Activation of Transcription by Progesterone Receptor Involves Derepression of Activation Functions by a Cofactor
Michael Klotzbücher,
Christian Schwerk,
Beatrix Holewa and
Ludger Klein-Hitpass
Institut für Zellbiologie (Tumorforschung),
Universitätsklinikum, D-45122 Essen, Germany
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ABSTRACT
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Hormone-induced progesterone receptors (PR) bound
to response elements stimulate transcription initiation at target
promoters through a mechanism that presumably involves cofactors or
coactivators. To allow identification of such cofactors of
transcriptional activation in a functional assay, we have established a
reconstituted transcription system that is characterized by a specific
loss of responsiveness to purified baculovirus-expressed wild type PR.
In contrast to wild type PR, a C-terminally truncated PR mutant
displayed strong activation potential in this system. As the purified
recombinant full-length PR is capable of DNA binding, our results
suggest that C-terminal sequences of PR mediate a
cis-repression of N-terminal activation functions.
Moreover, using this PR-nonresponsive transcription system, we
identified and partially purified an activity from rat liver, termed
COPRA (cofactor of PR activation), that restores transactivation by
full-length PR. Characterization of COPRA revealed that this cofactor
exhibits activator specificity and is not involved in basal
transcription. We postulate that COPRA acts by relieving the repression
of activation functions mediated by C-terminal sequences.
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INTRODUCTION
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Regulation of mRNA synthesis is achieved partly through the
coordinate action of general transcription factors (GTFs),
RNA-polymerase II (RNAPII), and activators. GTFs (TFIIA, TFIIB, TFIID,
TFIIE, TFIIF, and TFIIH) mediate binding of RNAPII to the promoters
(1), but also have important functions at later steps of the initiation
process. In cell-free transcription systems GTFs and RNAPII are
sufficient to initiate basal levels of transcription from TATA-only
promoters (2). Activators bind to promoters or enhancers in a
sequence-specific manner and thereby stimulate the rate of
transcription initiation at the target promoter above basal levels. The
underlying mechanisms of transcriptional stimulation are not well
understood, but many activators have been shown to interact with
various components of the initiation complex (1). These interactions
are thought to help to recruit RNAPII, GTFs, or the RNAPII holoenzyme
complex, to stabilize initiation complex intermediates, or to induce
conformational changes within components of the initiation complex that
may activate subsequent steps (for review, see Refs. 1 and 3).
There is increasing evidence that some activators, in order to
function, require another class of factors, so-called coactivators
(also termed positive cofactors, intermediary factors, adaptors, or
mediators; Refs. 1 and 4). By definition, coactivators do not
bind sequence-specifically to DNA and are not involved in basal
transcription. These factors may exert their effects on transcriptional
activation through various mechanisms, including stimulation of
DNA-binding of activators (5, 6), by affecting chromatin structure
(7, 8, 9) or by mediating activator-initiation complex interactions
(10, 11, 12). Although the TATA box-binding protein (TBP)-associated
factors (TAFs) are stably associated subunits of the TFIID complex,
they can also be regarded as coactivators because they are dispensable
for basal transcription but absolutely essential for activated
transcription in vitro (13, 14).
The progesterone receptor (PR) is a transcriptional activator of the
nuclear receptor superfamily whose function is strictly dependent on
the presence of progesterone (15, 16, 17). PR exhibits the modular
structure common to all steroid receptors (Fig. 1A
). The
DNA-binding domain (domain C) is located in the center of the molecule
and consists of two zinc-fingers. Initially, two independent activation
functions, AF1 and AF2, have been localized to the N- and C-terminal
domains of the receptor, respectively (18, 19). A more detailed
deletion analysis has shown that AF1 consists of a core region, which
displays activation potential when linked to a heterologous DNA-binding
domain, and a modulatory region that cannot act on its own but
modulates the activity of the core region (20). As we have recently
shown in cell-free transcription and cotransfections (21), the
DNA-binding domain of PR appears to contain another independent
activation function, AF3. The C-terminal part of PR also harbors the
hormone-binding domain, a dimerization motif (22), and sequences
involved in the interaction with chaperones that maintain the
unliganded receptor in an inactive state (23, 24). In vivo,
binding of progesterone induces high-affinity binding of the receptor
to progesterone response elements (PREs) through a complex activation
cascade that includes conformational changes, release of chaperones,
and multiple phosphorylation events (25, 26). Presumably, due to the
separation of chaperones, PR purified from chicken oviduct and
recombinant human PR purified from baculovirus-infected
Spodoptora cells bind to PREs in a hormone-independent
manner and thus display an activated state (27, 28). Consistently, in
cell-free transcription systems based on crude nuclear extracts, such
purified PR preparations efficiently stimulate transcription initiation
of PRE-containing reporter genes (27, 28). In vitro assays
revealed binding of PR to TFIIB and the TFIID subunit
dTAFII110 (29, 21), which possibly contribute to
transcriptional stimulation by PR. However, there is evidence from
squelching experiments in cell culture and cell-free transcription
systems that transactivation by PR might involve additional cofactors
(20, 30, 31, 32). Recently, several candidate genes, steroid receptor
coactivator-1 (SRC-1), hRPF1, transcriptional intermediary factor 2
(TIF2), and binding protein of CRE-binding protein (CBP) have been
identified that, when overexpressed, stimulate PR-dependent
transcription (33, 34, 35, 36). In the case of SRC-1 and TIF2, interactions
with the ligand-binding domain of PR have been demonstrated, suggesting
that they may act as mediators between PR and the transcription
machinery. However, the mechanisms by which these coactivators effect
transactivation by PR remains largely uncharacterized.

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Figure 1. N- and C-Terminal Activation Functions of
Baculovirus-Expressed hPR Are Active in Cell-Free Transcription
A, At the top a schematic description of the domain
structure of hPR form B (hPR0) is given. Below, the
structures of baculovirus-expressed full-length hPR
(MH6-hPR0) and deletion mutants
(MH6-hPR0 core and MH6-ABC) are shown. AF3
denotes the independent activation function located within the
DNA-binding domain (21). The N-terminal His-tag (MH6) is
depicted as a solid box, and the core domain of AF1 is
shown as a stippled box. B, Schematic representation of
the templates used in cell-free transcription to analyze basal (TATA)
and activated transcription (P2-TATA), respectively. TATA,
TATA-box; G400 and G300, G-free cassettes of 400 and 300 nt in length,
respectively. C, Autoradiogram of cell-free transcription reactions
with crude rat liver nuclear extract in the absence (lanes 1, 6, and
11) or presence of the indicated amounts of MH6-hPR0 (lanes
25), deletion mutants MH6-hPR0 core (lanes 710), or
MH6-ABC (lanes 1215). The positions of RNAs derived from
the TATA and from the P2-TATA templates are indicated. In
the bar diagram below, normalized fold activation
(activated/basal transcription) values are given for each lane.
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To identify cofactors of transcription of mammalian origin, two
basically different approaches have been mainly used. With the help of
the yeast two-hybrid system (37), cDNA libraries can be screened for
proteins that interact with the activator of choice and that may
ultimately turn out to act as cofactors of transcription. The immediate
access to the cloned genes for these proteins is, of course, the major
advantage of this approach. However, a large number of false positives
are frequently obtained and, more importantly, cofactors that act
through mechanisms that do not involve stable interactions with the
activator may not be recovered by this genetic assay. Moreover, final
proof for a functional role of the cloned gene in transcription
activation and the analysis of its mechanism of action requires
additional assays. Such efforts are, however, often hampered by the
lack of appropriate cell lines or in vitro transcription
systems containing limiting amounts of the candidate factor (38).
Most of these limitations are circumvented by the second type of
approach, which uses cell-free transcription systems to identify
cofactors of transcriptional activation. This biochemical approach is
based on the observation that in vitro transcription systems
reconstituted from highly purified and/or recombinant GTFs and RNAPII
often display reduced effects to activators of transcription (39, 40).
This loss of activator responsiveness can be attributed to the removal
of important cofactors during purification and offers the opportunity
to identify and characterize complementing activities in a functional
test system. We have chosen this biochemical approach to study a
possible role of cofactors of PR-mediated transcriptional activation.
Using partially purified GTFs and RNAPII from rat liver as well as
recombinant GTFs, we have established an efficient basal transcription
system that is characterized by a strongly reduced response to highly
purified recombinant full-length PR (form B). By screening various
chromatographic fractions from rat liver nuclear extract, we identified
and partially purified an activity that restored transactivation by PR
in vitro but did not influence DNA binding of the activator.
Because it stimulated PR activity but not two other activators tested,
we termed this activity cofactor of PR activation (COPRA). Comparison
of full-length PR and deletion mutants in the COPRA-deficient system
revealed that C-terminal sequences of PR impose an inhibitory effect on
the activation functions. We postulate a model in which COPRA acts by
relieving this repression.
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RESULTS
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Activation of Cell-Free Transcription by Recombinant Human PR
To obtain sufficient amounts of PR for in vitro
transcription studies, we overexpressed isoform B of human (h) PR as a
His-tagged fusion protein (MH6-hPR0) in Spodoptora
frugiperda cells with the help of a recombinant baculovirus. To
investigate the role of the two major activation functions, AF1 and
AF2, we also expressed two deletion mutants,
MH6-hPR0
core and MH6-ABC (Fig. 1A
; 21 .
Mutant MH6-hPR0
core was generated by deletion of the
core domain of AF1, while MH6-ABC was generated by deleting
C-terminal sequences containing AF2. Purification of the recombinant
proteins by nickel-nitrilotriacetic acid (NTA)-chromatography resulted
in highly purified protein preparations (21). As analyzed by
electrophoretic mobility shift assay (EMSA) and in accordance with
published data (27, 28), the purified wild type PR, as well as deletion
mutants, bound to DNA in a constitutive fashion.
The transcriptional activation potential of these activators was first
compared in a cell-free transcription system that is based on crude
nuclear extract from rat liver and does not contain significant amounts
of PR. To detect basal transcription, a TATA-only promoter linked to a
G-free cassette of 400 bp was used (TATA, Fig. 1B
). Human PR-mediated
activation was monitored using a construct that contains two PREs
upstream of the TATA-box and a G-free cassette of 300 bp
(P2-TATA, Fig. 1B
). In the absence of activators, basal
transcription could be observed from both templates (Fig. 1C
, lane 1).
Addition of increasing amounts of MH6-hPR0 stimulated
transcription from the P2-TATA-promoter up to 23-fold,
whereas transcription from the TATA-promoter remained unchanged (Fig. 1C
, lanes 25), indicating that transactivation by the recombinant PR
is strictly PRE-dependent. Specific stimulation of the
P2-TATA-promoter was also seen with the mutants
MH6-hPR0
core (Fig. 1C
, lanes 710) and
MH6-ABC (Fig. 1C
, lanes 1215). However, compared with
full-length PR, the deletion mutants lacking the core of AF1
(MH6-hPR0
core) or AF2 (MH6-ABC) showed
clearly decreased transcriptional stimulation, indicating that both the
core of AF1 and AF2 contribute to transactivation by full-length
receptor. To achieve nearly saturated transcription activation, about
5-fold higher molar amounts of MH6-ABC were required (Fig. 1C
, right panel). This correlates well with the reduced
DNA-binding activity of this mutant (data not shown), which most likely
results from the deletion of a dimerization motif located in the
C-terminal domain of steroid receptors (22).
Reconstitution of a Cell-Free Transcription System with Reduced PR
Response
As a convenient source for the purification of GTFs and RNAPII, we
used rat liver nuclear extracts, which are highly active and obviously
contain all factors required for PR-mediated transcription (Fig. 1
). As
indicated in Fig. 2A
, in a first purification step
nuclear extracts were fractionated by phosphocellulose chromatography.
Subsequently, fractions A, C, and D were subjected to diethylaminoethyl
(DEAE)-Sepharose chromatography, yielding a total of nine
chromatographic fractions (Fig. 2A
). At this stage of purification,
three fractions (AB, CC, and DA) were required to reconstitute
efficient basal transcription (data not shown). As summarized in Fig. 2A
, using various assays we determined that DA contained TFIIB, TFIIH,
and TFIID. CC contained TFIIE, TFIIF, RNAPII, and another TFIID
complex. Based on chromatographic behavior we assume that fraction AB
contains TFIIA.

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Figure 2. Reconstitution of Transcription
A, Purification scheme for GTFs, RNAPII and COPRA. Rat liver nuclear
extracts were separated by phosphocellulose (PC-11), DEAE-Sepharose
(DEAE), and MonoQ chromatography as described in Materials and
Methods. The presence of transcription factors is indicated and
was determined by Western blotting (TFIIB, E, F, H, TBP) or activity
assays (TFIIA, D, RNAPII, and COPRA). B, Cell-free transcription of the
TATA template was assayed in reactions containing a complete set of
transcription factors (lane 1) or lacking individual components (lanes
26). Relative activities were determined by densitometric scanning
and are given above each lane in percent of control
(lane 1).
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The reconstituted transcription system containing AB, CC, and DA showed
still a strong response to added PR (data not shown). To obtain a more
defined system, we thus replaced CC by recombinant TFIIE and TFIIF as
well as a RNAPII fraction that had been obtained by separation of CC on
MonoQ (Fig. 2A
). As documented in Fig. 2B
(lane 1), by combining AB,
DA, and RNAPII from rat liver with recombinant human TFIIE and TFIIF,
efficient basal transcription could be observed. Transcription proved
to be highly dependent on each of the five components (lanes 25).
Importantly, addition of saturating amounts of MH6-hPR0 did
not result in significant stimulation of the PRE-containing template in
this system (Fig. 3A
, compare lanes 1 and 2). Because increasing the
GTFs to promoter ratio by lowering the template concentration or
elevating the concentrations of the various GTFs in the reconstituted
system did not improve the response to added MH6-hPR0 (data
not shown), we can eliminate the possibility that the poor
responsiveness is due to a limiting concentration of a GTF. Therefore,
these results suggest that a cofactor essential for transactivation by
hPR became limiting in the reconstituted system.

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Figure 3. Transcription Activation by PR Is Diminished in the
Reconstituted Transcription System and Can Be Restored by Addition of
COPRA
A, Transcription from the TATA and P2-TATA templates was
assayed with the reconstituted transcription system containing either
no activator (lane 1) or a nearly saturating amount (4 pmol) of
MH6-hPR0 (lanes 2 and 3). Lane 3 additionally contained 0.8
µl fraction CC. B, In vitro transcription with the
same system as in panel A containing either no activator (lanes 1 and
6) or MH6-hPR0 (lanes 25) in the absence (lanes 1 and 2)
or presence of the indicated amount of COPRA (MonoQ fraction).
Transcripts derived from the TATA and P2-TATA templates are
indicated. Fold activation values for each lane are given in the
diagram on the right.
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Identification of COPRA
The dramatic loss of PR response observed by replacing CC with
more purified RNAPII and recombinant TFIIE and TFIIF immediately
suggested that CC might contain a cofactor important for PR activation.
Indeed, addition of low amounts of CC to the nonresponsive system
significantly increased fold activation by MH6-hPR0 from
1.4- to 9.3-fold while little effect on basal transcription was
observed (Fig. 3A
, lanes 2 and 3). We next screened the
fractions obtained from separation of CC on MonoQ for the
activity-enhancing PR activation. The activity eluted between 325 and
400 mM KCl (Fig. 2A
, data not shown) and was termed COPRA.
To characterize COPRA in more detail a titration experiment was
performed. As shown in Fig. 3B
, addition of increasing amounts of COPRA
to reactions containing MH6-hPR0 increased fold activation
by PR from 2.1-fold to 14.3-fold in a dose-dependent manner. Thus, at
the maximum dose of COPRA about 7-fold potentiation of PR activity was
obtained. Importantly, basal transcription of the TATA-only template
remained unchanged (Fig. 3B
, lanes 15). Moreover, in the absence of
the activator, COPRA did not influence transcription from the
P2-TATA-promoter (Fig. 3B
, lane 6), excluding the
possibility that COPRA might represent an activator that acts through
binding to PREs. Together, these experiments show that COPRA does not
affect basal transcription, but greatly stimulates transactivation by
hPR. Therefore, COPRA might represent a bona fide
coactivator involved in enhancement of transcription by hPR in
vitro. Further searches for additional fractions containing
activities with similar function or able to cooperate with COPRA in
transactivation of PR proved to be unsuccessful (data not shown).
Different Activators Exhibit Distinct COPRA Requirements
To determine whether transactivation by other activators
might also be dependent on COPRA in our reconstituted transcription
system, we investigated transcriptional activation by hepatic nuclear
factor-4 (HNF4), an orphan receptor of the nuclear receptor
superfamily, which is expressed in the liver (41). The template we used
consisted of three HNF4-binding sites in front of a TATA-box
(H43-TATA). To assay activation in vitro we used
purified recombinant HNF4 expressed as a His-tagged fusion protein in
the baculovirus system. By EMSA we could show that rat liver fraction
AB, which is required for efficient transcription in the reconstituted
system, formed some protein-DNA complexes with the HNF4- binding site
(data not shown). As determined by competition with oligonucleotides
containing HNF4-binding sites (H4), these endogenous DNA-binding
proteins proved to be responsible for a minor (1.6-fold) activation of
the reporter gene H43-TATA (Fig. 4A
, compare
lanes 1 and 2). In the absence of COPRA, reactions containing
MH6-HNF4 supported 6.7-fold activation of
H43-TATA (lane 3), whereas reactions containing COPRA
supported 8-fold activation (lane 4). Thus, COPRA potentiates
transactivation by HNF4 by a factor of only 1.2.

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Figure 4. Transactivation by HNF4 and HNF1 Is Not Dependent
on COPRA
A, Cell-free transcription from the TATA template (TATA) and a template
containing three HNF4-binding sites in front of the TATA-box
(H43-TATA) was analyzed in the reconstituted system.
Reactions received either no activator (lanes 1, 2, and 5) or a
saturating amount of purified baculovirus-expressed HNF4
(MH6-HNF4, 5 pmol) in the absence (lanes 13) or presence
of 1 µl COPRA (lanes 4 and 5). Lane 2 contained 20 ng H4 competitor
oligonucleotides (H4-comp.). B, Transcription from reporter plasmid
syn0-TG1 containing one HNF1-binding site (H1-TATA) was analyzed in the
same system as in panel A except that plasmid PL-TG was used to measure
basal transcription (TATA). As HNF1 is present in rat liver fraction
AB, all reactions contained the activator. Lane 2 received 1 µl
COPRA, whereas lane 3 received 70 ng oligonucleotides containing
HNF1-binding sites (H1-comp.). Fold activation values, as normalized to
the reaction containing H1 competitor, are given in the diagram
on the right.
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Next, we investigated the COPRA requirement of HNF1, a member of the
POU-homeobox family, which is also highly expressed in liver (42). By
EMSA, endogenous HNF1 from rat liver could be detected in fraction AB
and is, thus, present in our reconstituted transcription system (data
not shown). To analyze whether these endogenous amounts of HNF1 support
activation of the reporter gene H1-TATA, which contains a single
HNF1-binding site in front of a TATA-box, we performed oligonucleotide
competition. Clearly, addition of an excess of HNF1 competitor
oligonucleotides (H1) specifically decreased transcription of H1-TATA
by a factor of 5 (Fig. 4B
, compare lanes 1 and 3), indicating that
endogenous HNF1 is able to mediate about 5-fold activation of the
promoter in the absence of COPRA. Reactions supplemented with COPRA
supported about 7-fold activation (lane 2). Thus, addition of COPRA
to the system resulted onlyin a 1.4-fold potentiation of
HNF1-mediated transactivation.
Together, these data demonstrate that COPRA does not play a major role
in activation of transcription by HNF1 and HNF4 while PR activity is
strongly dependent on COPRA. Hence, COPRA is not a general cofactor of
transcriptional activation but displays activator specificity.
A PR Deletion Mutant Lacking C-Terminal Sequences Is Active in the
Absence of COPRA
To explore the role of the N- and C-terminal activation
functions of hPR in mediating COPRA action, we compared the
transactivation potential and COPRA requirement of full-length
MH6-hPR0 with the two deletion mutants,
MH6-hPR0
core and MH6-ABC (Fig. 1
), in the
reconstituted transcription system. In accordance with the results
presented in Fig. 3
, MH6-hPR0 displayed little activity in
the basal system, but transactivation could be clearly potentiated up
to 4.5-fold by addition of COPRA (Fig. 5
, lanes 38).
Potentiation of MH6-hPR0 activity was already evident at a
limiting dose of activator (lanes 3 and 4), excluding the possibility
that COPRA might relieve self-squelching caused by excess of activator.
MH6-hPR0
core also showed little activity in the absence
of COPRA, and addition of COPRA reproducibly resulted in about 2-fold
potentiation (lanes 1316). The apparently lower potentiation factor
observed with the mutant might indicate that the core of AF1 is
necessary for full COPRA action. Alternatively, it seems possible that
the lower potentiation factor observed with MH6-hPR0
core
is a consequence of the lower intrinsic activation potential of this
mutant. Unexpectedly, in contrast to MH6-hPR0, the deletion
mutant lacking the C-terminal part of hPR, MH6-ABC,
supported activation of transcription in the absence of COPRA, and no
significant potentiation was observed upon addition of COPRA (lanes
912). Importantly, COPRA-independent transactivation and the lack of
potentiation by COPRA was observed both at a limiting (lanes 9 and 10)
as well as a nearly saturating dose of MH6-ABC (lanes 11
and 12). First of all, these results indicate that the basal system is
obviously capable of mediating transactivation by the N-terminal
activation functions (AF1/AF3). Furthermore, the fact that in the
absence of COPRA transactivation by AF1/AF3 is not evident in the
context of full-length PR, although the recombinant full-length PR
binds efficiently to PREs, suggests that N-terminal activation
functions are repressed through the presence of C-terminal sequences.
Consequently, we postulate that COPRA might play a role in relieving
this repression.
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DISCUSSION
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A Basal Transcription System Exhibiting a Specific Loss of PR
Response
To establish a reconstituted transcription system allowing
identification of cofactors of PR-mediated transactivation on a
functional basis, we have used recombinant GTFs (TFIIE, TFIIF) as well
as partially purified GTFs and RNAPII from rat liver. Rat liver GTFs
and RNAPII had to be purified through two and three columns,
respectively, to observe a dramatic loss of PR response. As two other
activators tested, HNF1 and HNF4, were still able to mediate
transactivation in this system (Fig. 4
), it is clear that our system
lacks a component that plays a rather specific role in transcription
activation by PR. Because chromatographic purification of GTFs to
homogeneity requires far more than two or three chromatographic steps,
our basal transcription system most likely contains still a number of
cofactors or coactivators that have been described by other groups
using HeLa cell-derived transcription systems (11, 39, 40, 43, 44, 45).
Assuming similar chromatographic behavior of human and rat
coactivators, e.g. upstream stimulatory activity
(USA)-derived cofactors PC1 to PC4 (39) should be contained in
DEAE-Sepharose fraction DA, which supplies TFIIB, TFIID, and TFIIH to
our system. Therefore, our data do not exclude the possibility that PCs
or other factors with coactivator properties might play essential roles
in PR-mediated transcription, most likely at steps after COPRA-mediated
activation of PR.
COPRA, an Activator-Specific Cofactor of Transactivation
Using this system, which is characterized by a specific loss of PR
response, we have identified an activity from rat liver, termed COPRA,
which restores transcriptional activation by full-length PR
in vitro. COPRA alone is not capable of mediating a
transcriptional response through PREs (Fig. 3
) and does not form
specific protein-PRE complexes in EMSA (data not shown), suggesting
that it does not represent a protein that acts through binding to PREs.
As COPRA has no influence on PR-PRE complex formation in EMSAs (data
not shown), we determined that it cannot influence activation by
increasing the DNA-binding affinity of PR, e.g. by removing
chaperones or by stabilizing PR dimers. COPRA has no effect on basal
transcription and thus increases activation solely by a positive effect
on PR-mediated transcription. This property distinguishes COPRA from
cofactors such as DR2/PC3/Topoisomerase I or NC2/Dr1, which increase
relative stimulation by activators by specifically repressing basal
promoter activity (45, 46). In summary, COPRA displays several
hallmarks of a coactivator of transcriptional activation. However,
although COPRA clearly potentiated PR activity, it had little effect on
transactivation by HNF1, a member of the POU-homeodomain
transcription factor family. Moreover, COPRA also did not function in
conjunction with HNF4, which is an orphan member of the nuclear
receptor family and, thus, distantly related to PR. These observations
indicate that COPRA is a rather specific cofactor that functions with a
restricted set of activators only. However, we cannot rule out the
possibility that HNF1 and HNF4 possess higher affinity for COPRA than
PR and thus are saturated by small amounts of COPRA that might
contaminate components of the basal transcription system.
Western blot analysis indicated that COPRA copurifies with a
TFIID complex through three columns (Fig. 2
, data not shown). However,
as heat treatment for 10 min at 47 C known to destroy TFIID activity
(47) did not inactivate COPRA (data not shown), we consider it unlikely
that COPRA corresponds to a specific TFIID subtype (48) that, due to a
specific TAF or TAF combination, might be particularly responsive to
PR. We have not been able to demonstrate a stable association of COPRA
and PR in EMSAs (data not shown). Moreover, using antibodies against
CBP and SRC-1 (kindly provided by T.-P. Yao and D. Livingston,
Boston, MA) in Western blotting experiments we found no indication
that CBP or the major 160-kDa variant of SRC-1 is particularly enriched
in the COPRA fraction (data not shown), suggesting that COPRA might not
correspond to CBP or SRC-1 (33, 36). However, a relationship of COPRA
with the PR cofactor hRPF1 (34), which has been shown to act as a
positive cofactor of PR, TIF-2 (35), and other less characterized
interacting factors (49, 50) cannot be excluded at present and requires
further analysis.
Analysis of PR Mutants in the COPRA-Deficient System Reveals a
cis-Repression of N-Terminal Activation Functions
In contrast to MH6-hPR0 and
MH6-hPR0
core, the C-terminally truncated mutant
MH6-ABC efficiently activated transcription, presumably
through AF1 and AF3 (AF1/AF3), in the absence of COPRA, and no
potentiation of MH6-ABC activity was observed in the
presence of COPRA (Fig. 5
). From these results we conclude that all
factors required for AF1/AF3 activity are present in the basal system
and that COPRA does not act as a mediator of transactivation for these
activation functions. As noted above, EMSAs showed that
MH6-hPR0 and MH6-hPR0
core, as well as
MH6-ABC, are able to bind to DNA in a constitutive fashion,
when tested alone or after mixing with GTFs and RNAPII fractions (data
not shown). Thus, the two PR constructs containing C-terminal
sequences, MH6-hPR0 and MH6-hPR0
core, do not
reveal full AF1/AF3 activity, although the activators are in a
DNA-binding state and transactivation by AF1/AF3 is basically possible
under these conditions. This clearly suggests that in these constructs
AF1/AF3 is inactivated due to a repressive effect imposed or mediated
by C-terminal sequences. As depicted in Fig. 6A
, this
repression might be due to an intramolecular masking of AF1/AF3 by the
C-terminal sequences containing AF2, which possibly also eliminates AF2
activity. Alternatively, repression of PR activation functions might be
mediated by a repressor whose binding depends on the presence of
C-terminal PR sequences (Fig. 6B
). Consistent with the performance of
MH6-ABC, in both models deletion of C-terminal PR sequences
would allow transactivation through AF1/AF3 either by relieving the
intramolecular inhibition or by eliminating inhibitor binding (Fig. 6C
). Presumably, due to the presence of COPRA, the repressed state of
activation functions is not apparent in crude nuclear extracts,
inasmuch as MH6-hPR0 and MH6-hPR0
core
display transactivation potential (Fig. 1
). Consistently, addition of
COPRA did not further potentiate PR-mediated activation in crude
nuclear extracts (data not shown), indicating that they contain nearly
saturating amounts of COPRA. It is noteworthy that, based on the
analysis of a PR deletion mutant lacking the C-terminal 42 amino acids
that could be activated by the antagonist RU486, while full-length PR
remained inactive, Vegeto et al. (51) proposed a very
similar model in which sequences at the very C-terminal end of PR play
a role in silencing transactivation domains.

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|
Figure 6. Model for the Differential Effects of COPRA on
Full-Length PR and C-Terminally Deleted Receptor
In the absence of COPRA, activation functions of DNA-bound wild type PR
are repressed due to intramolecular masking mediated by the C-terminal
part of PR including AF2 (A) or due to the binding of a repressor that
interacts with the C-terminal part (B). COPRA relieves this repression
either through mechanisms that may involve binding of the coactivator
to the C terminus or transient interactions with the C-terminal end
and/or modifications of PR or repressor. In contrast, due to the
absence of intramolecular repression or to the lack of the
inhibitor-binding site, the C-terminally truncated PR lacking AF2 (ABC)
transactivates through AF1/AF3 in the absence and presence of COPRA
(C).
|
|
Possible Mechanisms of COPRA Action
Based on our findings that PR activation functions are repressed
in the absence of COPRA and that COPRA restores transactivation by
full-length PR as well as MH6-hPR0
core, we postulate
that COPRA functions by relieving the intramolecular or
repressor-mediated inhibition of activation functions (Fig. 6
, A and
B). Consistent with the result that MH6-ABC cannot be
stimulated by COPRA (Fig. 5
), this may occur through a conformational
change induced by binding of COPRA to the C-terminal part of PR. In
this scenario, the associated cofactor might not only cause
derepression of activation functions but also act as a mediator of
transactivation, which links AF2 to targets of the general
transcription apparatus. Due to the lack of evidence for a stable
complex of PR and COPRA, a more likely explanation might be that
derepression occurs through mechanisms that involve a transient
interaction of COPRA and PR or modifications of PR or repressor
mediated by COPRA. It is attractive to speculate that derepression of
activation functions might be the step that triggers stable association
of additional molecules (e.g. SRC-1 or TIF2) contributing to
the transactivation potential of AF2. In any case, we believe that our
report is the first one to suggest that transactivation by PR might
involve a cofactor-mediated derepression of activation
functions.
Because DNA-binding is constitutive and hormone does not affect
transactivation by purified PRs (27, 28), the role of the hormone
cannot be directly assessed in our system. However, Allan et
al. (52) have presented evidence that DNA binding of PR induced by
antibody-mediated dimerization is not sufficient for transactivation,
but rather hormone, in addition, is required. Because purified
baculovirus-expressed PR activates transcription in a
hormone-independent manner, we therefore speculate that its
conformation might resemble that of hormonally activated receptor. This
assumption is supported by the comparison of the activation potentials
of full-length PR and PR deletion constructs (Fig. 1
), which indicated
that AF2, which is thought to require hormone-induced conformational
changes for its activity, contributes to transactivation of full-length
PR. Another indication results from a recent study of Beck et
al. (1996), who demonstrated that the phosphorylation state of
unliganded baculovirus-expressed PR resembles that of hormonally
activated native PR (53). If baculovirus-expressed PR indeed
corresponds to a hormonally activated PR, then our results imply that
neither DNA binding nor hormone-induced conformational changes suffice
to render PR active, but that derepression of activation functions
through COPRA is another obligatory and separable step of the
activation cascade. Finally, although the physiological relevance is
not proven to date, the mechanism by which COPRA regulates
transactivation by PR may represent another level of regulatory
complexity for PR at the level of target genes, as variations in COPRA
concentrations and/or the ratio of coactivator/repressor might alter
the magnitude of the progesterone response in different PR-positive
target tissues.
 |
MATERIALS AND METHODS
|
---|
Baculovirus Expression
Construction of baculovirus transfer vectors encoding the
histidine-tagged fusion proteins MH6-hPR0,
MH6-hPR0
core, and MH6-ABC was described in
detail elsewhere (21). To generate a HNF4-encoding transfer vector the
complete open reading frame of the Xenopus laevis HNF4
cDNA (54) was amplified by PCR with primers generating BglII
sites at both ends. After BglII digestion, products were
ligated into pBlueBacHisB (Invitrogen, San Diego, CA). Construction of
baculovirus transfer vectors encoding histidine-tagged 56-kDa and
untagged 34-kDa subunits of human TFIIE will be described elsewere (M.
Klotzbücher and L. Klein-Hitpass, manuscript in preparation).
Generation of recombinant baculoviruses, infection of Spodoptora
frugiperda (Sf21) cells, preparation of
Sf21 cell extracts, and affinity purification
of overexpressed proteins on Ni-NTA-agarose (Qiagen,
Hilden, Germany) were performed as described (55) with minor
modifications. Cells were swollen in five packed cell volumes of buffer
I, Ni-NTA-agarose bound proteins were washed three times with 20
volumes of buffer III (20 mM HEPES, pH 7.9, 10% glycerol,
100 mM KCl, 1.5 mM MgCl, 0.5 mM
dithiothreitol, 5 mM NaF, 0.5 mg/ml aprotinin, 0.1
mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin A,
0.1 mM phenylmethylsulfonylfluoride) including 5
mM imidazole, transferred to a column, eluted in buffer III
containing 120 mM imidazole, and dialyzed against buffer
III containing 0.2 mM EDTA. In the case of TFIIE,
Sf21 cells were coinfected with viruses encoding both
subunits, and in vivo assembled TFIIE complexes (rTFIIE)
were isolated from Sf21-cell extracts by Ni-NTA
affinity chromatography.
Overexpression in Escherichia coli
TFIIF subunits RAP74 and RAP30 were overexpressed in E.
coli BL21(DE3) (Novagen, Madison, WI) using
pET23d/RAP74H6 and pET11d/RAP30 (56, 57). Expression,
purification, and assembly of recombinant TFIIF complexes (rTFIIF) by
renaturation were performed essentially as described by Wang et
al. (56).
Purification of GTFs, RNAPII and COPRA
Nuclear extracts were prepared from livers of 3- to 6-month-old
rats according to the procedure of Gorski et al. (58) with
modifications described by Döbbeling et al. (59). For
preparation of partially purified transcription factors, nuclear
extracts were adjusted to buffer D (25 mM HEPES, 10%
glycerol, 2 mM dithiothreitol, 0.2 mM EDTA, and
protease inhibitors as indicated above) containing 0.1 M
KCl and loaded on a phosphocellulose P11 (Whatman) column. The 0.1
M KCl flowthrough (fraction A) and proteins eluted between
0.30.475 M KCl (fraction C) and 0.4750.85 M
KCl (fraction D) were required for transcription and further purified.
Fraction A was loaded on a DEAE-Sepharose column (CL-6B, Pharmacia
Biotech, Uppsala, Sweden), and bound proteins were eluted with 0.3
M (fraction AB) and 0.5 M KCl (fraction AC).
P11 fraction C was dialyzed against buffer D/100 mM KCl
before DEAE-Sepharose chromatography. Bound proteins were eluted with
0.25 M (fraction CB) and 0.85 M KCl (fraction
CC). P11 fraction D was dialyzed against 0.18 M KCl before
DEAE-Sepharose chromatography. The 180 mM flowthrough
fraction (DA) as well as 0.32 M KCl (fraction DB) and 0.85
M KCl (fraction DC) eluates were collected. For further
purification of RNAPII and COPRA, DEAE-Sepharose fraction CC was
dialyzed against buffer D/100 mM KCl, loaded on a MonoQ
column (Pharmacia), and eluted with a 0.1 to 0.6 M KCl
gradient. COPRA activity was found in the range from 325 to 400
mM KCl, while RNAPII activity eluted in the range of
400500 mM KCl. Pooled fractions were dialyzed against
buffer D/100 mM KCl/20% glycerol, frozen in liquid
nitrogen, and stored at -80 C.
Western Blot Analysis
Fractions were subjected to SDS-PAGE in 0.1% SDS-10%
polyacrylamide gels. Proteins were electrotransferred to nitrocellulose
filters by semidry blotting. Blots were blocked with 2.5% blocking
reagent (Boehringer, Mannheim, Germany), followed by incubation with
primary antibodies and appropriate horseradish peroxidase-conjugated
anti-IgG in blocking solution. Bound antibodies were detected using the
ECL (enhanced chemiluminescence) system (Amersham,
Braunschweig, Germany). For detection of TFIIB, TFIIE, TFIIF, and TBP,
commercially available (UBI, Santa Cruz, CA) cross-reactive antibodies
were used. TFIIH was detected with the help of monoclonal antibody 3c9
directed against p62 (60).
RNAPII Assay
RNAPII activity was measured as described by Hodo and Blatti
(61).
Transcription Templates
Reporter plasmids P2-TATA
(PRE2TATA-G300) and TATA (TATA-G300 and TATA-G400)
containing the TATA-box of the ovalbumin promoter were as described
(31, 62). Transcription templates PL-TG, containing the TATA-box of the
Xenopus albumin gene in front of a 380 nucleotides (nt)
G-free cassette and Syn0-TG1 carrying the HNF1-binding site of the
albumin promoter in front of a 300 nt long G-free cassette, were as
described by Schorpp et al. (63). H43-TATA was
generated by inserting three copies of the HNF4-binding site of the
human
1-antitrypsin promoter (64) into the BglII-site of
TATA-G300.
Cell-Free Transcription
Transcription reactions with 4.5 µl of rat liver nuclear
extract and the indicated amounts of recombinant activators were
performed in a total volume of 20 µl as described previously (31).
Reconstituted transcription reactions contained 0.8 µl AB, 2.3 µl
DA, 1.2 µl RNAPII, 40 ng rTFIIE, and 60 ng rTFIIF. Sixty nanograms of
the TATA constructs and 90 ng of reporter constructs containing
activator-binding sites were used. Reactions with crude nuclear extract
additionally contained 200 ng sonicated salmon sperm DNA. The
transcription reactions were incubated, processed, and analyzed as
described (27). For quantitative analysis, autoradiograms were scanned
using a laser densitometer (Pharmacia Biotech). Fold activation values
(activated/basal) were normalized to the ratio obtained in reactions
without activator, which was set to be 1.
 |
ACKNOWLEDGMENTS
|
---|
We are thankful to P. Kastner and P. Chambon (Strasbourg,
France), D. Reinberg (Piscataway, NJ), T. Drewes (Essen, Germany), and
B. Q. Wang and Z. F. Burton (East Lansing, MI) for providing plasmids;
to J. M. Egly (Strasbourg, France) and D. Livingston and T.-P. Yao
(Boston, MA) for providing antibodies; to V. Ulber for excellent
technical assistance and U. Schmücker for oligonucleotide
synthesis and sequencing. We wish to thank M. Meisterernst (Munich,
Germany) for advice and help with the purification of RNAPII. We are
grateful to G. U. Ryffel and L. Vaßen (Essen, Germany) for helpful
discussions and critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Ludger Klein-Hitpass, Institut für Zellbiologie (Tumorforschung), Universitätsklinikum, Virchowstr. 173, D-45122 Essen, Germany.
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB354
(to L.K.-H.).
Received for publication November 6, 1996.
Revision received March 21, 1997.
Accepted for publication March 24, 1997.
 |
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