From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine and the Nashville Veterans Administration Hospital, Nashville, Tennessee 37232
Received for publication, October 13, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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In the liver, glucocorticoids induce a
10-15-fold increase in the rate of transcription of the
phosphoenolpyruvate carboxykinase (PEPCK) gene, which encodes a key
gluconeogenic enzyme. This induction requires a multicomponent
glucocorticoid response unit (GRU) comprised of four glucocorticoid
accessory factor (AF) elements and two glucocorticoid receptor binding
sites. We show that the AFs that bind the gAF1, gAF2, and gAF3 elements
(hepatocyte nuclear factor [HNF]4/chicken ovalbumin upstream
promoter transcription factor 1 and HNF3 Mammals must have a readily adaptable mechanism for maintaining
glucose homeostasis in the face of fluctuations in nutrient supply.
Hormones such as glucocorticoids control transcription of genes that
encode enzymes that help provide the adaptive response to these
environmental changes (3-5). These hormone responses were originally
thought to be mediated through a direct interaction of the hormone
receptor and a simple DNA element, the hormone response element
(HRE)1 (6-8). In recent
years it has become apparent that more complex structures are involved.
Hormone response units, comprised of HREs and accessory factor (AF)
elements have been found in many genes involved in metabolic regulation
(9-14). AFs are DNA binding transcription factors that act in concert
with the hormone receptor to provide the flexible regulation of gene
transcription required for metabolic homeostasis (5, 6, 15, 16).
In addition to their function in hormone-mediated gene transcription,
AFs also play a central role in determining how metabolites regulate
transcription. For instance, glucose-stimulated transcription of the
L-PK gene requires a glucose response element that binds transcription factors of the upstream stimulating factor family. This glucose response, however, is regulated positively or negatively by the binding of HNF4 or COUP-TF, respectively, at an adjacent accessory element (17-20). In addition, Osborne and co-workers show
that, in the absence of cholesterol, SREBP activates
transcription of the low density lipoprotein receptor and
hydroxymethylglutaryl-CoA reductase gene promoters (21-23). An SREBP
response element (SRE) alone, however, is not sufficient for activation
of either gene. The activation of the low density liproprotein receptor
gene promoter by SREBP requires not only a sterol response element but
also an SP1 binding element (21, 24). SP1 binding to this promoter correlates with transcription of the gene in the absence of cholesterol (9). Similarly, the activation of hydroxymethylglutaryl-CoA reductase
gene expression by SREBP requires both a sterol response element and an
element that binds nuclear factor Y (9, 22, 23).
The role of AFs is well characterized in the regulation of PEPCK gene
transcription by glucocorticoids. In adipose tissue, where PEPCK is
involved in glycerogenesis, glucocorticoids repress basal and
cAMP-induced PEPCK gene transcription (25, 26). By contrast, the PEPCK
gene is stimulated by glucocorticoids in the liver, where PEPCK is an
important control point for gluconeogenesis (16, 27). In the liver,
glucocorticoids induce an A mutation of any one of the accessory elements results in a 50-60%
reduction of glucocorticoid-stimulated PEPCK gene transcription in
H4IIE hepatoma cells (28, 32, 36, 37). Any combination of two mutations
of gAF1/gAF3 or gAF2 nearly abolishes the response (28, 37). The
physiologic importance of these gAFs is evident in their role in
glucose production. Overexpression of an HNF3 variant that reduces the
response of the PEPCK gene to glucocorticoids by ~50% nearly
abolishes glucocorticoid-induced glucose production in hepatoma cells
(14).
These gAF elements are not all functionally equivalent in the context
of the PEPCK gene promoter (28, 31, 38). gAF1 and gAF3 can be
exchanged, a finding consistent with the observation that COUP-TF binds
to both elements. However, gAF1/gAF3 cannot be replaced with gAF2 and
vice versa (37). Furthermore, the spacing between gAF2 and
GR1 must be maintained, whereas this is not as important for gAF1 and
gAF3 (37). HNF3, the AF that binds gAF2, interacts with GR in
vitro (37). Thus, direct contact between HNF3 and GR may be
important for stabilizing GR binding to the GR1 and GR2 elements of the
PEPCK gene promoter, which bind GR with low affinity relative to a
consensus GRE (36). In fact, these nonconsensus elements (GR1 and GR2),
either alone or in combination, cannot by themselves mediate a
glucocorticoid response when placed in the context of a heterologous
promoter (36). Thus, the glucocorticoid response of the PEPCK gene is completely dependent on the gAF elements and the factors that bind to
these sites. However, the mechanism by which AFs control the direction
and magnitude of PEPCK gene transcription in response to
glucocorticoids remains largely unknown.
The recruitment of coactivator molecules provides a crucial step in the
integration of the functions of steroid hormone receptors and AFs with
the basal transcription machinery (2, 39, 40). Coactivators generally
do not bind DNA, but instead associate with DNA-bound activator
proteins. Some coactivators such as CREB (cAMP-response element-binding
protein)-binding protein (CBP) mediate the functions of a diverse group
of transcription factors. Others, such as steroid receptor coactivator
1 (SRC1) and glucocorticoid receptor interacting protein (GRIP1), are
functionally more specific in that they interact with activator
proteins involved in steroid hormone-mediated gene transcription (41,
42).
In this study, we show that the AFs bound to the gAF1 and gAF2 elements
of the PEPCK gene promoter recruit the coactivator molecule SRC1. HNF4
and COUP-TF interact with SRC1 through the domains required for their
AF activity (1, 2). We show here that HNF3 also interacts with SRC1
through the same domain required for its AF activity in the PEPCK gene
promoter. Mutations of either gAF1 or gAF2 that prevent HNF4/COUP-TF or
HNF3 Plasmid Construction--
The reporter construct PEPCK-LUC was
made by excising the PEPCK gene promoter (from Transient Transfection--
The maintenance and transfection of
H4IIE cells and the measurements of luciferase activity were performed
as described previously (44, 45), except that H4IIE cells were
maintained in alpha-modified Eagle's medium 10% fetal bovine
serum growth medium during incubation with calcium
phosphate-precipitated DNA for 3 h before the Me2SO shock. Subsequently, the cells were washed and incubated overnight in
serum-free medium with or without 500 nM dexamethasone, as described in the figure legends. In experiments performed with H4IIE
cells, the glucocorticoid response varied from 7- to 20-fold (with
dexamethasone/without dexamethasone). The variation is passage- and
batch-dependent. For this reason, we expressed the data in each experiment as a percentage of the wild-type glucocorticoid response, which allowed for comparison of experiments done in cells
grown at different times. For experiments that involved mapping the
domains of GAL4-SRC1 required for accessory factor activity, 5 µg of
the reporter construct (gAF1 Gel Mobility Shift Assay--
The gel mobility shift assays were
performed as described previously, except that 7.5 µg of nuclear
extracts were used for incubations rather than whole-cell extracts
(43). Nuclear extracts were prepared as described (30, 46). Briefly,
extracts were incubated in the presence or absence of GAL4-DNA binding
domain (GAL4-DBD) antibody or Lex A antibody as a control (Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min at room temperature in a
10-µl reaction and then mixed with 10 µl of a reaction buffer containing 30,000 cpm of a 32P-labeled DNA probe
(5'-CACACGGAGGACTGTCCTCCGACCA-3'), 20 mM HEPES, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 10 µM ZnS04, 6% glycerol, 0.6 µg
poly(dI-dC) (Amersham Pharmacia Biotech), and 2 µg of salmon sperm DNA for another 15 min at room temperature. The reaction mixtures
were loaded onto a 6% polyacrylamide gel in 0.5× Tris-buffered EDTA and electrophoresed at 20 mA for 150 min at room
temperature. The gels were dried and exposed to x-ray film (Eastman
Kodak Co.).
Direct Recruitment of a Coactivator, GAL4-SRC1, Can Replace the AF
Function of HNF4 or COUP-TF at gAF1--
Previous studies of the PEPCK
gene GRU show that each gAF element is specifically required and that a
precise organization of these elements in the GRU is necessary for a
full glucocorticoid response (28, 37). Glucocorticoids cause a 15-fold
induction of reporter enzyme activity when a PEPCK gene
promoter-luciferase reporter construct that contains all of the gAF
elements is expressed transiently in H4IIE hepatoma cells (Fig.
1). H4IIE cells have significant amounts
of endogenous accessory factors and potential coregulators such as SRC1
to provide for this
response.2 The presence of
large amounts of these wild-type proteins, however, makes it difficult
to analyze the effects of their transiently expressed variants on PEPCK
gene transcription. Mutations of the gAF1 element that prevent
HNF4/COUP-TF from binding, including replacement of gAF1 with a GAL4
binding site (gAF1
The reduction of the glucocorticoid response caused by replacement of
the gAF1 element with a GAL4 element cannot be restored by expression
of the GAL4-DBD, indicating that the GAL4-DBD cannot act as an AF
through this surrogate gAF1 element (Fig. 1 and Ref. 43). By contrast,
the binding of chimeric proteins consisting of the GAL4-DBD and either
HNF4 or COUP-TF restores the glucocorticoid response (1, 43). It is
possible that HNF4 and COUP-TF recruit coactivator molecules to form a
glucocorticoid-specific enhanceosome, whereas the GAL4-DBD does not.
These coactivators, in turn, could stabilize the enhanceosome complex
and/or provide necessary activation to the promoter region (47). In
support of this model, we have shown that HNF4 and COUP-TF (AFs which
bind gAF1 and gAF3) interact with SRC1 through the domains demonstrated
to be required for their accessory factor activity (1, 2).
We hypothesized that, if the role of HNF4/COUP-TF at gAF1 is to recruit
coactivators such as SRC1, then direct recruitment of the latter to the
gAF1 element by making a chimeric protein with the GAL4-DBD and
full-length SRC1 (GAL4-SRC1) should also mediate the glucocorticoid
response. Indeed, the binding of GAL4-SRC1 completely restores the
glucocorticoid induction through a gAF1
In a set of experiments similar to those shown in Fig. 1, we found that
GAL4-SRC1 can also replace COUP-TF at gAF3 in a construct where the
gAF3 element was changed to a GAL4 binding element, gAF3 Coactivator Recruitment Does Not Relieve the Requirement for Active
Involvement of Ligand-bound GR in the Glucocorticoid Response--
The
ligand-bound GR could serve primarily as a signal transducer for
recruitment of coactivators and assembly of a glucocorticoid-specific enhanceosome. If so, ligand-bound GR would no longer be required after
this enhanceosome is assembled. The low basal reporter gene activity in
cells expressing GAL4-SRC1 was surprising, as the coactivator is
constitutively bound to the promoter, yet the reporter gene is inactive
in the absence of glucocorticoid treatment (Fig. 1). We therefore
reasoned that there must be active involvement of ligand-bound GR for
GAL4-SRC1 to function as a surrogate AF. In a set of experiments
performed concomitantly with those described in Fig. 1, PEPCK promoter
reporter gene constructs were made in which block mutations were
created in either GR2 or both of the GREs (Fig.
2, inset). Since GR1 is more
important than GR2 in the glucocorticoid response (36), we were able to
create a graded system in which only ~40% (GR2 mutated) or all of
the function of GR is eliminated (GR1 and GR2 mutated), respectively.
Mutation of GR2 or both GREs severely blunts or eliminates the
glucocorticoid response, respectively, even when GAL4-SRC1 is tethered
to the promoter through the surrogate gAF1 element (Fig. 2).
GAL4-SRC1 Can Also Replace the AF Function of the gAF3-bound
Factor--
The gAF1/3 and gAF2 elements are not interchangeable in
the PEPCK gene GRU (36, 37). As expected, about 60% of the
glucocorticoid response is lost when gAF2 is changed to a GAL4 binding
element (gAF2 HNF3
The region of HNF3 Promoter- and Element-specific Requirements for the bHLH/PAS and
HAT Domains of GAL4-SRC1--
It is difficult to analyze the effect of
transiently expressed SRC1 variants in the presence of significant
amounts of the endogenous, wild-type protein. However, the system
described in the previous figures, in which GAL4-SRC1 is recruited to
the GAL4 element of the gAF
SRC1 was originally identified from a cDNA that lacked the region
encoding the 41-most N-terminal amino acids (40). The protein encoded
by this cDNA does not function as a coactivator for
HNF4,2 although it still presumably binds HNF4
through the nuclear receptor binding motifs, NRBI-IV, which are present
in the truncated protein. By contrast, full-length SRC1 does serve as a
coactivator for HNF4, and as a GAL4-chimeric protein, it can replace
HNF4 as an AF on a gAF1
SRC1, with its resident acetyltransferase activity and protein-protein
interaction surfaces, could serve both as a structural adapter molecule
in the GRU enhanceosome and also to promote transcription by the
acetylation of chromatin components and transcription factors (49-51).
A deletion construct of GAL4-SRC1, in which the acetyltransferase domain was deleted (GAL4-SRC
The expression of the GAL4-SRC1 variants was confirmed in a gel
mobility shift experiment in which nuclear extracts prepared from
GAL4-SRC1 fusion protein-expressing cells were tested. The empty vector
was used as a control to ensure that no endogenous mammalian proteins
bind to the GAL4 binding element. COS cells were used for this
experiment because of the very low transfection efficiency of H4IIE
hepatoma cells. All of the structural variants of GAL4-SRC1 are
expressed efficiently and at approximately equal levels (Fig.
5b). The identity of these protein-DNA complexes as the
GAL4-SRC1 variants was determined by preincubation of the nuclear
extract with a polyclonal antibody raised against the GAL4-DBD. This
preincubation with the anti-GAL4-DBD antibody prevents each complex
from forming, but an unrelated antibody (anti-Lex A) is without effect
(Fig. 5b). No protein-DNA complexes are seen with nuclear
extracts prepared from COS cells transfected with pRC-RSV, the vector
control for GAL4-SRC1 (Fig. 5b). It is not known why
GAL4-SRC1-
The gAF1 and gAF2 elements are functionally distinct in the PEPCK gene
GRU (36, 37); thus, we speculated that there might be different
requirements for SRC1 when it is recruited through these respective
sites. As seen at gAF1, the acetyltransferase activity of SRC1 is also
required at gAF2, since GAL4-SRC1
The element-specific requirements for GAL4-SRC1 at gAF1 and gAF2
demonstrate that multiple functions of coactivators are required for
the PEPCK glucocorticoid response. These requirements may be determined
by the local promoter environment to which the coactivator is
recruited. This high degree of specificity may also reflect promoter-specific requirements of the bHLH/PAS and HAT domains of SRC1
between different promoters. None of the GAL4-SRC1 variants significantly activate basal PEPCK gene promoter-reporter activity (Figs. 1 and 3); however, GAL4-SRC1 has low basal transactivation potential through a simple promoter that contains multimerized GAL4
binding elements (53). We used this system to test the inherent
capacity of the GAL4-SRC1 variants to activate basal transcription. The
coexpression of GAL4-SRC1 and the (GAL4)5-E1b-LUC reporter
in HeLa cells results in about a 10-fold activation of expression from
the reporter as compared with that achieved when the GAL4-DBD alone is
cotransfected (Fig. 7). GAL4-SRC1-
By contrast to the PEPCK gene GRU, where GAL4-SRC The accessory factors HNF4, COUP-TF, and HNF3 The ability of GAL4-SRC1 to replace the function of the AFs bound to
gAF1, gAF2, or gAF3 in the GRU shows that the precise factor that binds
these elements is less critical than is the ability of these proteins
to assemble a functional enhanceosome. There is, however, specificity
associated with these AFs in the GRU. The observation that the PEPCK
gene GRU uses liver-enriched factors such as HNF4 and HNF3 The precise role of ligand-bound GR in mediating this response is less
clear. A previous in vivo footprint analysis showed that all
of the gAF elements in the GRU are occupied in the absence of
glucocorticoids (56). Here we show that GAL4-SRC1 is only able to
function as a surrogate AF when GR is present (Figs. 1-3). Upon
exposure to the ligand, the GR may help promote the formation of a
functional enhanceosome by interacting with SRC1 to recruit components
of the basal transcription machinery. GR may also activate SRC1 once
the latter has been recruited to the GRU by AFs such as HNF4, COUP-TF,
and HNF3 All of the p160 family of coactivators contain three central
LXXLL motifs (NRBI-III) that are important for interaction
with nuclear receptors (58, 59). SRC1a, used in these experiments, has
an additional C-terminal LXXLL motif (NRBIV) that exhibits strong in vitro binding to class I nuclear receptors such as
estrogen receptor and GR (39, 52, 59, 60). One might imagine that the
NRBIV domain could help stabilize GR binding to the low affinity GR1
and GR2 elements. If so, deletion of the NRBIV region of SRC1 (GAL4-SRC1 The recruitment of GAL4-SRC1 or variants of this protein to promoter
constructs with GAL4 binding elements (gAF The acetyltransferase domain of GAL4-SRC1 is required at both gAF1 and
gAF2 (Figs. 5a and 6); however, the target of this acetyltransferase activity in the PEPCK gene GRU is unknown.
Acetylation of chromatin components and transcription factors
correlates with activation of gene transcription (49, 50, 63-65). The
chromatin structure of the PEPCK gene promoter is already free of
nucleosomes before exposure to hormone in H4IIE cells (66); thus, it is unlikely that this is the target of the acetyltransferase activity required of SRC1. There are several other potential targets for the
acetyltransferase activity of SRC1 in the GRU. HNF4 can be acetylated
by CBP, a reaction that promotes DNA binding and nuclear retention of
HNF4 (64). The androgen receptor can also be acetylated by CBP (67);
thus, it is possible that other nuclear receptors, such as GR, may also
be acetylated. Different targets of the acetyltransferase activity of
SRC1 (chromatin components versus transcription factors) may
also explain why GAL4-SRC1 The use of the GAL4-SRC1 chimeric protein to recruit this coactivator
to specific DNA elements allowed us to demonstrate both promoter-specific requirements of SRC1 (between the simple and PEPCK
gene promoters) and also element-specific requirements for domains of
SRC1 at gAF1 and gAF2 in the PEPCK gene GRU. A summary of these
differences is illustrated in Fig. 8.
HNF4/COUP-TF and HNF3) all interact with steroid
receptor coactivator 1 (SRC1). This suggests that the AFs function in
part by recruiting coactivators to the GRU. The binding of a GAL4-SRC1
chimeric protein completely restores the glucocorticoid induction that
is lost when any one of these elements is replaced with a GAL4 binding site. Thus, when SRC1 is recruited directly to gAF1, gAF2, or gAF3, the
requirement for the corresponding AF is bypassed. Surprisingly, glucocorticoid receptor is still required when SRC1 is recruited directly to the GAL4 site, suggesting a role for the receptor in
activating SRC1 in the context of the GRU. Structural variants of
GAL4-SRC1 were used to identify requirements for the
basic-helix-loop-helix and histone acetyltransferase domains of
SRC1, and these are specific to the region of the promoter to which the
coactivator is recruited.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10-fold increase in the rate of
transcription of the PEPCK gene through a multicomponent glucocorticoid
response unit (GRU) located in the promoter region (28). The GRU
consists of a tandem array of four AF elements (gAF1-3 and the cAMP
response element) that bind HNF4/COUP-TF, HNF3
, COUP-TF, and
C/EBP
, respectively, and two nonconsensus glucocorticoid receptor
binding sites (GR1, GR2) (13, 28-32). Interestingly, neither HNF3 nor
HNF4 is expressed in adipose tissue, whereas both are expressed at high
levels in the liver (33-35). Thus, the accessory factors HNF3 and HNF4
appear to play an additional important role in facilitating
tissue-specific induction of PEPCK gene transcription.
from binding, including replacement of the gAF element with a
GAL4 binding site (gAF
GAL4), result in a 50-60% reduction of the
response of the PEPCK gene to glucocorticoids (43). The binding of a
GAL4-SRC1 chimeric protein to a gAF1
GAL4 or a gAF2
GAL4 element in
a PEPCK luciferase reporter gene construct completely restores the
glucocorticoid induction. Thus, when SRC1 is recruited directly to gAF1
or gAF2, the requirement for the corresponding AF is functionally
bypassed. Surprisingly, even when SRC1 is recruited to the PEPCK gene
promoter in this manner, active involvement of ligand-bound GR is still required, suggesting a role of the receptor in activating SRC1 in the
context of this enhanceosome. Finally, we show that the bHLH and HAT
domains of SRC1 function differently according to the component of the
PEPCK gene GRU to which the coactivator is recruited.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
467 to +65) from
pPL32-CAT (44) with AflIII/BglII and inserting it
into the MluI/BglII sites of pGL3 basic
(Promega). The same procedure was used to generate the gAF1
GAL4 PEPCK-LUC, gAF2
GAL4 PEPCK-LUC, and gAF3
GAL4 PEPCK-LUC reporter constructs from their respective chloramphenicol
acetyltransferase reporters, as described previously (43). The
gAF1
GAL4 PEPCK-LUC constructs with block mutations in the GR1 and
GR2 elements were constructed using the Quick Change site-directed
mutagenesis kit (Stratagene). The GAL4-SRC1 deletion constructs were
also made by the Quick Change site-directed mutagenesis kit using a
GAL4-SRC1 construct as the template. Oligonucleotides used to generate
these constructs were made by an Expedite 8909 oligonucleotide
synthesizer (Perspective Biosystems, Framingham, MA). The sequences of
all oligonucleotides used in this study are shown in Table
I. All constructs were verified by
sequencing. The reporter plasmid (GAL4)5-E1b-LUC has been
described previously (1, 2). The GAL4-HNF3
constructs were also
described previously and are expressed at approximately equal levels in
transient transfection experiments (43).
Oligonucleotides used; sense strand 5' to 3'
GAL4 PEPCK-LUC or gAF2
GAL4 PEPCK-LUC)
were cotransfected with 2.5 µg of GR expression vector and 0.25, 0.5, or 1.0 µg of expression plasmid encoding the various GAL4-SRC1
structural variants. The total amount of DNA transfected in each
experiment was kept constant by using varying amounts of the empty
vector. These experiments were repeated 3-5 times, and the optimal
hormonal responses from each titration experiment were pooled for the
results shown in Figs. 5a and 6. The optimal concentration
of GAL4-SRC was consistently 0.25 µg, but the optimal concentration
of the structural variants varied from 0.25 to 1.0 µg. Luciferase
activity was determined using the Promega luciferase assay system and
normalized to the protein concentration of whole cell extracts.
Transient transfections of HeLa and COS cells were performed as
described previously (2, 43). The expression vector for GAL4-SRC1 was
provided by Dr. Peter Kushner, University of California, San Francisco.
Statistical analysis was performed using Student's t test,
assuming two-tailed variance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GAL4), result in a 50-60% reduction of the
response of the PEPCK gene to glucocorticoids, as noted previously
(Fig. 1 and Refs. 28 and 43). The GAL4 binding site can bind chimeric
proteins that contain domains of interest and, thus, can serve as a
surrogate gAF element. This is useful because the endogenous PEPCK AFs
cannot function through this surrogate gAF element; thus, they are
effectively removed from the analytical system.
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Fig. 1.
GAL4-SRC1 mediates accessory factor activity
through the gAF1 element. H4IIE cells were transfected with 5 µg
of the PEPCK-LUC reporter construct (wild-type PEPCK gene promoter,
from position 467 to +65 relative to the transcription start site,
fused to LUC) and 2.5 µg of an expression plasmid for GR.
Alternatively, cells were transfected with 5 µg of a reporter
construct based on PEPCK-LUC in which the gAF2 element has been mutated
to a GAL4 DNA binding element (gAF1
GAL4). These cells were
cotransfected with 0.5 µg of an expression plasmid encoding either
the GAL4-DBD or GAL4-SRC1. Transfected cells were incubated in
serum-free medium with or without hormone overnight. Cells were
harvested, and luciferase assays were performed as described under
"Experimental Procedures." These results represent the mean ± S.E. of at least three experiments. Dex,
dexamethasone.
GAL4 PEPCK-LUC construct
(Fig. 1). Thus, the requirement for the AF is bypassed when SRC1 is
recruited directly to gAF1. Surprisingly, there is little increase in
basal promoter-reporter activity with GAL4-SRC1 expression in the
absence of glucocorticoids (Fig. 1). This implies that, even when SRC1
is constitutively recruited by a GAL4 binding element, active
involvement of ligand-bound GR is still required, suggesting a role of
the receptor in activating SRC1 in the context of the enhanceosome. We
and others also show that the coactivator cAMP-response element-binding
protein (CREB)-binding protein (CBP) interacts with HNF4 (2, 48). A
fusion construct expressing a GAL4-CBP chimeric protein also restores
the lost glucocorticoid response through the gAF1
GAL4 PEPCK-LUC
construct (data not shown). Thus, one role of the AF bound at the gAF1
element appears to be recruitment of its cognate coactivators.
GAL4
PEPCK-LUC (data not shown). This result is not surprising since the
gAF1 and gAF3 elements are interchangeable, and both bind COUP-TF. The
amino acid region from 408-423 of COUP-TFI is necessary for this
protein to function as an AF in the PEPCK gene GRU (1). This same
region of COUP-TFI is also essential for the interaction with SRC1 (1).
These observations suggest that GAL4-SRC1 replaces some essential
function of the AFs that bind to these elements in the native promoter.
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Fig. 2.
The PEPCK gene glucocorticoid response
requires GR binding even after coactivator recruitment. These
experiments were performed as described in Fig. 1, except that the
reporter constructs illustrated in the inset were used. In
the inset, crosses through the GR1 or GR2 elements represent
block mutations in these elements that prevent GR form binding. For
simplicity, results are represented relative to the fold induction of
wild-type PEPCK-LUC by glucocorticoids, taken as 100%. These results
represent the mean ± S.E. of at least three experiments.
Dex, dexamethasone.
GAL4 PEPCK-LUC) (Fig. 3
and Ref. 43). This AF activity cannot be restored by coexpression of
the GAL4-DBD but is completely restored by GAL4-HNF3
(43). Despite
the functional distinction between gAF1/3 and gAF2, GAL4-SRC1
recruitment to gAF2
GAL4 PEPCK-LUC completely restores the
glucocorticoid response lost by mutation of this element (Fig. 3).
Thus, GAL4-SRC1 can act as a surrogate AF through gAF2 as well, and in
so doing, it provides a functional bypass of the requirement for
HNF3
.
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Fig. 3.
GAL4-SRC1 mediates accessory factor activity
through the gAF2 element. Experiments were performed as described
in Fig. 1, except that a reporter plasmid in which the gAF2 element of
PEPCK was been mutated to a GAL4 DNA binding element (gAF2 GAL4
PEPCK-LUC) was used. These results represent the mean ± S.E. of
at least three experiments. Dex, dexamethasone.
Interacts with SRC1 in Vivo--
As mentioned previously,
HNF4 and COUP-TF, the AFs that bind gAF1 and gAF3, both interact with
SRC1. We proposed that GAL4-SRC1 could serve as a surrogate AF through
gAF1 and gAF3 when these elements are changed to a GAL4 binding
element, respectively, because SRC1 is recruited to the promoter at an
appropriate position (this paper and Refs. 1 and 2). If SRC1 replaces a
distinct function of HNF3
in the GRU, it is possible that HNF3
also interacts with SRC1. To test this, an expression vector for SRC1
was cotransfected into HeLa cells with GAL4-HNF3
or variants thereof
and a luciferase reporter plasmid that contained a minimal E1b promoter
positioned downstream of five GAL4 binding elements
[(GAL4)5-E1b-LUC]. All GAL4 constructs were expressed at
low levels (0.5 µg of vector) to allow for maximal coactivation by
SRC1. Cotransfection of SRC1 potentiates transactivation mediated by
GAL4-HNF3
an additional 7-fold relative to expression of an empty
control vector (Fig. 4). Coexpression of
SRC1 does not coactivate through the GAL4-DBD, as seen in previous work
(Fig. 4 and Refs. 1 and 2).
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Fig. 4.
SRC1 potentiates the activity of
GAL4-HNF3 fusion proteins. Expression
plasmids that encode SRC1 (5 µg) and GAL4-HNF3
fusion proteins
(0.5 µg) were cotransfected into HeLa cells along with the reporter
construct (GAL4)5-E1b-LUC (2.5 µg). The additional
increase of activity provided by SRC1 is presented relative to the
activity of the GAL4-DBD and is expressed as fold coactivation when
equal amounts of DNA from the control vectors were transfected. These
results represent the mean ± S.E. of at least three
experiments.
encompassing amino acids 361-458 is required for
the AF activity of this protein (43). The interaction of SRC1 with
GAL4-HNF3
361-458 results in a 12-fold coactivation,
whereas GAL4-HNF3
361-442, which cannot act as an AF
(43), is ineffective (Fig. 4). Thus, the region of HNF3
required for
AF activity in the GRU may act by recruiting SRC1, a finding consonant
with the ability of GAL4-SRC1 to replace the function of HNF3 on the
gAF2
GAL4 PEPCK-LUC reporter construct.
GAL4 PEPCK and (GAL4)5-E1b
promoters, respectively, is useful because variants of GAL4-SRC1 can be
specifically targeted to these promoters, effectively removing
endogenous SRC1 from the analytical system. This system can be used to
identify the minimal functional domains of SRC1 required for the PEPCK
glucocorticoid response.
GAL4 PEPCK-LUC construct (Fig. 1). Expression
of a variant of GAL4-SRC1 that lacks the N-terminal 45 amino acids (GAL4-SRC1-
bHLH) does not replace HNF4 as an AF (Fig.
5a). This GAL4-SRC1-
bHLH
protein provides no more AF activity than does the GAL4-DBD alone or a
deletion of the gAF1 element (Fig. 5a and Ref. 28). Thus,
the ability of GAL4-SRC1 to serve as a surrogate AF through the gAF1
element correlates with its ability to coactivate HNF4, the AF that
binds this element in the wild-type promoter (30, 43).
View larger version (24K):
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Fig. 5.
The bHLH and HAT domains of SRC1 are required
at gAF1 for the glucocorticoid response. Panel A,
experiments were performed as described in Fig. 1, except that equal
amounts of GAL4-DBD, GAL4-SRC1, or the structural variants indicated
were used. The results are represented relative to the fold induction
of wild-type PEPCK-LUC by glucocorticoids, which is taken as 100%.
These results represent the mean ± S.E. of at least three experiments.
The asterisks represent statistically significant
differences (p < 0.05) from GAL4- SRC1.
Dex, dexamethasone. Panel B, the expression and
DNA binding of various GAL4-SRC1 fusion proteins was determined in COS
cells transfected with 20 µg of expression plasmids that encode
various GAL4-SRC1 fusion constructs. Nuclear extracts were prepared,
and the gel mobility shift assay was used to analyze expression levels
(as described under "Experimental Procedures"). The
arrows indicate the various GAL4-SRC1 fusion proteins. Each
protein complex was eliminated by preincubation of the extracts with a
polyclonal antibody directed against the GAL4-DBD but not an unrelated
antibody raised against Lex A.
HAT), does not restore the
glucocorticoid responsiveness of the reporter gene (Fig.
5a). A chimeric construct in which the five C-terminal amino
acids encompassing NRBIV were deleted (GAL4-SRC1
IV) provides a
glucocorticoid response comparable with that of GAL4-SRC1 (Fig.
5a). This region is important for the interaction of SRC1
with GR, although NRBI-III also contributes to this physical
interaction (52).
bHLH migrates slightly more slowly. All of these proteins
migrate as expected, as determined by Western blot analysis, when the
gel is run under denaturing conditions (data not shown).
HAT does not mediate AF activity
(Fig. 6). As at the gAF1 element, the
NRBIV region of GAL4-SRC1 is also dispensable at gAF2 (Fig. 6).
However, by contrast to the functional requirements of GAL4-SRC1 at
gAF1, where the bHLH/PAS domain is critical to the surrogate-AF activity provided by this protein, the bHLH/PAS region is completely dispensable at gAF2 (Fig. 6). The distinct requirements of various regions of GAL4-SRC1 at gAF1, as compared with gAF2, supports the
contention that the gAF1 and gAF2 elements have distinct roles in the
glucocorticoid response (36). This result also suggests that GAL4-SRC1
replaces some essential, but distinct function of both HNF4/COUP-TF and
HNF3
at gAF1 and gAF2, respectively.
View larger version (21K):
[in a new window]
Fig. 6.
The HAT domain of SRC1 is required for the
glucocorticoid response at gAF2, but the bHLH domain is not.
Experiments were performed as described in Fig. 3, except that equal
amounts of GAL4-DBD (+G4-DBD), GAL4-SRC1
(+G4-SRC1), or the structural variants indicated were used.
This result demonstrates element-specific requirements of SRC1 in the
PEPCK gene GRU, and it further confirms that GAL4-SRC- bHLH is not
transcriptionally inactive. Results are represented relative to the
fold induction of wild-type PEPCK-LUC by glucocorticoids, which is
taken as 100%. These results represent the mean ± S.E. of at
least three experiments. The asterisk represents a
statistically significant difference (p < 0.05) from
GAL4-SRC1.
bHLH activates this reporter only 4-fold (Fig. 7). This region appears to be
a general activation domain of SRC1, since it is important both for AF
activity at PEPCK gAF1 and in the context of a simple promoter (Figs.
5a and 7 and Ref. 53).
View larger version (15K):
[in a new window]
Fig. 7.
Basal transcriptional activity of structural
variants of GAL4-SRC1. HeLa cells were transfected with 2.5 µg
of (GAL4)5-E1b-LUC along with 5 µg of the indicated
GAL4-SRC1 construct or the GAL4-DBD. This result identifies
promoter-specific requirements of SRC1 acetyltransferase activity in
PEPCK gene transcription, and it further demonstrates that
GAL4-SRC1 HAT is not transcriptionally inactive. Results are
presented relative to the activity of the GAL4-DBD and represent the
average ± S.E. of at least three experiments. The gAF1 activity
of each GAL4-SRC fusion protein is also shown. wt, wild
type.
HAT does not act as
an AF at either gAF1 or gAF2, this protein increases expression through
the simple promoter about 100× more effectively than does the DBD
(Fig. 7). The heightened activation produced by deletion of the HAT
domain may reflect different protein targets of this enzyme activity in
the two promoters. Deletion of NRBIV has no effect on basal
transcription mediated by GAL4-SRC1 (Fig. 7).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
are required for
maximal induction of PEPCK gene transcription by glucocorticoids (13,
28-32), and each interacts with the coactivator SRC1 through exactly
the same regions required for their AF function (this paper and Refs. 1
and 2). This series of observations strongly suggests that recruitment
of SRC1 is important for the glucocorticoid response. Recruitment of
SRC1, however, does not exclude recruitment of other coactivators such
as CBP to, or by, the same AFs. We envision that, together, the AFs
provide a surface for recruitment of a coactivator complex that
includes SRC1, CBP, and probably other proteins. The PEPCK gene GRU
shares DNA elements with the hormone response units that provide a
response to retinoic acid, cAMP, and insulin (38), but different
transcription factors are recruited to the AF elements in response to
these hormones (37, 54, 55). It is probable that the constellation of
coregulators recruited by each of these effectors is also different,
yet somehow this array of hormone response units provides a coordinated
response to the metabolic challenges imposed by hypoglycemia, when the PEPCK gene is activated, and hyperglycemia, when the gene is repressed.
to
recruit coregulatory molecules rather than ubiquitously expressed
factors provides insight into how tissue specificity of gene
transcription in response to glucocorticoids may arise. In this regard,
it is noteworthy that in adipose tissue, which does not express HNF4 or
HNF3
but does express GR, glucocorticoids repress basal and
cAMP-induced PEPCK gene transcription (25, 26). Thus, tissue-specific
expression of the AFs may provide directionality to gene transcription
in response to glucocorticoids, as well as contributing to the
magnitude of the response.
. The activation of coactivators by nuclear receptors is not
unprecedented. The function of the peroxisome proliferator-activated
receptor-
coactivator, PGC1, is stimulated by its association with
peroxisome proliferator-activated receptor-
(57). This
protein-protein interaction promotes a conformational change in PGC1
that permits recruitment of additional coactivators SRC1 and CBP/p300
(57). We propose that the binding of GR to the PEPCK gene GRU promotes
activation of SRC1 and assembly of a functional enhanceosome in a
similar fashion. The interaction between GR and SRC1 appears to be
facilitated by localization of GR to its DNA binding elements. When
both the GR1 and GR2 elements are intact, GAL4-SRC1 is able to serve as
a surrogate AF at gAF1. As the ability of GR to be recruited to the
promoter is reduced or lost by introducing mutations in the GR2 or GR1
and GR2 elements, respectively, the ability of GAL4-SRC1 to serve as a
surrogate AF is reduced or lost in a coordinate manner (Fig. 2).
IV) should reduce the effectiveness of GAL4-SRC1 as a
surrogate AF. Deletion of this region is without effect, however, as
GAL4-SRC1
IV functions comparably with the full-length chimeric protein (Figs. 5a and 6). Kalkhoven et al. show
that, although NRBIV is the nuclear receptor interaction motif that
interacts most strongly with estrogen receptor in vitro,
deletion of this region has little functional consequence in estrogen
receptor activation, providing the NRBI-III region is intact (60). This suggests that multiple LXXLL motifs facilitate the in
vivo effects of SRC1 on nuclear receptors. Rather than stabilizing
GR binding, the SRC1-GR interaction may be more important for the
activation of SRC1. By this model, AFs such as HNF4, COUP-TF1, and
HNF3
would serve to recruit SRC1, which in turn is only activated by the additional presence of ligand bound GR.
GAL4 PEPCK and
(GAL4)5-E1b promoters, respectively) allowed us to look for promoter- and DNA element-specific requirements of SRC coactivation. The bHLH region of GAL4-SRC1 is required for AF activity at the gAF1
element (Fig. 5a). GAL4-SRC1-
bHLH also provides a low
rate of basal transcriptional activation through the simple E1b
promoter (Fig. 7). The bHLH/PAS domain of SRC1 is highly conserved
among the p160 family of coactivators, and the PAS motif serves
as a protein-protein interaction surface for the transcription factors, Per, ARNT, and SIM (61, 62). The importance of the bHLH/PAS domain of
SRC1 in the context of the PEPCK gene GRU may indicate the requirement
for interaction with an additional PAS-containing factor that is
necessary for SRC1 function. Interestingly, this region is not required
when GAL4-SRC1 is recruited to gAF2 (Fig. 6). This result supports the
idea that these gAF elements and associated factors have distinct roles
in the response of the PEPCK gene to glucocorticoids.
HAT strongly activates a simple promoter
but is not functional at any of the PEPCK gAF elements tested (Figs.
5a, 6 and 7).
mediate distinct functions as AFs, yet they
are both capable of interacting with the same coactivator, SRC1. SRC1
may serve both as a scaffolding molecule and an activator through its
acetyltransferase activity. Which of these functions is required seems
to be determined by the local promoter environment to which SRC1 is
recruited. Furthermore, the function of SRC1 as a coactivator is not
simply determined by the site of its recruitment to the GRU but also depends upon activation by GR. This role of recruitment and activation of coactivators may have implications for other genes that employ AFs
to mediate responses to metabolic signals. Indeed, the AFs required for
the glucocorticoid response of the PEPCK gene are involved in
regulating the hormone responses of many genes that encode enzymes
required for metabolic homeostasis (10-14, 68, 69). Thus, the
functional interactions between HNF3
and HNF4 with SRC1, as
described in the PEPCK gene GRU, may have a broad role in metabolic
adaptation.
View larger version (15K):
[in a new window]
Fig. 8.
A comparison of accessory factor activity and
basal transactivation as mediated by GAL4-SRC structural
variants.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Peter Kushner (University of California, San Francisco) for providing the expression vector for GAL4-SRC1, Dr. Jen-Chywan Wang for helpful advice, Cathy Caldwell for technical assistance, and Deborah Caplenor Brown for helping prepare this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK20593 (Vanderbilt Diabetes Research and Training Center), DK35107, and GM07347 (Vanderbilt Medical Scientist Training Program) and the Veterans Administration Research Service.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Physiology and Biophysics, 707 Light Hall, Vanderbilt University School
of Medicine, Nashville, TN 37232. Tel.: 615-322-7004; Fax: 615-322-7236; E-mail: daryl.granner@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M009389200
2 J. M. Stafford, M. Waltner-Law, and D. K. Granner, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HRE, hormone response element; AF, accessory factor; PEPCK, phosphoenolpyruvate carboxykinase; GRU, glucocorticoid response unit; CBP, cAMP-response element-binding protein (CREB)-binding protein; SRC1, steroid receptor coactivator 1; DBD, DNA binding domain; GR, glucocorticoid receptor; HNF, hepatocyte nuclear factor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; bHLH, basic-helix-loop-helix; HAT, histone acetyltransferase; SREBP, sterol-response element binding protein.
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