Regulation of Estrogen Receptor Activation of the Prolactin Enhancer/Promoter by Antagonistic Activation Function-2-Interacting Proteins
Fred Schaufele
Metabolic Research Unit University of California San
Francisco, California 94143-0540
 |
ABSTRACT
|
---|
Transcriptional responses to estrogens are
controlled by the cell- and gene-specific interactions of the nuclear
estrogen receptor (ER) with cofactors and other transcription factors.
The pituitary-specific PRL enhancer/promoter is regulated by estrogens
only when it is bound by both ER and the pituitary-specific
transcription factor, Pit-1. Cooperative ER/Pit-1 activation of the
dormant PRL enhancer/promoter in pituitary progenitor cells
requires the estrogen-dependent activation function-2 (AF-2) of ER, but
is inhibited by one AF-2-interacting cofactor, RIP140. Here, the
complex actions of RIP140 and other AF-2-interacting proteins at the
PRL enhancer/promoter were shown to operate via ER itself. RIP140
inhibition of ER/Pit-1 activation in the absence of AF-1 and RIP140
inhibition of both ER
and ERß cooperative activation with Pit-1
suggested a conserved ER site for RIP140 action, possibly AF-2.
Coexpression of other AF-2-interacting proteins, including the p160
factors, steroid receptor coactivator-1a (SRC-1a) and glucocorticoid
receptor interacting protein-1 (GRIP1), had negligible effects on
ER
/Pit-1 cooperative activation, but partially relieved RIP140
inhibition. Relief of RIP140 inhibition required the AF-2-binding,
LXXLL motifs in SRC-1a and GRIP1. An ER AF-2 mutant that selectively
blocked ER interaction with p160s, but not RIP140, still cooperated
with Pit-1 and was inhibited by RIP140, but was not relieved by SRC-1a
or GRIP1 expression. Thus, SRC-1a and GRIP1 binding to AF-2
counteracted the inhibition of ER/Pit-1 activation by another
AF-2-interacting protein, RIP140. Complex, sometimes antagonistic,
actions of different classes of AF-2-interacting proteins may play an
important role in the cell- and gene-specific estrogen regulation of
PRL and other genes.
 |
INTRODUCTION
|
---|
Estrogens regulate cellular response, differentiation, and
proliferation in a variety of tissues. The postmenopausal reduction in
estrogen synthesis is associated with significant declines in bone and
cardiovascular health, which can be countered via estrogen replacement
therapy. However, estrogen therapy is also associated with a higher
estrogen-mediated risk of breast and uterine cancer. The development of
estrogen analogs that mimic only the beneficial effects of estrogens in
selected tissues would significantly improve hormone replacement
therapy and breast cancer treatment. To do so, it will be necessary to
understand the molecular nature of the tissue-specific estrogen
responses.
Many estrogen responses are mediated by the estrogen receptor (ER), a
DNA-binding transcription factor of the nuclear receptor superfamily
that generally is more effective at promoting transcription when bound
by estrogens (1, 2, 3, 4, 5, 6). ER activation is associated with the
estrogen-dependent interaction of certain receptor-interacting proteins
(RIPs) with activation function-2 (AF-2) (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), which is conserved in
the carboxyl-terminal ligand-binding domain (LBD) of ER and most
nuclear receptors (20). A separable amino-terminal domain of ER
contains the more poorly conserved AF-1, which in most promoter
contexts is dormant unless released by agonist binding to the LBD (2, 4, 21, 22). Ligand dependence of AF-1 may be related to relief of
corepression at many nuclear receptors (23, 24, 25, 26, 27) or may be associated
with the cobinding of the same receptor interacting coactivator to AF-1
and AF-2 (28, 29, 30).
AF-1 and AF-2 have been described mostly in studies of ER action at
short, isolated DNA sequences to which ER binds. Estrogen regulation of
natural genes is likely to use many of the same ER activities in
conjunction with the activities provided by other promoter-binding
factors. Synthesis of PRL declines with age in females and is strongly
regulated by estrogens. Estrogen activation of pituitary-specific PRL
gene transcription provides an excellent model in which to study
cell-specific modulation of ER action by other promoter-binding
factors.
ER regulates PRL gene transcription only if the PRL enhancer/promoter
is bound by the pituitary-specific transcription factor Pit-1 (31, 32, 33, 34, 35).
This cooperative activation by ER and Pit-1 explains why estrogen
regulation of PRL gene transcription is pituitary specific. ER/Pit-1
cooperative activation requires AF-2 (35). However, RIP140, a 140-kDa
cofactor that interacts with AF-2 of ER and a number of nuclear
receptors in a ligand-dependent fashion (18), inhibits ER/Pit-1
activation (35). RIP140 may inhibit any of the PRL
enhancer/promoter-binding proteins including ER. Understanding how
RIP140 and other AF-2-interacting proteins modulate AF-2-dependent
ER/Pit-1 cooperative activation will be crucial for understanding
cell-specific estrogen regulation of gene transcription.
In the present study, it was observed that RIP140 inhibition of
ER/Pit-1 PRL enhancer/promoter activation operated completely via ER.
Within ER, AF-1 was not required for RIP140 inhibition. RIP140
inhibition of the PRL enhancer/promoter activated by Pit-1 and either
of the
- or ß-isoforms of ER suggested a conserved site for RIP140
action, possibly AF-2. Relief of RIP140 inhibition by the coexpression
of other AF-2-interacting factors supported AF-2 as the target of
RIP140 action. Disrupting interaction of those counteracting factors
with AF-2 blocked their ability to relieve RIP140 inhibition. Thus,
AF-2 dependent promoter- and cell-specific ER regulation of the PRL
enhancer arises from the cooperative and antagonistic interactions of
Pit-1, ER, and AF-2-interacting proteins docking directly to ER
AF-2.
 |
RESULTS
|
---|
p160 Coactivator Regulation of ER/Pit-1 Activation
We previously studied ER regulation of the rat PRL
enhancer/promoter attached to the luciferase gene in transiently
transfected mouse pituitary progenitor GHFT15 cells (34, 35).
GHFT15 cells are ideal for studying cell-specific PRL
enhancer/promoter activation because they are derived from an embryonic
pituitary transformed immediately after the onset of low level Pit-1
expression (36) but before commitment to the PRL, GH, or TSH cell types
that derive from the Pit-1-containing progenitor (37).
In GHFT15 cells, PRL enhancer/promoter activity was low unless
an expression vector for the cDNA of Pit-1 was cotransfected (34, 35)
(Fig. 1A
). Expression of the cDNA for the
-isoform of human ER (ER
) had a marginal effect on PRL
enhancer/promoter activity. In contrast, activation was substantial
when ER
and Pit-1 were coexpressed, averaging 25.1 ± 5.1-fold
activation over the uninduced PRL enhancer/promoter.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 1. RIP140 Is a Dose-Dependent Inhibitor of the
Cooperative Activation of the 3-kb Rat PRL Enhancer/Promoter by Human
ER and Rat Pit-1 Tagged with the FLAG Epitope in Mouse GHFT15
Pituitary Progenitor Cells
A, ER/Pit-1 cooperative activation was not affected by the expression
of SRC-1a, GRIP1, TIF1 , or CBP, but was inhibited by RIP140
coexpression. B, Titration of RIP140 inhibition by cotransfection with
1, 3, or 10 µg RIP140 expression vector. C, Representative Western
blot of an experiment in B, probed for ectopically expressed human ER
(blotted with human-specific anti-ER antibody) or FLAG-Pit-1 expression
(blotted with anti-FLAG antibody) demonstrated that RIP140 inhibited
PRL promoter activity, not Pit-1 or ER expression. The FLAG
epitope-tagged Pit-1 was used in all subsequent experiments and behaves
exactly as native Pit-1 in cooperative activation with nuclear
receptors (35 ). Three independent experiments were normalized to the
activity of the ER/Pit-1-coactivated PRL enhancer/promoter (100%), and
results are plotted as the mean ± SD in A and B.
|
|
ER/Pit-1 cooperative activation of the PRL enhancer/promoter was
blocked by a point mutation in AF-2 of ER (35). This suggested that one
or more of the known AF-2-interacting proteins may act as coactivators
of cooperative ER/Pit-1 activation. Expression vectors encoding the
cDNAs for the AF-2-interacting proteins, steroid receptor
coactivator-1a (SRC-1a) (also called NCoA-1) (10),
glucocorticoid receptor interacting protein (GRIP1, also called
TIF2 or NCoA-2) (15), TIF1
(13), and RIP140 (18) were
individually cotransfected along with the ER
and Pit-1 expression
vectors and the PRL enhancer/promoter reporter into GHFT15 cells
(Fig. 1A
). PRL enhancer/promoter activities in the presence of ER,
Pit-1, and SRC-1a, GRIP1, or TIF1
were 128 ± 29%, 129 ±
31%, and 129 ± 13%, respectively, as active as PRL
enhancer/promoter activity in the absence of coactivator. Similarly,
the coactivator CREB-binding protein (CBP) (38), which also
participates in nuclear receptor action (39), did not significantly
alter ER/Pit-1 activation of the PRL enhancer/promoter (120 ±
14%).
RIP140 Is a Dose-Dependent Inhibitor of ER/Pit-1 Synergy
Expression of RIP140, which, like SRC-1a, GRIP1, and TIF1
,
binds AF-2 in a ligand-dependent fashion, strongly reduced ER/Pit-1
cooperative activation of the PRL enhancer/promoter (35) (Fig. 1A
).
RIP140 inhibition was dose dependent; transfection of 110 µg of the
RIP140 expression vector caused a gradual decrease in ER/Pit-1
cooperative activation (Fig. 1B
). ER/Pit-1 activation was effectively
abolished to the level of Pit-1 activation by transfecting 10 µg of
the RIP140 expression vector. In contrast, cotransfection of 10 µg of
the RIP140 expression vector had no effect on C/EBP
activation of
the PRL enhancer/promoter (35). Transfection of lower amounts of the
same RIP140 expression vector was previously reported to enhance ER
activation at a minimal promoter containing an estrogen response
element (ERE) (18), but in the current studies had no activating or
inhibitory effect on ER
/Pit-1 cooperative activation of the PRL
promoter (data not shown).
The levels of ER and Pit-1 expressed were determined by Western blots
of nuclear extracts of the same transfected cells (Fig. 1C
).
Ectopically expressed human ER
and Pit-1 (with the FLAG epitope
fused to the Pit-1 amino-terminus) were detected with antibodies
specific for human ER and the FLAG epitope. Mouse GHFT15 cells
contain some endogenous ER (35) that were not detected by the
anti-human ER antibodies used here. The nuclear levels of ectopically
expressed ER
or Pit-1 were not changed by the coexpression of Pit-1
or ER (Fig. 1C
) by RIP140 (Fig. 1C
) or by SRC-1a, GRIP1, or TIF1
expression (not shown). Similarly, endogenous ER and Pit-1 levels were
not changed (35). This showed that ER/Pit-1 activation and its
regulation by RIP140 were not artifacts of effects on the expression
vectors themselves. Thus, the AF-2-interacting protein RIP140 directly
inhibited estrogen-dependent, cooperative activation by ER and
Pit-1.
ER Is Required for RIP140 Inhibition
RIP140 may inhibit the PRL enhancer/promoter by acting directly at
the ER. Conversely, RIP140 may inhibit other factors, including Pit-1,
that bind to other PRL enhancer/promoter sites (40, 41, 42, 43, 44, 45, 46). We determined
that RIP140 did not affect PRL enhancer/promoter activity in the
absence of ER (Fig. 2
). Because GHFT15
cells contain endogenous ER (35), we conducted these experiments under
conditions in which endogenous ER was selectively inactivated by
incubating the transfected cells with 10-6 M
ICI 164,384. ICI 164,384 is an inactive estrogen mimic that antagonizes
estrogen action by competitive binding to the ligand-binding pocket of
ER. A concentration of 10-6 M ICI 164,384 was
sufficient to completely block ER/Pit-1 cooperative activation of the
PRL enhancer/promoter (data not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Figure 2. RIP140 Does Not Inhibit PRL Enhancer/Promoter
Activity in the Absence of Active ER
Incubation of GHFT15 cells with the estrogen antagonist ICI 164,384
showed that the observed Pit-1 activation of the PRL enhancer/promoter
was not due to the cooperative action of expressed Pit-1 with
endogenous GHFT15 cell ER. Three independent experiments were
normalized to the activity of the Pit-1-activated PRL enhancer/promoter
(100%), and the results are plotted as the mean ±
SD.
|
|
In the presence of 10-6 M ICI 164,384, the PRL
enhancer/promoter was still activated 3.26 ± 0.43-fold by the
expression of Pit-1. This showed that Pit-1 activated PRL
enhancer/promoter action in the absence of active ER. With ER
inactivated by ICI 164,384, RIP140 coexpression did not inhibit Pit-1
activation of the PRL enhancer/promoter (Fig. 2
). Thus, RIP140
inhibition of PRL enhancer/promoter specifically required the presence
of active ER.
RIP140 Directly Inhibits ER Action
We showed above that ER is an essential participant in RIP140
inhibition of ER/Pit-1 cooperative activation at the PRL
enhancer/promoter. To determine whether ER itself could be the site of
RIP140 action, we studied ER action in isolation from all PRL
enhancer/promoter elements. The well characterized vitellogenin ERE was
appended to a minimal promoter containing the TATA box of the herpes
simplex thymidine kinase gene (ERE-TATA). Like the PRL ERE, the
vitellogenin ERE operates through what is referred to as the classical
ER pathway (47) (see Fig. 4
). The vitellogenin ERE can also replace the
PRL ERE to support ER cooperative activation with Pit-1 (33).

View larger version (25K):
[in this window]
[in a new window]
|
Figure 4. Both Human ER (A) and Rat ERß (B) Activation
of the PRL Enhancer/Promoter Are Pit-1 Dependent and Require AF-2
In GHFT15 cells grown in medium reduced in estrogens, the selective
AF-2 antagonists/AF-1 agonists tamoxifen and raloxifene did not support
Pit-1 cooperative activation with either ER isoform. Three independent
experiments were normalized to the activity of the Pit-1-activated PRL
enhancer/promoter (1-fold), and the results are plotted as the
mean ± SD.
|
|
When transfected into GHFT15 cells grown in culture medium deficient
in estrogens, the vitellogenin ERE-TATA promoter was activated, on the
average, 33.6 ± 8.4-fold by the addition of 10-8
M estradiol (E2; Fig. 3
). E2 activation was
completely inhibited by 10-6 M ICI
164,384 (ICI+E2; Fig. 3
). Therefore, E2
activation of the ERE-TATA promoter operated via endogenous ER
and/or ERß, both of which are detected by Western blots of nuclear
extracts of GHFT15 cells (35) (our unpublished data).

View larger version (16K):
[in this window]
[in a new window]
|
Figure 3. RIP140 Directly Represses ER Action in the Absence
of Other PRL Enhancer/Promoter-Binding Factors
GHFT15 cells were grown in medium reduced in estrogens. RIP140
inhibited estrogen (E2) activation of the vitellogenin ERE
controlling the activation of the minimal herpes simplex thymdine
kinase TATA box. Estrogen (E2) activation of this promoter
was marginally (1.3-fold) affected by the coexpression of 0.2 (shown)
or 1.0 or 5.0 µg (not shown) human ER expression vector. ICI 164,384
antagonized estrogen activation (ICI + E2). Three
independent experiments were normalized to the activity of the
E2-activated ERE-TATA promoter (100%), and the results are
plotted as the mean ± SD.
|
|
Cotransfection of 0.2 (Fig. 3
) to 5 µg (data not shown) of ER
expression vector only modestly enhanced E2 activation of
the ERE-TATA promoter 1.3-fold, on the average. In contrast,
E2 induction of the PRL enhancer/promoter depended on the
ectopic expression of ER
along with Pit-1 (Fig. 4A
); maximum levels of ER/Pit-1
cooperative activation of the PRL enhancer/promoter were not reached
even at 10 µg of the same ER
expression vector (35). Thus,
E2 activation was saturated at much lower levels of ER at
the ERE-TATA promoter than at the PRL enhancer/promoter. The
differential response of different promoters to cellular ER levels may
represent an overlooked component of the cell- and promoter-specific
actions of ER.
Cotransfection of the RIP140 expression vector reduced E2
activation of the vitellogenin ERE-TATA promoter (Fig. 3
). In the
presence of 0.2 µg of cotransfected ER, RIP140 reduced
E2-dependent ERE-TATA promoter activity to 60.8 ±
7.6%. Therefore, RIP140 inhibition was not limited to the PRL
enhancer/promoter and even occurred at a minimal promoter at which the
effects of promoter binding factors other than ER were minimized.
RIP140 is likely to directly inhibit ER action at a variety of EREs in
a variety of enhancer/promoter contexts.
AF-2 Dominates ER/Pit-1 Activation of the PRL Enhancer/Promoter
The strong inhibition of ER/Pit-1 cooperative activation of the
PRL enhancer/promoter by RIP140 might reflect RIP140 inhibition via ER
AF-2 to which RIP140 is known to bind. Conversely, RIP140 might target
a site in ER other than or in addition to AF-2. A second ER activation
function, AF-1, is located in a domain separable from the LBD in which
AF-2 resides. Some AF-2-interacting proteins have been observed to
interact also with AF-1 of some nuclear receptors, including ER (28, 29). In the context of the full-length ER, AF-1 is dormant until
released by ligand binding to the LBD. To study which domains of ER
were required for RIP140 inhibition, we initially distinguished the
relative contributions of AF-1 and AF-2 to ER/Pit-1 activation of the
PRL enhancer/promoter pharmacologically.
GHFT15 cells were grown in medium containing low levels of estrogens
and then transfected with ER
and/or Pit-1 expression vectors.
Incubation with 10-8 M E2
resulted in significant activation of the PRL enhancer/promoter by ER
and Pit-1 (Fig. 4A
). E2 had little effect on the PRL
enhancer/promoter activated only by Pit-1 expression (Fig. 4A
). The
small, but reproducible, activation observed with ER/Pit-1 expression
in the absence of any ligand was due to residual estrogens in the
medium, as it was blocked by 10-6 M ICI
164,384, which prevents both AF-1 and AF-2 activities.
Other ligands, including tamoxifen and raloxifene, that bind the
E2-binding pocket of ER, commonly activate AF-1 (22, 28, 47, 48) without allowing the ER LBD to adopt the
E2-dependent conformation in which AF-2 is available for
coactivator binding (49). Tamoxifen and raloxifene are thereby partial
agonists at promoters in which AF-1 is sufficient for activation.
Incubation of the transfected cells with 10-6
M tamoxifen or 10-6 M raloxifene
did not activate PRL enhancer/promoter activity. This suggested that
ER/Pit-1 activation of the PRL enhancer/promoter is relatively AF-2
driven, with little or no contribution of AF-1 in the absence of
AF-2.
AF-1 Is Not Required for RIP140 Inhibition of ER/Pit-1 Cooperative
Activation
The lack of ER/Pit-1 activation in the presence of tamoxifen and
raloxifene suggested that active AF-1 alone was insufficient for
estrogen regulation of the PRL enhancer/promoter. However, AF-2 was
also inhibited by these ligands. To examine whether any portion of
ER/Pit-1 cooperative activation was affected by AF-1, we studied the
ability of an ER deleted of its entire amino-terminal domain, including
AF-1 (ER-
N), to activate the PRL enhancer/promoter. ER-
N was as
effective as full-length ER in its ability to cooperate with Pit-1 and
activate the PRL enhancer/promoter (Fig. 5
). Thus, AF-1 makes no contribution to
ER/Pit-1 activation. Moreover, RIP140 also inhibited ER-
N/Pit-1
activation as efficiently as ER/Pit-1 activation (Fig. 5
). Therefore,
despite the contribution of some AF-2-interacting proteins to AF-1/AF-2
cooperation (28, 29), RIP140 inhibition is complete in the absence of
AF-1.
PRL Enhancer/Promoter Activation via the Classical ER Pathway
The recently described ß-isoform of ER (50) is present in
pituitaries (51) and pituitary cell lines (S. Gong and F. Schaufele,
unpublished data), but its contribution to PRL enhancer/promoter
activation is unknown. PRL synthesis in ER
knockout mice is reduced,
but not eliminated (52), suggesting a role for ER
in PRL gene
regulation that might be partially compensated by pituitary ERß. Like
ER
(Fig. 4A
), ERß activation of the PRL enhancer/promoter in
GHFT15 cells grown in E2-deficient medium depended upon
Pit-1 and E2 (Fig. 4B
).
Estrogen regulation results from classical ER interactions with an ERE
in the PRL enhancer. Estrogen regulation also may arise from ER
interactions with other factors, including AP-1 (47), which is a potent
regulator of the PRL promoter (44, 53). We used differences in the
pharmacology of the estrogen response via the classical and AP-1
pathways to determine their respective contributions to Pit-1
cooperative activation with both ER
and ERß. In general, ER
and
ERß activation via AP-1 are mechanistically distinct and
distinguishable by differing agonist/antagonist profiles (48). At AP-1,
tamoxifen acts as an agonist for both isoforms, and raloxifene
selectively activates ERß. Both ER
/Pit-1 and ERß/Pit-1
activation of the PRL enhancer/promoter were insensitive to tamoxifen,
raloxifene, and ICI 164,384 and were only activated by estradiol (Fig. 4
). The lack of any agonist activity by either tamoxifen or raloxifene
suggested that the AP-1 pathway did not contribute to estrogen
regulation of Pit-1 cooperation with either ER isoform.
RIP140 Inhibits Pit-1 Cooperation with ERß
The similar pharmacology and similar Pit-1 dependence of ER
and
ERß suggest that the two ER isoforms play similar roles in the
estrogen regulation of the PRL gene. ER
and ERß are homologous
over the DNA-binding domain and LBD (50), which are sufficient
for RIP140 inhibition of ER
/Pit-1 cooperative action (Fig. 5
). We,
therefore, examined whether RIP140 would inhibit ERß cooperative
activation with Pit-1.
Like ER
, the coexpression of RIP140 completely blocked ERß/Pit-1
cooperative activation (Fig. 6
). RIP140
expression also did not change the relative abilities and inabilities
of E2, tamoxifen, raloxifene, or ICI 164,384 to promote
ER
or ERß cooperative activation with Pit-1 at the PRL
enhancer/promoter (data not shown). Thus, ER
and ERß behave
similarly with respect to RIP140 inhibition of ER/Pit-1 cooperativity
at the PRL enhancer/promoter.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 6. ERß/Pit-1 Cooperative Activation of the PRL
Enhancer Promoter Is Inhibited by RIP140
Four independent experiments were normalized to the activity of the
Pit-1-activated PRL enhancer/promoter (1-fold), and the results are
plotted as the mean ± SD.
|
|
AF-2 Interacting Coactivators Counteract RIP140 Inhibition
The similar effects of RIP140 on ER
and ERß action at the PRL
enhancer/promoter most likely indicated that they possess a common
target for RIP140 action. Both ER
and ERß contain AF-2, which is
required for direct RIP140 binding to ER and other nuclear receptors.
Within AF-2, the binding sites for some of the AF-2-interacting
proteins overlap, albeit with AF-2 amino acid requirements specific for
each interacting protein (8, 16, 54, 55, 56, 57). Consistent with binding site
overlap, at least RIP140 and SRC-1a have been shown to compete for
binding to PPAR
(58).
If AF-2 were a target for RIP140 inhibition, other AF-2-interacting
proteins that did not inhibit ER/Pit-1 cooperative interaction (Fig. 1
)
should compete with RIP140 binding and reduce RIP140 inhibition of the
PRL enhancer/promoter. Expression of SRC-1a, GRIP1, and TIF1
shifted
the dose response of RIP140 inhibition to the right (Fig. 7A
). Western blots showed that these
effects were not due to changes in the expression of ER or Pit-1 (Fig. 7B
). PRL enhancer/promoter activity progressively declined with higher
RIP140 amounts in the presence of each coactivator (Fig. 7A
). However,
there were some differences in the coactivator responses.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 7. Other AF-2-Interacting Proteins Relieve RIP140
Inhibition of Human ER/Pit-1 Cooperative Activation of the PRL
Enhancer/Promoter
A, Cotransfection of 10 µg of the expression vectors for the
AF-2-interacting proteins SRC-1a, GRIP, or TIF1 partially relieved
dose-dependent RIP140 inhibition, albeit with differing
characteristics. Three independent experiments were normalized to the
activity of the ER/Pit-1-coactivated PRL enhancer/promoter (100%), and
the results are plotted as the mean ± SD. B, ER and
Pit-1 expressions, detected by Western blot, were not affected by
expression of the AF-2-interacting proteins.
|
|
Relief of RIP140 inhibition by TIF1
became less effective and was
eventually eliminated at higher RIP140 expression levels. Intersection
of the RIP140 dose-response curves in the presence and absence or
TIF1
at high RIP140 levels would be expected if RIP140 competed for
and occluded TIF1
binding to the same site. At high RIP140 levels,
the RIP140 dose-response curve in the presence of GRIP1 also approached
that in the absence of GRIP1, although it did not intersect it. The
difference in the degree to which GRIP1 and TIF1
counteracted RIP140
inhibition may reflect relative differences in TIF1
and GRIP1
expression levels, interaction with AF-2, or ability to coactivate the
PRL enhancer/promoter.
In contrast, the slope of the RIP140 dose-response curve in the
presence of coexpressed SRC-1a diverged from the RIP140 dose-response
curve. This would not be anticipated if the only effect of SRC-1a was
to occlude AF-2 and prevent RIP140 from binding. This may suggest that
the SRC-1a-containing complex was a strong coactivator of ER/Pit-1
action at the PRL enhancer/promoter or that the SRC-1a-containing
complex was more stable to RIP140 competition than its GRIP1 or
TIF1
-containing counterparts. An alternative possibility was that
SRC-1a acted through a different site on the ER than did GRIP1 or
TIF1
.
Relief of RIP140 Inhibition Requires SRC-1a and GRIP1 Interaction
with AF-2
Inhibition of ER/Pit-1 cooperative activation of the PRL
enhancer/promoter by the AF-2-interacting protein RIP140 was therefore
counteracted by the expression of AF-2-binding coactivators. To
determine whether AF-2 itself was the site of action for either or both
SRC-1a and GRIP1, mutations that disrupt SRC-1a and GRIP1 interaction
with ER AF-2 were examined for their effect on the relief of RIP140
inhibition. SRC-1a and GRIP1 bind to AF-2 via a conserved sequence
motif, LXXLL (59), present in multiple copies in SRC-1a and GRIP1.
Point mutants changing all four copies of LXXLL in SRC-1a or two of the
three copies of LXXLL in GRIP1 to LXXAA block interaction of those
coactivators with ER AF-2 (57, 59). SRC-1a or GRIP1 containing those
same mutants were no longer capable of relieving RIP140 inhibition of
the PRL enhancer/promoter (Fig. 8
). Upon
coexpression of RIP140 and mutant SRC-1a or GRIP1, PRL
enhancer/promoter activity remained at 38.8 ± 18.3% or 34.4
± 16.8% that of the ER/Pit-1-activated PRL enhancer/promoter. This
was not statistically different (P > 0.10) from the
41.8 ± 13.0% activity upon coexpression of RIP140. In contrast,
a statistically significant relief of RIP140 inhibition was observed
with the coexpression of either wild-type SRC-1a (P =
0.01) or GRIP1 (P = 0.03) to levels that were not
significantly different (P > 0.10) from those of the
enhancer/promoter activity in the absence of RIP140 or coactivator.
Thus, SRC-1a and GRIP1 relieve RIP140 inhibition through a target that
binds the LXXLL motifs.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 8. Mutation of the LXXLL Boxes in SRC-1a and GRIP1 to
LXXAA Eliminates SRC-1a and GRIP1 Relief of RIP140 Inhibition
A dose of RIP140 expression vector within the linear inhibitory range
(5 µg) was transfected in these experiments. Ten micrograms of each
wild-type (wt) or mutant coactivator were cotransfected. Four
independent experiments were normalized to the activity of the
ER/Pit-1-coactivated PRL enhancer/promoter (100%), and the results are
plotted as the mean ± SD.
|
|
The suggestion that the LXXLL-binding site targeted by SRC-1a and GRIP1
was AF-2 in ER was confirmed by using a mouse ER containing the K366A
point mutation that selectively disrupts binding to SRC-1a without
affecting RIP140 binding (56). The K366A mutant ER remained capable of
cooperating with Pit-1 to activate the PRL enhancer/promoter, albeit at
a level 50.0 ± 9.8% that of the wild-type mouse ER (Fig. 9A
). This activity was still
significantly above (P < 0.05) the level of PRL
enhancer/promoter activity that would have been obtained by the
addition of the independent Pit-1 and ER K366A activation levels.

View larger version (35K):
[in this window]
[in a new window]
|
Figure 9. The SRC-1a- and GRIP1-Binding Site in ER Is
Required for SRC-1a and GRIP1 Relief of RIP140 Inhibition
A, The K366A mutant of the mouse ER , which blocks SRC-1a binding
without affecting RIP140 binding (56 ), cooperated with Pit-1 to
activate the PRL enhancer/promoter. PRL enhancer/promoter activation by
the K366A ER mutant was less than that of wild-type (wt) mouse ER under
saturating and subsaturating ER conditions. B, PRL enhancer/promoter
activation by Pit-1 and the K366A ER mutant was inhibited by the
cotransfection of 5 µg RIP140 expression vector; this was not
relieved by the cotransfection of 10 µg SRC-1a or GRIP1 expression
vector. Three independent experiments were normalized to the activity
of the wild-type mouse ER/Pit-1-coactivated PRL enhancer/promoter
(100%), and the results are plotted as the mean ± SD
in A and B.
|
|
PRL enhancer/promoter activity cooperatively activated by ER K366A and
Pit-1 was reproducibly reduced by RIP140 expression to a level
61.0 ± 17.9% of the activity in the absence of RIP140. In
contrast to the wild-type mouse ER, RIP140 inhibition of cooperative
activation of ER K366A with Pit-1 was not relieved by expression of
SRC-1a and GRIP1 (Fig. 9B
). In the presence of RIP140 and SRC-1a or
GRIP1, ER-K366A/Pit-1-activated PRL enhancer/promoter activity was not
statistically different from and even tended to be lower than the
activity in the absence of coactivator. Thus, cooperative activation by
Pit-1 and the K366A mutant mouse ER was still inhibited by RIP140, but
was no longer relieved by SRC-1a or GRIP1 coexpression. Together, the
results shown in Figs. 8
and 9
conclusively demonstrated that
counteraction of RIP140 inhibition of ER/Pit-1 cooperative activation
of the PRL enhancer/promoter by SRC-1 and GRIP1 required the
interaction of both coactivators with ER AF-2.
 |
DISCUSSION
|
---|
Most natural estrogen-regulated promoters contain not only
ER-binding sites, but also sites for other transcription factors.
Indeed, estrogen regulation of many natural promoters is cell specific,
suggesting that other factors affect ER action. Pituitary-specific
estrogen regulation of the PRL enhancer/promoter results from ER
dependence on the pituitary-specific PRL enhancer/promoter-binding
factor, Pit-1 (31, 32, 33, 34, 35). Thus, at the PRL enhancer/promoter, Pit-1 may
even be thought of as a sequence-specific DNA-binding coactivator for
ER. To fully understand cell-specific estrogen regulation, it will be
important to define how ER activities are used in the context of Pit-1
and other promoter-binding factors.
ER contains at least two transcriptional activation functions, AF-1 and
AF-2, which regulate basal promoter activity (3, 4). Deletion of AF-1
had no effect on ER/Pit-1 cooperative activation at the PRL
enhancer/promoter (Fig. 5
). Consistent with this, the thyroid hormone
receptor, which contains no or poor AF-1 activity (60), also cooperates
with Pit-1 to activate the pituitary-specific GH gene promoter (61). In
contrast, null mutations within ER AF-2 (35) or blocking ER AF-2
activity with the selective AF-2 antagonists tamoxifen or raloxifene
(Fig. 4
) did not allow ER cooperative activation with Pit-1. Similarly,
thyroid hormone receptor cooperative activation with Pit-1 required
AF-2 (35). Thus, Pit-1 cooperative activation with at least two nuclear
receptors seems to be relatively AF-2 driven. Selective use of AF-2 may
be related to activities provided by Pit-1 that supercede, mimic, or,
in ER, preclude the use of AF-1.
Part of AF-2 consists of a conserved sequence (20) that in all nuclear
receptors forms an amphipathic
-helix on one side of a
ligand-induced hydrophobic pocket. This pocket acts as a docking site
(62) for certain ER-interacting proteins, including SRC-1a, GRIP1,
TIF1
, and RIP140. In mammalian cells, at least SRC-1a, GRIP1, and
RIP140 can potentiate the activities of artificial promoters in which
an ERE is placed in close proximity to the transcription start (18, 56, 57, 59) and are thus bona fide ER coactivators. However,
expression of GRIP1, SRC-1a, or TIF1
did not substantially affect
ER/Pit-1 activation of the PRL enhancer/promoter (Fig. 1
). Perhaps
GRIP1, SRC-1a and TIF1
are already present in excess in GHFT15
cells, or Pit-1 cooperative activation with ER mutated such that it
cannot bind SRC-1 (Fig. 9A
) might indicate that other known (7, 8, 9, 11, 12, 14, 17, 19, 23) or unknown AF-2-interacting coactivators
participate in ER action at the PRL enhancer/promoter.
The putative coactivator is not RIP140, which strongly inhibited
ER/Pit-1 cooperative activation at the PRL enhancer/promoter (35) (Fig. 1
). RIP140 directly repressed ER in GHFT15 cells (Fig. 3
), but did
not inhibit the PRL enhancer/promoter when ER was selectively
inactivated (Fig. 2
). Thus, RIP140 inhibition of PRL gene transcription
operated via ER and not via any of the other transcription factors that
bind to the PRL enhancer/promoter. Within ER, the target of RIP140
inhibition appeared to be common to both ER isoforms (Figs. 4
and 6
)
and was not affected by the deletion of AF-1.
Competition by TIF1
for RIP140 binding to a common site in ER seemed
to explain partial relief of RIP140 inhibition by TIF1
and probably
GRIP1 and SRC-1a (Fig. 7
). In support of direct competition at AF-2,
the relief of RIP140 inhibition by both SRC-1a and GRIP1 was dependent
on AF-2 (Figs. 8
and 9
). Similarly, RIP140 inhibition of ER/Pit-1
cooperative activation of the PRL enhancer/promoter in GHFT15 cells
may arise from RIP140 competitive inhibition of the binding of the
putative endogenous AF-2-interacting coactivator. The prevention of
activation by mass action interference of the binding of a strong
coactivator by the binding of a poorer coactivator, such as RIP140, is
emerging as a mechanism of corepression that is distinct from the
active mechanisms involving the recruitment of enzymatic activities.
Mass action competition at AF-2 is, however, likely to be modified by
the recruitment of activating or inhibitory complexes by the competing
factors, which may explain the observed differences in coactivator
relief of RIP140 inhibition (Fig. 7A
).
Therefore, in pituitary cells, estrogen regulation of the PRL
enhancer/promoter is a synthesis of mutual, complementary, and/or
counteracting activities possessed by ER, its AF-2 interacting
cofactors, and Pit-1. The estrogen responses of other promoters might
similarly be regulated by the amount and binding preferences of
competing AF-2-interacting proteins in a cell. This would also be
influenced by the amount of ER and the differential sensitivity of some
promoters to ER levels (Figs. 3
and 4
). Thus, the relative effects and
amounts of ER, different AF-2-interacting proteins, and cooperating
transcription factors will probably determine the level of
cell-specific estrogen response at many natural promoters. The
development of ER ligands that specifically affect any of those
interactions may one day provide a means to selectively activate or
inhibit estrogen responses in specific tissues.
 |
MATERIALS AND METHODS
|
---|
Transfection and Analysis
GHFT15 cells were grown in DMEM-H21 supplemented with 10% FCS
(Figs. 1
, 2
, and 5
7). The agonist/antagonist response was determined
in cells that had been grown for 24 h before transfection in
medium lacking phenol red and containing 10% iron-supplemented newborn
calf serum tested for reduced amount of estrogens (Figs. 3
and 4
).
Transfected cells were grown in the estrogen-deficient medium and
treated with 10-8 M estradiol,
10-6 M ICI 164,384, 10-6
M tamoxifen, 10-6 M raloxifene,
and/or delivery vehicles 1 day after transfection, then collected the
following day. Transfection was performed by electroporation using the
conditions and buffers previously described (45).
Collected cells were lysed in reporter lysis buffer (Promega Corp., Madison, WI). Luciferase (Figs. 1
, 2
, and 4
7) and
chloramphenicol acetyltransferase (Fig. 3
) activities in these extracts
were determined as previously described (34, 35, 45, 61). Data from
multiple independent experiments were normalized to specific reference
points (see figure legends for n and points), and the mean ±
SD were determined for each.
For Western blots (Figs. 1
and 7
), the cell pellet posttreatment with
reporter lysis buffer was resuspended in 50 mM Tris
buffered to pH 7.8 with 2-[N-morpholino]ethanesulfonic
acid, 1 mM dithiothreitol, and 0.1% Triton X-100;
pelleted; resuspended in the same buffer; then pelleted again. The
resulting crude nuclei were resuspended three times with 20 µl 20
mM HEPES (pH 7.9), 300 mM KCl, 200
mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, and
15% glycerol, and the extracts were pooled. Equivalent amounts of
extract protein (520 µg depending on the experiment) were loaded
onto 10% SDS-polyacrylamide gels and probed with either the FLAG
M2
mouse monoclonal antibody (ICI-Kodak, Rochester, NY) or the
human-specific ER HC-20 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing away the
primary antibodies, horseradish peroxidase-linked antimouse IgG
(Life Technologies, Gaithersburg, MD) or antirabbit IgG
(Amersham, Arlington Heights, IL) secondary antibodies
were incubated with the filters, washed, and detected with enhanced
chemiluminescence reagents (Amersham).
Plasmids
Levels of 2.5 µg (Figs. 1
, 2
, and 4
7) and 2.0 µg (Figs. 8
and 9
) of the 3-kb PRL enhancer/promoter cloned in front of the
luciferase gene (63) were transfected in each experiment shown. The
plasmid containing the vitellogenin ERE inserted in front of the
thymidine kinase TATA box controlling the bacterial chloramphenicol
acetyltransferase gene was previously described (47); 1 µg of this
reporter was transfected in Fig. 3
.
Ten micrograms of the human ER
(64), 10 µg rat ERß (48), 5 µg
FLAG-tagged rat Pit-1 (35), and 110 µg RIP140 (18) expression
vectors were transfected into GHFT15 cells (Figs. 1
, 2
, and 4
7) as
previously described (35). Expression vectors were under the control of
the simian virus-40 (human ER
), cytomegalovirus (ERß, Pit-1), and
elongation factor-1
(RIP140) promoters. A level of 0.2 µg human
ER
expression vector was transfected in Fig. 3
; 0.33 µg
wild-type or K366A mutant of the mouse ER (56) were transfected in Fig. 9
. Activation of the PRL enhancer/promoter saturated at 1 µg mouse ER
expression vector, whereas saturation was not reached even with 15 µg
human ER expression vector. This presumably reflected a much higher
transcriptional activity in GHFT15 cells of the adenovirus major late
promoter used to express the mouse ER.
Expression vectors containing CBP (38) under the control of the Rous
sarcoma virus promoter (Fig. 1
), wild-type SRC-1a (10) under the
control of the cytomegalovirus promoter (Figs. 1
and 7
), wild-type
SRC-1a and SRC-1a mutated in its four LXXLL motifs (L636A/L637A,
L693A/L694A, L752A/L753A, and L1438A/L1439A; Figs. 8
and 9
), wild-type
GRIP1 (15), GRIP1 mutated in its two LXXLL motifs (L693A/L694A and
L748A/L749A; Figs. 1
and 7
9) (57), and TIF1
(Figs. 1
and 7
) (13),
all under the control of the simian virus-40 promoter, were previously
described. For points in which a particular cDNA was not expressed in a
matched experiment, the "empty" expression vector not containing
the specific cDNA was cotransfected.
 |
ACKNOWLEDGMENTS
|
---|
I thank Dr. Paul Webb for critical reading of the manuscript,
Dr. Tom Scanlon for providing raloxifene, and the following for the
indicated expression vectors: Drs. V. Cavaillès and M. Parker
(RIP140, wild-type and K366A mutant of mouse ER, wild-type and LXXLL
mutants of SRC-1a); Drs. S. Oñate, M.-J. Tsai, and B. OMalley
(SRC-1a); Drs. H. Hong and M. Stallcup (wild-type GRIP1); Carol
Anderson and Dr. Peter Kushner (LXXLL mutant of GRIP1); Drs. B.
LeDouarin and R. Losson (TIF1
); Drs. R. Kwok and R. Goodman (CBP);
and K. Paech and Dr. P. Webb (ERß).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Fred Schaufele, Metabolic Research Unit, University of California, San Francisco, California 94143-0540. E-mail: freds{at}metabolic.ucsf.edu
This work was supported by Grant RPG-94028-TBE from the American
Cancer Society (to F.S.).
Received for publication September 30, 1998.
Revision received March 15, 1999.
Accepted for publication March 19, 1999.
 |
REFERENCES
|
---|
-
Shibata H, Spencer TE, Oñate SA, Jenster G, Tsai
SY, Tsai MJ, OMalley BW 1997 Role of co-activators and co-repressors
in the mechanism of steroid/thyroid receptor action. Recent Prog Horm
Res 52:141164[Medline]
-
Ribeiro RC, Kushner PJ, Baxter JD 1995 The nuclear hormone
receptor gene superfamily. Annu Rev Med 46:443453[CrossRef][Medline]
-
Parker MG 1998 Transcriptional activation by oestrogen
receptors. Biochem Soc Symp 63:4550[Medline]
-
Katzenellenbogen JA, OMalley BW, Katzenellenbogen BS 1996 Tripartite steroid hormone receptor pharmacology: interaction with
multiple effector sites as a basis for the cell- and promoter-specific
action of these hormones. Mol Endocrinol 10:119131[Medline]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor
coactivators. Curr Opin Cell Biol 9:222232[CrossRef][Medline]
-
vom Baur E, Harbers M, Um SJ, Benecke A, Chambon P, Losson R 1998 The yeast Ada complex mediates the ligand-dependent activation
function AF-2 of retinoid x and estrogen receptors. Genes Dev 12:12781289[Abstract/Free Full Text]
-
vom Baur E, Zechel C, Heery D, Heine MJ, Garnier JM, Vivat V,
Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential
ligand-dependent interactions between the AF-2 activating domain of
nuclear receptors and the putative transcriptional intermediary factors
mSUG1 and TIF1. EMBO J 15:110124[Abstract]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Oñate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone
receptor superfamily. Science 270:13541357[Abstract]
-
Lee JW, Choi HS, Gyuris J, Brent R, Moore DD 1995 Two classes
of proteins dependent on either the presence or absence of thyroid
hormone for interaction with the thyroid hormone receptor. Mol
Endocrinol 9:243254[Abstract]
-
Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid-hormone receptor with a conserved
transcriptional mediator. Nature 374:9194[CrossRef][Medline]
-
LeDouarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat P,
Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of
TIF1, a putative mediator of the ligand-dependent activation function
(AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein
T18. EMBO J 14:20202033[Abstract]
-
Huang N, vom Baur E, Garnier JM, Lerouge T, Vonesch JL, Lutz
Y, Chambon P, Losson R 1998 Two distinct nuclear receptor interaction
domains in NSD1, a novel SET protein that exhibits characteristics of
both corepressors and coactivators. EMBO J 17:33983412[Abstract/Free Full Text]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a
transcriptional coactivator for the AF-2 transactivation domain of
steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:27352744[Abstract]
-
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Cavaillès V, Dauvois S, LHorset F, Lopez G, Hoare S,
Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates
transcriptional activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
Ichinose H, Garnier JM, Chambon P, Losson R 1997 Ligand-dependent interaction between the estrogen receptor and the
human homologues of SWI2/SNF2. Gene 188:95100[CrossRef][Medline]
-
Danielian PS, White R, Lees JA, Parker MG 1992 Identification
of a conserved region required for hormone dependent transcriptional
activation by steroid hormone receptors [published erratum appears in
EMBO J 1992 Jun;11(6):2366]. EMBO J 11:10251033[Abstract]
-
Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG,
Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor
transactivational capacity is determined by both cellular and promoter
context and mediated by two functionally distinct intramolecular
regions. Mol Endocrinol 8:2130[Abstract]
-
Kraus WL, McInerney EM, Katzenellenbogen BS 1995 Ligand-dependent, transcriptionally productive association of the
amino- and carboxyl-terminal regions of a steroid hormone nuclear
receptor. Proc Natl Acad Sci USA 92:1231412318[Abstract]
-
Sande S, Privalsky ML 1996 Identification of TRACs (T3
receptor-associating cofactors), a family of cofactors that associate
with, and modulate the activity of, nuclear hormone receptors. Mol
Endocrinol 10:813825[Abstract]
-
Zamir I, Harding HP, Atkins GB, Hörlein A, Glass CK,
Rosenfeld MG, Lazar MA 1996 A nuclear hormone receptor corepressor
mediates transcriptional silencing by receptors with distinct
repression domains. Mol Cell Biol 16:54585465[Abstract]
-
Seol W, Mahon MJ, Lee YK, Moore DD 1996 Two receptor
interacting domains in the nuclear hormone receptor corepressor
RIP13/N-CoR. Mol Endocrinol 10:16461655[Abstract]
-
Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE,
Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a
complex containing SMRT, mSin3A, and histone deacetylase. Cell 89:373380[Medline]
-
Blanco JC, Minucci S, Lu J, Yang XJ, Walker KK, Chen H, Evans
RM, Nakatani Y, Ozato K 1998 The histone acetylase PCAF is a nuclear
receptor coactivator. Genes Dev 12:16381651[Abstract/Free Full Text]
-
McInerney EM, Tsai MJ, OMalley BW, Katzenellenbogen BS 1996 Analysis of estrogen receptor transcriptional enhancement by a nuclear
hormone receptor coactivator. Proc Natl Acad Sci USA 93:1006910073[Abstract/Free Full Text]
-
Norris JD, Fan D, Stallcup MR, McDonnell DP 1998 Enhancement
of estrogen receptor transcriptional activity by the coactivator GRIP-1
highlights the role of activation function 2 in determining estrogen
receptor pharmacology. J Biol Chem 273:66796688[Abstract/Free Full Text]
-
Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO 1998 Functional interactions of the AF-2 activation domain core region
of the human androgen receptor with the amino-terminal domain and with
the transcriptional coactivator TIF2 (transcriptional intermediary
factor 2). Mol Endocrinol 12:11721183[Abstract/Free Full Text]
-
Day RN, Koike S, Sakai M, Muramatsu M, Maurer RA 1990 Both
Pit-1 and the estrogen receptor are required for estrogen
responsiveness of the rat prolactin gene. Mol Endocrinol 4:19641971[Abstract]
-
Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS,
Rosenfeld MG, Swanson LW 1990 Pituitary cell phenotypes involve
cell-specific Pit-1 mRNA translation and synergistic interactions with
other classes of transcription factors. Genes Dev 4:695711[Abstract]
-
Holloway JM, Szeto DP, Scully KM, Glass CK, Rosenfeld MG 1995 Pit-1 binding to specific DNA sites as a monomer or dimer determines
gene-specific use of a tyrosine-dependent synergy domain. Genes Dev 9:19922006[Abstract]
-
Chang W, Zhou W, Theill LE, Baxter JD, Schaufele F 1996 An activation function in Pit-1 required selectively for synergistic
transcription. J Biol Chem 271:1773317738[Abstract/Free Full Text]
-
Chuang FM, West BL, Baxter JD, Schaufele F 1997 Activities in
Pit-1 determine whether receptor interacting protein 140 activates or
inhibits Pit-1/nuclear receptor transcriptional synergy. Mol Endocrinol 11:13321341[Abstract/Free Full Text]
-
Lew D, Brady H, Klausing K, Yaginuma K, Theill LE, Stauber C,
Karin M, Mellon PL 1993 GHF-1-promoter-targeted immortalization of a
somatotropic progenitor cell results in dwarfism in transgenic mice.
Genes Dev 7:683693[Abstract]
-
Li S, Crenshaw E Bd, Rawson EJ, Simmons DM, Swanson LW,
Rosenfeld MG 1990 Dwarf locus mutants lacking three pituitary cell
types result from mutations in the POU-domain gene pit-1. Nature 347:528533[CrossRef][Medline]
-
Kwok RP, Lundblad JR, Chrivia JC, Richards JP,
Bächinger HP, Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB.
Nature 370:223226[CrossRef][Medline]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Iverson RA, Day KH, dEmden M, Day RN, Maurer RA 1990 Clustered point mutation analysis of the rat prolactin promoter. Mol
Endocrinol 4:15641571[Abstract]
-
Harvey C, Jackson SM, Siddiqui SK, Gutierrez-Hartmann A 1991 Structure-function analysis of the rat prolactin promoter: phasing
requirements of proximal cell-specific elements. Mol Endocrinol 5:836843[Abstract]
-
Bach I, Rhodes SJ, Pearse RV, 2nd, Heinzel T, Gloss B, Scully
KM, Sawchenko PE, Rosenfeld MG 1995 P-Lim, a LIM homeodomain factor, is
expressed during pituitary organ and cell commitment and synergizes
with Pit-1. Proc Natl Acad Sci USA 92:27202724[Abstract]
-
Bradford AP, Wasylyk C, Wasylyk B, Gutierrez-Hartmann A 1997 Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1
reconstitutes pituitary-specific gene expression. Mol Cell Biol 17:10651074[Abstract]
-
Farrow KN, Manning N, Schaufele F, Gutierrez-Hartmann A 1996 The c-Jun delta-domain inhibits neuroendocrine promoter activity in a
DNA sequence- and pituitary-specific manner. J Biol Chem 271:1713917146[Abstract/Free Full Text]
-
Schaufele F 1996 CCAAT/enhancer-binding protein alpha
activation of the rat growth hormone promoter in pituitary progenitor
GHFT15 cells. J Biol Chem 271:2148421489[Abstract/Free Full Text]
-
Szeto DP, Ryan AK, OConnell SM, Rosenfeld MG 1996 P-OTX: a
PIT-1-interacting homeodomain factor expressed during anterior
pituitary gland development. Proc Natl Acad Sci USA 93:77067710[Abstract/Free Full Text]
-
Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation
of the estrogen receptor/AP-1 pathway: potential origin for the
cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443456[Abstract]
-
Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner
PJ, Scanlan TS 1997 Differential ligand activation of estrogen
receptors ER
and ERß at AP1 sites. Science 277:15081510[Abstract/Free Full Text]
-
Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T,
Engström O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen
receptor. Nature 389:753758[CrossRef][Medline]
-
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary.
Proc Natl Acad Sci USA 93:59255930[Abstract/Free Full Text]
-
Kuiper GG, Carlsson B, Grandien K, Enmark E, Häggblad J,
Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding
specificity and transcript tissue distribution of estrogen receptors
and ß. Endocrinology 138:863870[Abstract/Free Full Text]
-
Scully KM, Gleiberman AS, Lindzey J, Lubahn DB, Korach KS,
Rosenfeld MG 1997 Role of estrogen receptor-alpha in the anterior
pituitary gland. Mol Endocrinol 11:674681[Abstract/Free Full Text]
-
Caccavelli L, Manfroid I, Martial JA, Muller M 1998 Transcription factor AP1 is involved in basal and okadaic
acid-stimulated activity of the human PRL promoter. Mol Endocrinol 12:12151227[Abstract/Free Full Text]
-
Thénot S, Henriquet C, Rochefort H, Cavaillès V 1997 Differential interaction of nuclear receptors with the putative
human transcriptional coactivator hTIF1. J Biol Chem 272:1206212068[Abstract/Free Full Text]
-
Lazennec G, Ediger TR, Petz LN, Nardulli AM, Katzenellenbogen
BS 1997 Mechanistic aspects of estrogen receptor activation probed with
constitutively active estrogen receptors: correlations with DNA and
coregulator interactions and receptor conformational changes. Mol
Endocrinol 11:13751386[Abstract/Free Full Text]
-
Henttu PM, Kalkhoven E, Parker MG 1997 AF-2 activity and
recruitment of steroid receptor coactivator 1 to the estrogen receptor
depend on a lysine residue conserved in nuclear receptors. Mol Cell
Biol 17:18321839[Abstract]
-
Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ,
Stallcup MR 1998 Nuclear receptor-binding sites of coactivators
glucocorticoid receptor interacting protein 1 (GRIP1) and steroid
receptor coactivator 1 (SRC-1): multiple motifs with different binding
specificities. Mol Endocrinol 12:302313[Abstract/Free Full Text]
-
Treuter E, Albrektsen T, Johansson L, Leers J, Gustafsson JA 1998 A regulatory role for RIP140 in nuclear receptor activation. Mol
Endocrinol 12:864881[Abstract/Free Full Text]
-
Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
Ribeiro RC, Apriletti JW, West BL, Wagner RL, Fletterick RJ,
Schaufele F, Baxter JD 1995 The molecular biology of thyroid hormone
action. Ann NY Acad Sci 758:366389[Medline]
-
Schaufele F, West BL, Baxter JD 1992 Synergistic activation of
the rat growth hormone promoter by Pit-1 and the thyroid hormone
receptor. Mol Endocrinol 6:656665[Abstract]
-
Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW,
Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent
coactivator binding to a hydrophobic cleft on nuclear receptors.
Science 280:17471749[Abstract/Free Full Text]
-
Nelson C, Crenshaw E Bd, Franco R, Lira SA, Albert VR, Evans
RM, Rosenfeld MG 1986 Discrete cis-active genomic sequences dictate the
pituitary cell type-specific expression of rat prolactin and growth
hormone genes. Nature 322:557562[Medline]
-
Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941951[Medline]