M oligonucleotide is shown by an arrow.
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Fig. 4.
Purified GR interacts with Oct-1 in
vitro on the mouse GnRH distal nGRE. 2 µl of purified
Oct-1 was incubated with 0.05 pmol of 32P-labeled L7 or L7M
oligonucleotide (lanes 1 and 3, respectively) in 1× Oct-1 binding buffer for 15 min at room
temperature as described under "Experimental Procedures." For
interaction of purified GR with Oct-1, 0.5 µl of purified GR was
first incubated with 2 µl of pure Oct-1 for 15 min at room
temperature and then for 5 min on ice. 0.05 pmol of
32P-labeled L7 or L7M oligonucleotide (lanes
2 and 4, respectively) was then added and then
reaction continued for another 15 min at room temperature. The
protein-DNA complexes were resolved on a 10% polyacrylamide gel (75:1)
at 4 °C. This gel was electrophoresed for a relatively long period
of time (relative to other gels in this report) in order to maximize
the resolution between closely spaced complexes. Complexes C1-L7 and
C1-L7M, shown by arrows, represent Oct-1-bound L7 and L7M
oligonucleotides, respectively. The supershifted complexes on the L7
oligonucleotide, S1-L7 and S2-L7, respectively, are shown by
arrows. The supershifted complex on the L7M oligonucleotide,
S1-L7M, is also shown by an arrow.
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Fig. 5.
Purified Oct-1 adopts a different
conformation on the distal nGRE and the consensus Oct-1 binding
site. A, 2 µl of human Oct-1 expressed in
Drosophila cells (S2) was incubated with 0.05 pmol of
32P-labeled L7 (lanes 1-6) or L7M
oligonucleotide (lanes 7-12) in 1× Oct-1
binding buffer for 15 min at room temperature as described under
"Experimental Procedures." Increasing amounts of trypsin were then
added and the reaction incubated on ice for an additional 10 min.
Lanes 1 and 7 contain the L7 and L7M
oligonucleotide probes alone; lanes 2 and
8 contain probe and Oct-1 but no trypsin. Oct-1 purified
from the Drosophila cell expression system forms two
complexes, complex 1 and complex 2, with both both the L7 and L7M
oligonucleotide probes. Lanes 3 and 9 contain 1.25 ng/ml trypsin, lanes 4 and
10 contain 2.5 ng/ml, lanes 5 and
11 contain 3.75 ng/ml, and lanes 6 and
12 contain 5 ng/ml. The protein-DNA complexes were resolved
on a 10% polyacrylamide gel (75:1) at 4 °C. The protein-DNA
complexes formed with the L7 probe upon digestion with trypsin are
labeled A and B. The protein-DNA complexes formed
with the L7M probe upon digestion with trypsin are labeled
a-e. B, lanes 5 and
11 from panel A were spliced together
to generate lanes 1 and 2,
respectively.
471 was used in transient
transfection assays since this deletion had previously been shown to
retain full repression by dexamethasone (25). Both a wild type distal
nGRE (
471GnRH) and a mutant nGRE containing a consensus Oct-1
sequence (
471MSGnRH) were used in these analyses. GT1-7 cells,
transiently transfected with luciferase reporter plasmids possessing
these nGREs, were treated with dexamethasone and promoter activity
assessed by measuring luciferase activity. As shown in Fig.
6, dexamethasone treatment significantly
decreased luciferase activity generated from the wild type
471GnRH
promoter (p = 0.03) and the mutant
471MSGnRH promoter
(p = 0.06). The differences in the extent of
dexamethasone repression mediated by the wild type promoter
(i.e. 60% repression; Fig. 6) versus the mutant
promoter (i.e. 29% repression; Fig. 6) was statistically
significant (p = 0.004).
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Fig. 6.
Effect of dexamethasone and TPA on
transcriptional activity of mouse GnRH promoters containing the wild
type GnRH distal nGRE and a consensus Oct-1 binding site at the distal
nGRE. GT1-7 cells were transiently transfected with 5 µg of the
plasmid 471GnRH along with 5 µg of herring sperm carrier DNA or 5 µg of the plasmid
471MSGnRH along with 5 µg of herring sperm
carrier DNA. The plasmid
471GnRH contains 471 base pairs of the GnRH
promoter inserted into a luciferase reporter vector, and the plasmid
471MSGnRH contains 471 base pairs of the GnRH promoter with mutations
at positions
206 and
207, creating a consensus Oct-1 binding site
at the GnRH distal nGRE, inserted into a luciferase vector. Cells were
transfected by the calcium phosphate procedure as described under
"Experimental Procedures." The cells were washed after 16 h
and then treated with either 10
6 M
dexamethasone or 100 ng/ml TPA. Untreated and treated cells were
harvested 20 h later for protein and luciferase assays. Luciferase
activities shown (mean ± S.E.) are expressed as a percentage of
control untreated cultures. The differences in dexamethasone (*) and
TPA (
) effects mediated by wild type 471GnRH versus
mutant
471MSGnRH promoters are statistically significant (see
"Results").
471GnRH promoter (p = 0.06; Fig. 6) but
increased luciferase activity generated from the mutant
471MSGnRH
promoter (p = 0.06; Fig. 6). The differences in TPA
effects mediated by the wild type promoter (i.e. 35%
repression; Fig. 6) versus the mutant promoter
(i.e. 1.5-fold induction; Fig. 6) was statistically significant (p = 0.007). Thus, TPA and glucocorticoid
regulation converge at a site within the GnRH promoter that is
recognized by Oct-1. Altering the strength of Oct-1 binding at the
nGRE·nTRE exerts dramatically different effects on these distinct
signaling pathways.
DISCUSSION
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Fig. 7.
Model of protein complexes bound to the
distal nGRE and the consensus Oct-1 site. A,
protein-DNA complexes at the distal nGRE. B, protein-DNA
complexes at the consensus Oct-1 site. See "Discussion" for
detailed description of model.
In some cases where POU domain transcription factors participate in steroid receptor-mediated transcriptional regulation, altering the POU domain protein target site has been found to influence hormonal responsiveness. Appropriate androgen receptor function within the Sex-limited protein (Slp) gene enhancer, appears to require Oct-1 binding to a nonconsensus site (56). Analogous to our results with the GnRH distal nGRE, increasing Oct-1 affinity within the Slp enhancer alters androgen responsiveness in transfected cells. As direct interactions between Oct-1 and the androgen receptor at the Slp enhancer has not been demonstrated, the mechanism responsible for altered hormone response is unknown. DNA sequences which flank those directly contacted by Oct-1, or other POU domain proteins also appear to influence the association of accessory factors. For example, sequences that flank an Oct-1 recognition site influence the ability of DNA-bound Oct-1 to discriminate between the VP16 protein of herpes simplex virus and its bovine homologue (36, 37). Furthermore, the POU domain transcription factor Pit-1 interacts with the estrogen receptor only at certain DNA sites and not others (57).
The consequences of altering the strength of Oct-1 interactions and its conformation at the distal nGRE are even more dramatic when TPA repression mediated by this sequence was examined. A fully functional GnRH promoter segment containing the mutant distal nGRE with the consensus Oct-1 site becomes activated in transfected GT1-7 cells upon TPA treatment. Transcription from the wild type GnRH promoter is well documented to be repressed by TPA in GT1-7 cells (8-10, 40). The mechanism of TPA repression of GnRH promoter activity remains controversial. Evidence that support and refute an involvement of AP-1 transcription factors in this repression has been presented (40, 58). The nTRE of the GnRH promoter, which overlaps with the nGRE, does not appear to be bound directly by AP-1 homodimers or heterodimers (40). The fact that the responsiveness of the promoter to TPA is reversed upon altering the strength of Oct-1 binding to this element suggests that, analogous to GR action at this element, the function of the TPA-responsive transcription factor at this site may be influenced by the nature of its interactions with DNA-bound Oct-1. The phosphorylation state of Oct-1 changes during the cell cycle, which dramatically alters its interaction with a consensus Oct-1 site located within the histone H2b promoter (59). It is not known whether changes in Oct-1 phosphorylation occur upon TPA treatment of GT1-7 cells, although it is premature to propose that Oct-1 itself is the direct target of TPA effects.
The regulation of GnRH gene transcription is clearly complex and
brought about by the action of multiple promoter- and enhancer-binding factors. Based upon our results, we hypothesize that an essential aspect of this promoter's function is not just the unique
constellation of DNA-bound transcription factors, but the precise
conformation that these factors adopt (Fig. 7). We suggest that
alternative conformations of GnRH promoter-bound transcription factors
will be dictated by the precise DNA sequence with which they interact and this may in turn affect the recruitment of additional regulatory factors.
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FOOTNOTES |
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* This work was supported by Research Grant RO1 DK47938 from the National Institutes of Health.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.
** To whom correspondence should be addressed: Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260. Tel.: 412-624-4259; Fax: 412-624-4759; E-mail: dod1{at}vms.cis.pitt.edu.
The abbreviations used are: GnRH, gonadotropin-releasing hormone; GR, glucocorticoid receptor; nGRE, negative glucocorticoid response element, EMSA, electrophoretic mobility shift assay; TPA, 12-O-tetradecanoylphorbol-13-acetate; TBE, Tris borate, EDTA; BSA, bovine serum albumin; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; POU, Pit-1, Oct-1, unc-86.
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