From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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ABSTRACT |
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The cyclic AMP response element (CRE) of the rat
phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for
a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The
first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed p Phosphoenolpyruvate carboxykinase
(PEPCK)1 (EC 4.1.1.32),
a rate-controlling gluconeogenic enzyme, is expressed in a
tissue-specific manner and is regulated at the transcriptional level by
various hormones and nutrients (1, 2). Rat liver PEPCK gene
transcription is stimulated by glucocorticoids, glucagon (via cyclic
AMP), and retinoic acid and is inhibited by insulin (1-5).
Glucocorticoid induction of the PEPCK gene is mediated through a
complex glucocorticoid response unit (GRU). This unit consists of (from
5' to 3') two accessory factor elements (AF1 and AF2), two
glucocorticoid receptor-binding sites (GR1 and GR2), a third accessory
element (AF3), and the cyclic AMP response element (CRE) (see Fig. 1).
The glucocorticoid response decreases by 50-60% when any one of the
accessory elements is mutated, and it is abolished when any combination
of two are disrupted (6, 7). We have identified the proteins that bind
to the three accessory elements. Hepatocyte nuclear factor 4 (HNF4) or
chicken ovalbumin upstream promoter-transcription factor (COUP-TF)
confers accessory factor activity by binding to the AF1 element, and
COUP-TF binds to and confers activity from the AF3 element (7-9).
Members of the CCAAT/enhancer-binding protein (C/EBP) and hepatocyte
nuclear factor 3 (HNF3) families bind to AF2, but only the latter
provide accessory factor activity for the glucocorticoid response from
this element (10, 11).
An intact CRE is also required for a complete glucocorticoid response,
because deletion of this element results in a 50% decrease of the
glucocorticoid response (12). The CRE is therefore a multifunctional
element, as it is also necessary for basal transcription and the cAMP
response (4, 13). Multiple members of the leucine zipper transcription
factor family bind to the CRE in vitro, including cyclic AMP
response element-binding protein (CREB), C/EBP family members, D
site-binding protein, and activator protein 1 (14-18). We, and others,
have shown previously that CREB and C/EBP family members are involved
in the cAMP response of the PEPCK gene (4, 13, 18-20). However, the
roles played by these CRE-binding factors in the glucocorticoid
response were unclear. We have previously reported that GR physically
interacts with CREB in vitro and that both C/EBP family
members and CREB bind to the PEPCK gene CRE (12, 21, 22).
The aim of the present study was to determine which protein(s) provides
accessory factor activity for the glucocorticoid response through the
CRE. A very good glucocorticoid response was retained when the CRE was
replaced with a consensus C/EBP-binding site, suggesting that C/EBP
family members contribute accessory activity. A PEPCK
promoter/chloramphenicol acetyltransferase (CAT) reporter gene
construct, in which the CRE was replaced by a GAL4-binding site, was
co-transfected with GAL4 DNA-binding domain (DBD)-fusion protein
expression vectors to determine which protein(s) potentiates the
glucocorticoid response from the CRE. We found that C/EBP Electrophoretic Mobility Shift Assay (EMSA)--
The conditions
of the EMSA have been described previously (22). The sequences of the
oligonucleotides used in the EMSA are listed in Table I. The TNT T7
Quick Coupled Transcription/Translation System (Promega Corp., Madison,
WI) was used for the in vitro translation of C/EBP Plasmids--
Construction of the plasmid pPL32, which contains
the PEPCK promoter region from
pRSV-GR, a glucocorticoid receptor expression vector, was obtained from
Dr. Keith R. Yamamoto (University of California, San Francisco, CA).
pBR322, a standard cloning vector, was obtained from Promega. The
plasmid pSG424 expresses the yeast transcription factor GAL4 DBD driven
by the SV40 promoter (27).
The plasmid pcDNA1-GAL4-CREB
The GAL4·C/EBP
The DNA sequence of each construct was verified by dideoxy sequencing.
All of the plasmids used in this study were purified by the
CsCl/ethidium bromide density gradient ultracentrifugation method.
Cell Culture, DNA Transfections, and CAT Assays--
H4IIE
cells, a rat hepatoma cell line, were cultured as described previously
(21). HeLa cells, a human cervical carcinoma cell line, were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% calf
serum. For the analysis of the glucocorticoid response in H4IIE cells,
10 µg of reporter plasmid and 5 µg of pRSV-GR, with or without
GAL4-fusion protein expression vectors, were co-transfected using the
calcium-phosphate method, as described previously (6). Four hours after
transfection, the cells were subjected to a 20% Me2SO
shock for 4 min and then maintained in serum-free DMEM for an
additional 16 h. This additional incubation was not performed for
the transfections involving PKI and PKImut. The medium was
then changed to serum-free DMEM, with or without 0.5 µM
dexamethasone, and the cells were cultured for an additional 24 h.
A titration using 0.5, 1, or 2.5 µg of the expression vector was
performed when the cells were co-transfected with the GAL4-fusion protein expression vectors. The greatest dexamethasone response was
employed to calculate the fold induction. The measurement of CAT
activity was carried out as described previously (32).
The GAL4-fusion protein expression vectors were transfected into HeLa
cells to determine the relative expression of the GAL4-fusion proteins.
The medium was changed 16 h after transfection, and after an
additional 32 h, nuclear extracts were prepared according to the
method of Schreiber et al. (33). EMSAs or Western blots were
used to measure the relative expression of these proteins (28, 30).
Analysis of CRE Region-substituted Constructs--
CREB and C/EBP
family members are the only proteins from rat liver nuclear extracts
that bind specifically to the PEPCK CRE (22), making them obvious
candidates for providing accessory factor activity to the
glucocorticoid response. Transient transfections of H4IIE cells were
performed using wild type (pPL32) and mutated PEPCK promoter/CAT
reporter plasmids (Fig. 1). In these
experiments, pPL32 conferred about a 10-fold glucocorticoid response,
which is taken as the maximal, or 100%, response (Fig. 1, top
row, right). In contrast, when the PEPCK CRE was replaced with a
GAL4-binding site (p
EMSAs were performed to confirm that the wild type PEPCK CRE and the
two mutant sequences described above bind the appropriate transcription
factors (Fig. 2). As expected, no
protein-DNA complex was formed when the wild type CRE (Table
I, CREwt) oligonucleotide was used as the
32P-labeled probe and incubated with an unprogrammed
reticulocyte lysate (Fig. 2, lane 1). However, a protein-DNA
complex was formed when this probe was mixed with a reticulocyte lysate
programmed to express CREB (Fig. 2A, lane 2). The addition
of a specific antiserum directed against CREB abolished the complex,
whereas a nonspecific antiserum directed against Tyk2 did not affect
the formation of the complex (Fig. 2A, lanes 3 and
4). In competition experiments, the formation of this
complex was prevented by the addition of a 100-fold molar excess of an
unlabeled CREwt oligonucleotide but not by equivalent amounts of
unlabeled oligonucleotides that contained binding sites for C/EBP,
GAL4, or USF (Table I; Fig. 2A, lanes 5-8). The formation
of the complex was not prevented even by the addition of a 500-fold
molar excess of the unlabeled C/EBP oligonucleotide (data not shown);
furthermore, no shifted complex was observed when the in
vitro translated CREB protein was incubated with a labeled C/EBP
oligonucleotide (Table I; data not shown). Together, these data
reaffirm that although CREB binds specifically to the PEPCK CRE, it
binds to neither the consensus C/EBP site nor the GAL4 DNA-binding
site. Therefore, in the context of p
Shifted protein-DNA complexes were formed when the labeled CREwt
oligonucleotide was incubated in binding reactions that contained either in vitro translated C/EBP C/EBP
When H4IIE cells were co-transfected with p
When GAL4·C/EBP
The possible role of C/EBP
As shown in Fig. 3A, the activation domain of C/EBP
To test whether the amino terminus of C/EBP
When GAL4·CREB 283, which lacks the bZIP domain of CREB, was
co-transfected with p PKI Inhibits the Glucocorticoid Response of PEPCK--
To test
whether endogenous cAMP-dependent protein kinase (PKA)
activity is necessary for glucocorticoid stimulation of PEPCK gene
transcription, H4IIE cells were co-transfected with the pPL32 reporter
plasmid, pRSV-GR, and increasing amounts of either RSV-PKI or
RSV-PKImut. We utilized a heat-stable inhibitor of PKA
(PKI), as well as an inactive mutant of the PKA inhibitor
(PKImut). As depicted in Fig.
4, there is a dose-dependent
decrease in dexamethasone-induced PEPCK transcription with increasing
amounts of PKI. Interestingly, the dexamethasone response is decreased to a little less than 50% of its original value, reminiscent of what
happens when the CRE is mutated in the context of the wild type PEPCK
promoter. In contrast to PKI, when the inactive mutant of the
heat-stable inhibitor (RSV-PKImut) is co-transfected, there
is no statistically significant difference observed between
glucocorticoid induction in the presence or absence of the mutant.
These results hold true at higher concentrations of PKI and
PKImut (data not shown).
Simple hormone response elements are DNA sites that bind
transcription factors that, directly or indirectly, alter the rate of
transcription of a specific gene. As classically defined, hormone response elements mediate hormone responses when placed in the context
of heterologous promoters, and they function in a manner that is
independent of orientation or position. In recent years, it has become
apparent that, in natural promoter contexts, this description of
hormone response elements is an oversimplification. Indeed, multiple
arrays of transcription factors in a very specific, ordered arrangement
are required for the proper regulation of genes. The operation of the
PEPCK gene promoter in mediating a glucocorticoid response provides a
case in point. Four accessory factor elements, including the CRE, and
two GR-binding sites are required for a complete glucocorticoid
response, and the proper order and placement of these elements is
necessary for this effect (35). Schüle et al. (36)
explored the role accessory factors play in the glucocorticoid response
mediated by a simple GRE in a minimal promoter system, and they found
that virtually any transcription factor that binds near the GR-binding
site augments the glucocorticoid response. By contrast, in the context
of the PEPCK promoter, specific transcription factors mediate accessory
activity only from particular accessory elements. For example, the AF2
element of the PEPCK gene promoter binds HNF3 and members of the C/EBP
family. When the AF2 element is changed to the sequence of the AF1
element, which binds HNF4 or COUP-TF, or to a consensus Sp1-binding
site, there is a reduction in the glucocorticoid response equivalent to
that observed when the AF2 element is functionally eliminated (11).
These transcription factors, at least, will not replace HNF3.
Furthermore, the insertion of a consensus HNF3-binding site in place of
the AF2 element provides a complete glucocorticoid response, whereas
mutations that prevent the binding of HNF3, but allow C/EBP family
members to bind, abolish AF2 accessory factor activity. Hence, HNF3,
rather than C/EBP family members, mediates accessory activity from the
AF2 element (11). In addition, accessory activity from AF1 and AF3 is
mediated by specific sets of transcription factors. For example, HNF3
cannot mediate AF1 or AF3 activity, but HNF4 and/or COUP-TF serve this
function (35). In the present study, we demonstrate that C/EBP C/EBP A complete restoration of the glucocorticoid response was not observed
with the experimental strategies employed in these studies. For
example, the glucocorticoid response is only 71% of wild type when the
CRE is replaced with a consensus C/EBP-binding site. However, because
the expression of C/EBP The presence of a cell line in which the glucocorticoid response can be
restored by the provision of one or more missing accessory factors
would be very helpful. Unfortunately, there is no such cell line.
Although not ideal, the GAL4 system affords us the power not only to
approximate the functions of various factors but also to manipulate
these factors in such a way as to specifically dissect their different
activities and assign these activities to different modules within the
protein. Similar to that seen with the first approach, the
GAL4·C/EBP Yet another possibility is that, in vivo, there may be
another factor that acts as a better accessory factor for the
glucocorticoid response through the CRE than does C/EBP Our results with PKI correlate well with work done by Angrand et
al. (49) to demonstrate how important endogenous PKA activity is
to the glucocorticoid response of the PEPCK gene. Moreover, they
suggest that the PEPCK GRU and cAMP response unit are intertwined not
only at the level of DNA elements but also at higher signaling levels.
Such a cooperative integration of signaling and hormone response
elements between hormone response units (HRUs) may provide the complex
mechanism through which synergism occurs when two hormones act
simultaneously to regulate the transcription of a single gene. Future
studies will be directed toward determining whether or not C/EBP The identification of C/EBPCREC/EBP, binds C/EBP
and
but not CREB, yet it
confers a nearly complete glucocorticoid response when transiently
transfected into H4IIE rat hepatoma cells. These results suggest that
one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in
which the CRE was replaced with a GAL4-binding sequence
(p
CREGAL4), and various GAL4 DNA-binding domain (DBD)
fusion protein expression vectors. Although chimeric proteins
consisting of the GAL4 DBD fused to either CREB or C/EBP
are able to
confer an increase in basal transcription, they do not facilitate the
glucocorticoid response. In contrast, a fusion protein consisting of
the GAL4 DBD and amino acids 1-118 of C/EBP
provides a significant
glucocorticoid response. Additional GAL4 fusion studies were done to
map the minimal domain of C/EBP
needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid
regions 1-84, 52-118, or 85-118 of C/EBP
fused to the GAL4 DBD do
not mediate a glucocorticoid response. We conclude that the amino
terminus of C/EBP
contains a multicomponent domain necessary to
confer accessory factor activity to the glucocorticoid response from
the CRE of the PEPCK gene promoter.
INTRODUCTION
Top
Abstract
Introduction
References
mediates
accessory factor activity through the CRE, whereas C/EBP
and CREB do
not. Furthermore, the amino-terminal domain of C/EBP
is required for
accessory factor activity.
MATERIALS AND METHODS
,
C/EBP
, and CREB using the plasmids pGEM-C/EBP
, pGEM-C/EBP
, and
pET-CREB, respectively (22). The pET-CREB plasmid was constructed by
inserting the rat CREB coding sequences into the
EcoRI/NdeI sites of the pET-3a vector (23).
Nonspecific, single-stranded DNA-binding proteins were competed away
with unlabeled, single-stranded CRE wild type (CREwt) antisense
oligonucleotide, added at a 10-fold molar excess of the labeled probe.
For supershift analyses, anti-CREB1, anti-C/EBP
, anti-C/EBP
, and
anti-Tyk2 antisera from Santa Cruz Biotechnology, Inc. were used.
467 to +69 bp relative to the
transcription start site, has been described previously (21). pPL32 was
used as a template in polymerase chain reactions (PCRs) to create
mutations in the CRE. The nucleotide sequences of all the PCR primers
used in this study are listed in Table I. A downstream primer (CAT, Table I) and the CRE mutant primers, C/EBP or GAL4, were used in PCRs
to generate two megaprimers (24-26). The megaprimers were gel purified
and used with an upstream primer (Table I, PLF) in a second PCR to
generate a fragment that, after digestion withHindIII and
BglII, was ligated into the reporter plasmid pPLF to
generate the plasmids p
CREC/EBP and
p
CREGAL4, respectively.
bZIP(4-283), which encodes a
GAL4·CREB fusion protein that lacks the basic region and leucine zipper (bZIP) domain of CREB, was a gift from Dr. Richard A. Maurer (28). An EcoRI/XbaI fragment from this vector,
which encodes the CREB
bZIP fusion protein, was subcloned into the
EcoRI/XbaI sites of the pSG424 plasmid to produce
the mammalian expression vector pGAL4·CREB 283. Dr. Maurer also
provided RSV-PKI and RSV-PKImut vectors that encode the
heat-stable inhibitor of the cyclic AMP-dependent protein
kinase and a mutant of the inhibitor, respectively (29).
358, 317, 108 and GAL4·C/EBP
276, 118, 84, GAL4 DBD-C/EBP
and GAL4 DBD-C/EBP
fusion protein expression vectors were gifts from Dr. Peter F. Johnson (30). To make
GAL4·C/EBP
52-118 and GAL4·C/EBP
85-118, the DNA fragments
that encode amino acids 52-118 and 85-118 in the C/EBP
gene were
amplified by the reverse transcription-PCR method. One of two upstream
primers,
52 or
85, and a downstream primer,
118, were used in
these reactions (Table I). Total RNA was isolated from H4IIE cells using the acid guanidine-phenol-chloroform method (31). Reverse transcription was performed at 37° C for 1 h in a 50-µl
reaction mixture that contained 50 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM dithiothreitol, 35 units
of RNase inhibitor, 1 mM dNTPs, 50 units of AMV reverse
transcriptase (Promega) in the presence of 1.5 µg of total RNA and 25 pmol of the
118 oligonucleotide. PCR was carried out using the
118 oligonucleotide and either the
52 or the
85
oligonucleotide as primers (Table I). Briefly, a 100-µl reaction
mixture contained 10 mM Tris-HCl, pH 8.3, 50 mM
KCl, 1.5 mM MgCl2, 0.001% gelatin, 2 mM dNTPs, 10 pmol of each primer, 10 µl of the reverse
transcription reaction as a template, and 5 units of AmpliTaq DNA
polymerase (Perkin-Elmer). The PCR conditions used were as follows:
denaturation for 3 min at 98° C; 5 cycles of 1 min at 98° C,
30 s at 55° C, and 30 s at 72° C; and another 25 cycles
of 1 min at 98° C, 30 s at 58° C, and 30 s at 72° C.
The amplified DNA fragments were digested with EcoRI and
XbaI, purified by agarose gel electrophoresis, and then
subcloned into the EcoRI/XbaI sites of the
plasmid pGAL4·C/EBP
118.
RESULTS
CREGAL4) (Fig. 1, middle
row), the basal transcriptional activity and the glucocorticoid
response decreased to 61 and 45% of the wild type activity,
respectively. Both responses are equivalent to those observed when
either an internal deletion or a block mutation of the CRE is made
(Ref. 12 and data not shown). However, when the CRE was replaced with a
consensus C/EBP-binding site (p
CREC/EBP) (Fig. 1,
bottom row), basal transcription increased 2-fold, and the
absolute dexamethasone response was substantially greater than that
conferred by the wild type promoter (cf. Fig. 1, top
row). When corrected for the increase in basal expression, however, the dexamethasone response from p
CREC/EBP was
71% that of wild type (Fig. 1, compare bottom row with
top two rows). These results suggest that a member of the
C/EBP family confers accessory factor activity to the glucocorticoid
response.
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Fig. 1.
Comparison of basal and
glucocorticoid-induced PEPCK transcription between wild type and mutant
plasmids. Schematic diagrams of the reporter constructs are
depicted on the left. pPL32 is the wild type PEPCK-CAT
reporter plasmid. In this diagram of pPL32, boxes denote
those elements required for basal transcription, circles
denote the glucocorticoid receptor binding sites, and ovals
denote the accessory elements essential for the glucocorticoid
response. Note that the CRE is involved in both processes.
p CREGAL4 is a mutant of pPL32 in which the CRE is
replaced with a GAL4-binding site. p
CREC/EBP is a mutant
of pPL32 in which the CRE is replaced with a C/EBP-consensus binding
sequence. Both basal and glucocorticoid (dexamethasone
(Dex))-induced CAT activities are shown on the
right. CAT activity from the wild type plasmid (pPL32) was
set at 100%. Glucocorticoid induction is defined as the quotient
between the dexamethasone response and the basal activity. The level of
glucocorticoid induction for the wild type plasmid (pPL32) was set at
100%. All values shown represent the mean ± S.E. of at least six
separate experiments.
CREC/EBP, CREB does
not provide accessory factor activity to the glucocorticoid
response (cf. Fig. 1, bottom row).
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Fig. 2.
Electrophoretic mobility shift assay analysis
of binding to the CRE. In three separate experiments, the
32P-labeled CREwt probe was mixed with in vitro
translated CREB, C/EBP , or C/EBP
(designated lysates are shown on
the left). Lane 1, unprogrammed lysates did not
form shifted complexes. Lane 2, programmed lysates formed
protein-DNA complexes with the radiolabeled probe. Lanes 3 and 4, specific (sp) antisera (anti-CREB,
anti-C/EBP
, or anti-C/EBP
) supershifted protein-DNA complexes,
whereas nonspecific (ns) antiserum (anti-Tyk2) did not.
Lanes 5-8, competition analyses were performed with a
100-fold molar excess of unlabeled oligonucleotides (CREwt, USF E-box,
C/EBP, or GAL4) added to the binding reactions as competitor DNA.
Oligonucleotides used in this study
or C/EBP
(Fig. 2,
B and C, respectively, lane 2). These
complexes were supershifted by specific antisera directed against
C/EBP
or C/EBP
, respectively (Fig. 2, B and C,
lane 3). By contrast, nonspecific antiserum directed against Tyk2
did not affect the formation of the C/EBP-DNA complexes (Fig. 2,
B and C, lane 4). Additionally, the C/EBP
antiserum did not cross-react with in vitro translated
C/EBP
, nor did the C/EBP
antiserum cross-react with C/EBP
(data not shown). In competition experiments, the formation of these
complexes was prevented by the addition of a 100-fold molar excess of
the unlabeled CREwt and C/EBP oligonucleotides, but not an equimolar
amount of either the GAL4 or USF E-box oligonucleotides (Table I; Fig. 2, B and C, lanes 5-8). Together, these results
indicate that the CREwt oligonucleotide is bound by CREB, C/EBP
, and
C/EBP
; that the C/EBP oligonucleotide is bound by C/EBP
and
C/EBP
but not CREB; and that the GAL4 oligonucleotide does not bind
any of these proteins in vitro.
Is an Accessory Factor for the Glucocorticoid
Response--
H4IIE cells were co-transfected with the
p
CREGAL4 reporter plasmid and various GAL4 DBD fusion
protein expression vectors to determine whether C/EBP
, C/EBP
, or
CREB (Fig. 3A) can confer accessory factor activity to the glucocorticoid
response. The
and
isoforms of the C/EBP family were tested
because both proteins are highly expressed in the liver and bind to the
consensus C/EBP-binding site that confers partial accessory activity
from the CRE (cf. Fig. 1, bottom row). The
cellular expression of the GAL4 fusion proteins was found to be
essentially equivalent, as determined by Western blotting or EMSA
(Refs. 28 and 30; data not shown).
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Fig. 3.
Identification of the transcription factor
mediating the glucocorticoid response from the PEPCK CRE.
A, linear representations of the transcription factors that
are able to bind the PEPCK CRE (i.e. C/EBP , C/EBP
, and
CREB) are depicted above. C/EBP
contains two activation domains
(AD1 and AD2) and a carboxyl-terminal bZIP
domain. C/EBP
contains an amino-terminal activation domain
(AD) composed of three subdomains, two repressor domains
(RD1 and RD2), and a carboxyl-terminal bZIP
domain. CREB contains three activation domains (Q1, P-box,
and Q2) and a carboxyl-terminal bZIP domain. B,
the reporter constructs used for the GAL4-fusion experiments are
p
CREGAL4 and (GAL4)5E1bCAT.
p
CREGAL4 is a mutant of pPL32 in which the CRE was
replaced with a GAL4-binding sequence. (GAL4)5E1bCAT is
a heterologous CAT reporter plasmid driven by an E1b promoter into
which five tandem GAL4-binding sites have been inserted. C,
p
CREGAL4 was co-transfected with various GAL4-fusion
protein expression vectors into rat hepatoma (H4IIE) cells to determine
basal and glucocorticoid-induced PEPCK transcription. The GAL4-fusion
constructs are depicted on the left. The wild type PEPCK-CAT
plasmid (pPL32) was used as an internal control for all experiments,
and its CAT activity was set at 100%. The left data column
shows the basal CAT activity mediated by each construct relative to
pPL32. The middle data column shows the
glucocorticoid-induced response of each construct relative to pPL32. To
ascertain basal transcriptional activity mediated by the GAL4-fusion
proteins in a heterologous context, 10 µg of the
(GAL4)5E1bCAT reporter construct and 0.2 µg of the
GAL4-fusion protein expression vectors were co-transfected into H4IIE
cells. CAT activity from the H4IIE cells co-transfected with the
(GAL4)5E1bCAT reporter construct and the GAL4 DBD was set
at 1. All values shown represent the mean ± S.E. of three
or more experiments.
CREGAL4 (Fig.
3B) and the GAL4 DBD expression vector, the basal and
glucocorticoid responses were 78 and 42% of those conferred by the
wild type pPL32 construct, respectively (as shown in Fig. 3C, row
1). These responses are similar to those obtained when the
reporter plasmid alone was transfected (data not shown). It is
important to reiterate the fact that a glucocorticoid response of 42%
is equivalent to that obtained when mutations are made that abolish
accessory factor activity from the CRE (Ref. 12; data not shown).
358, a vector that expresses the full-length
C/EBP
·GAL4-DBD fusion protein, was co-transfected with
p
CREGAL4, basal transcription was restored to that of
wild type, but the glucocorticoid response was unaffected at 50% of
the control (Fig. 3C, row 2). These results were expected
because the full-length C/EBP
·GAL4 DBD fusion protein contains two
bZIP domains that are known to interfere with transactivation (30). In
fact, when this construct was co-transfected with the
(GAL4)5E1bCAT heterologous reporter (34), a construct that
contains a TATA box and five tandem GAL4-binding sites (Fig.
3B), transcription activity was almost the same as that
provided by the GAL4 DBD alone (Fig. 3C, rows 1 and 2, right column). Therefore, we next tested GAL4·C/EBP
317, which lacks the leucine zipper domain, and GAL4·C/EBP
108, which
lacks both the basic region and the leucine zipper domain. When these
constructs were co-transfected into H4IIE cells with the
p
CREGAL4 reporter, basal transcription increased, but
the glucocorticoid response remained at 39 and 45% of wild type,
respectively. We also tested the ability of these constructs to confer
transcriptional activity to (GAL4)5E1bCAT. In this
heterologous system, the constructs that expressed the truncated forms
of C/EBP
provided significantly greater transcriptional activity
than did the full-length, bZIP-containing construct (Fig. 3C,
rows 3 and 4). In fact, as the fusion proteins were
truncated to leave only the amino-terminal activation domain, a greater
level of basal transcription was observed for both the p
CREGAL4 reporter construct and the
(GAL4)5E1bCAT heterologous promoter (Fig. 3C, row
4). This is in agreement with previous observations (30). Thus,
although these C/EBP
constructs are expressed well in H4IIE cells
and are capable of mediating transactivation, C/EBP
does not provide
accessory factor activity for the glucocorticoid response.
as an accessory factor was examined using
the same system. We used GAL4·C/EBP
276, a plasmid that encodes
the full-length C/EBP
, and GAL4·C/EBP
118, a plasmid that lacks
the bZIP region and the two repressor domains, RD1 and RD2, of C/EBP
(see Fig. 3A and Ref. 30). When the full-length bZIP-containing GAL4·C/EBP
276 construct was co-transfected with p
CREGAL4, basal transcription and the glucocorticoid
response were almost the same as those seen with the GAL4 DBD alone
(Fig. 3C, compare rows 1 and 5).
Additionally, the GAL4·C/EBP
276 construct did not confer
transcriptional activity in the (GAL4)5E1bCAT heterologous system, an observation that is consistent with previous results (30).
As noted before with C/EBP
, this result was anticipated because this
fusion protein retains the bZIP domain of C/EBP
. In contrast,
GAL4·C/EBP
118 conferred an increase of both basal transcription
and the glucocorticoid response, to 100 and 74% of wild type,
respectively (Fig. 3C, row 6). Therefore, this C/EBP
construct, containing amino acids 1-118, is able to confer accessory factor activity to the glucocorticoid response.
is
located in the region spanning amino acids 1-84. To determine whether this activation domain alone is capable of mediating accessory activity, GAL4·C/EBP
84 was co-transfected with
p
CREGAL4 into H4IIE cells. As was the case for C/EBP
,
truncation of the C/EBP
construct down to its amino-terminal
activation domain increased basal transcription from both the
p
CREGAL4 promoter and the (GAL4)5E1bCAT promoter (Fig. 3C, rows 5-7). However, the amino-terminal
activation domain of C/EBP
was not sufficient to confer accessory
activity to the glucocorticoid response (Fig. 3C, row 7).
This suggests that the region between amino acids 85 and 118 plays a
role in conferring accessory activity to the glucocorticoid response.
is required for
accessory activity, we next constructed the GAL4 DBD fusion proteins GAL4·C/EBP
52-118 and GAL4·C/EBP
85-118, wherein two
segments of the amino-terminal activation domain of C/EBP
were
deleted. Neither of these constructs conferred accessory factor
activity to the glucocorticoid response (Fig. 3C, rows 8 and
9). Taken together, these results suggest that the accessory
factor activity of C/EBP
requires the 1-84 activation domain but
that one or more amino acids in the 85-118 segment are also required.
In addition, these amino-terminal truncation constructs were unable to
confer transcriptional activation to the (GAL4)5E1bCAT
reporter plasmid, a result that was expected because these deletions
affected the activation domain (see Fig. 3A).
CREGAL4, basal transcription was
increased, but the glucocorticoid response remained at 46% of wild
type. Once again, this is equivalent to the GAL4 DBD alone or a
deletion of the CRE (12) (Fig. 3C, compare rows 1 and 10). Although GAL4·CREB 283 conferred neither
accessory activity from the p
CREGAL4 reporter nor basal
activity from the (GAL4)5E1bCAT reporter, co-transfection with a protein kinase A (PKA) expression vector increased
transcriptional activity in the latter system (data not shown). This
PKA-dependent stimulation is consistent with previous
observations (28). Taken together, these results suggest that C/EBP
,
rather than C/EBP
or CREB, provides accessory factor activity for
the glucocorticoid response from the PEPCK CRE.
View larger version (31K):
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Fig. 4.
Inhibition of the glucocorticoid response by
PKI. The wild type PEPCK reporter plasmid (pPL32) and an
expression vector for the glucocorticoid receptor (pRSV-GR) were
co-transfected into H4IIE cells with increasing amounts of either PKI
or PKImut. The total amount of DNA transfected into the
tissue culture cells in each experiment was equalized using the empty
cloning vector pBR322. As indicated in the key, the
black bars denote basal PEPCK transcription levels in the
presence of PKI, the dark gray bars denote
dexamethasone-induced PEPCK transcription in the presence of PKI, the
light gray bars denote basal PEPCK transcription levels in
the presence of PKImut, and the white bars
denote dexamethasone-induced PEPCK transcription in the presence of
PKImut. CAT reporter activity is expressed in terms of
percentage relative to dexamethasone-induced PEPCK transcription levels
in the absence of either PKI or PKImut. All values shown
represent the mean ± S.E. of five or more separate experiments.
Statistical significance was calculated using Student's t
test, so that differences are designated as highly significant for
p < 0.01 (*) and very highly significant for
p < 0.001 (**).
DISCUSSION
,
rather than C/EBP
or CREB, confers accessory activity to the
glucocorticoid response from the CRE. This is consistent with the idea
that accessory factor activity for native promoters requires specific
transcription factors.
interacts with a variety of transcription factors to mediate
the transcriptional activation of many genes. Both the carboxyl-terminal leucine zipper and the amino-terminal activation domains of C/EBP
are required for the interaction with the
glutamine- and serine/threonine-rich activation domains of Sp1, an
association that is necessary for transcription of the rat
CYP2D5 gene (37). The leucine zipper domain of nuclear
factor-interleukin 6, the human homologue of C/EBP
, interacts with
the glucocorticoid receptor in vitro, and these interactions
are presumed to result in increased transcription of the
1-acid
glycoprotein gene by glucocorticoids in F9 cells (38, 39). In addition,
the same domain of C/EBP
interacts with the Rel-homology domain of
nuclear factor-
B to regulate transcription of the interleukin-6 and
interleukin-8 genes (40-43). The amino-terminal region of nuclear
factor-M, the chicken homologue of C/EBP
, interacts with the
E1A-binding domain of CREB-binding protein/p300 to regulate
transcriptional activation of the chicken mim-1 gene (44).
Our data suggest that amino acids 1-118 of C/EBP
, a region that
includes the CREB-binding protein-binding domain and the amino-terminal
activation domain (see Fig. 3A), are required for the
maximal transcriptional stimulation of the PEPCK gene by
glucocorticoids. The activation domain (amino acids 1-84), although
necessary, is not by itself sufficient to provide accessory factor
activity. This implies that the region from amino acids 84-118 must
contain residues that, along with the activation domain, provide
accessory factor activity. The idea that a transcription factor, within
the context of a hormone response unit, would contain distinct modular
domains for basal transcriptional activation and accessory
transcriptional activation provides a mechanism for integrating
multiple hormonal signals and providing an adaptive response. Just such
a separation of activation domains was reported recently by Merika
et al. (45), who found both a "synergism-specific"
domain and a basal transcriptional activation domain within the
carboxyl terminus of nuclear factor-
B. In an observation that was
strikingly similar to the results that we found with C/EBP
, they
observed that the small region of p65 that is adjacent to yet distinct
from the basal activation domain was not required for basal activation
alone, but was nevertheless essential for synergistic effects.
and C/EBP
, the two most abundant C/EBP
isoforms in the liver (46), are induced by glucocorticoids to
equivalent levels in H4IIE cells (47), and because C/EBP
and
C/EBP
have the same binding affinity for the consensus C/EBP-binding
site (22), it might be argued that C/EBP
, which does not confer
accessory activity, may act as a competitive antagonist to C/EBP
at
the C/EBP site, and so blunt the glucocorticoid response.
Alternatively, the position of C/EBP
on the consensus C/EBP-binding
site in the context of the PEPCK promoter may be a few base pairs out
of register from its location on the wild type PEPCK CRE, because the
core binding site for C/EBP family members is not the same size as the
CRE (48). This could result in a reduced glucocorticoid response,
because there is a very stringent requirement for spacing between
accessory elements in the GRU (35).
118 construct provides significant but incomplete
restoration of activity to the glucocorticoid response. There are a
number of possible explanations for this observation. First, the
GAL4-fusion protein may not have the exact conformation of native
C/EBP
in vivo, due either to the fusion itself or to
allosteric effects mediated by the GAL4-binding site. Another
possibility is that the leucine zipper domain, which interferes with
transactivation in the GAL4 system (30) and, therefore, was removed in
order to unmask accessory activity, may be required by the native
protein for complete accessory factor activity.
. If so,
other CRE-binding factors, such as members of the Fos, Jun, CREB
modulator, or activating transcription factor families, could confer
the activity in vivo. However, we find this scenario
unlikely, because the major rat liver nuclear extract CRE-binding
proteins found by EMSA are all members of the C/EBP family (22). In
fact, our conclusions with C/EBP
concur with several previous
studies that independently associated this particular transcription
factor with the induction of PEPCK by hormones (e.g. cAMP
and glucocorticoids) at the CRE. In 1993, Park et al. (18)
reported a role for C/EBP
, and not C/EBP
, at the CRE in the
induction of PEPCK by cAMP. The following year, in accordance with
previous work published by this lab, Angrand et al. (49)
found that a full glucocorticoid response for PEPCK requires the CRE.
Soon afterward, in a set of in vivo studies using transgenic
mice, Friedman and colleagues (50, 51) correlated the dramatic increase
in C/EBP
levels with the CRE-dependent glucocorticoid
induction of PEPCK gene transcription during exercise.
plays a role in the integration of these two hormonal signals at the
PEPCK CRE.
as an accessory factor for the
glucocorticoid response completes the cataloging of accessory factors
for the complex PEPCK GRU that began a number of years ago (3, 6-8, 9,
11, 12). Several HRUs of the PEPCK gene have been described in some
detail, including the GRU, the retinoic acid response unit, the insulin
response unit, and the cAMP response unit. These HRUs share a common
feature in that they each contain several multifunctional elements able
to bind more than one set of proteins and thereby mediate responses to different signals. For example, the retinoic acid response unit is
composed of two elements: retinoic acid response elements 1 and 2 (which bind RAR/RXR heterodimers), containing sequences that are
coincident with AF1 and AF3 of the GRU (which bind COUP-TF/HNF4 and
COUP-TF, respectively). The CRE is also a pleiotropic element that is
crucial for basal transcription, the cAMP response, and the
glucocorticoid response. CREB binds to the CRE to mediate the cAMP
response, whereas C/EBP
binds to the same sequence to confer
accessory activity to the glucocorticoid response. This arrangement of
the PEPCK promoter, with its constellation of overlapping HRUs, is
termed a metabolic control domain. It is the unique structure of the
metabolic control domain that permits the integration of multiple
hormonal stimuli, thereby facilitating a multifaceted response of the
PEPCK gene to a variety of environmental challenges.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Richard A. Maurer, Peter F. Johnson, Richard L. Printz, and Keith R. Yamamoto for providing vectors. We thank Catherine Caldwell for maintaining the H4IIE cells and Deborah Brown for assistance in the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK35107, the Vanderbilt Diabetes Research and Training Center (DK20593), and the Vanderbilt University School of Medicine Medical Scientist Training Program (GM07347).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.
These authors contributed equally to this work.
§ Current address: Department of Biochemistry, Fukui Medical University, Fukui, Japan.
¶ To whom correspondence should be addressed. Tel.: 615-322-7000; Fax: 615-322-7236; E-mail: Daryl.Granner{at}mcmail.vanderbilt.edu.
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ABBREVIATIONS |
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The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; GRU, glucocorticoid response unit; AF, accessory factor; GR, glucocorticoid receptor; CRE, cyclic AMP response element; HRUs, hormone response units; HNF, hepatocyte nuclear factor; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; C/EBP, CCAAT/enhancer-binding protein; CREB, cyclic AMP response element-binding protein; CAT, chloramphenicol acetyltransferase; DBD, DNA-binding domain; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; bZIP, basic and leucine zipper; PKA, cyclic AMP-dependent protein kinase A; PKI, heat-stable inhibitor of the cyclic AMP-dependent protein kinase; PKImut, mutant of PKI; CREwt, CRE wild type; USF, upstream stimulatory factor.
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REFERENCES |
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