From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
Received for publication, November 20, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Although many genes are regulated by the
concerted action of several hormones, hormonal signaling to gene
promoters has generally been studied one hormone at a time.
The phosphoenolpyruvate carboxykinase (PEPCK) gene is a case in point.
Transcription of this gene is induced by glucagon (acting by the second
messenger, cAMP), glucocorticoids, and retinoic acid, and it is
dominantly repressed by insulin. These hormonal responses require the
presence of different hormone response units (HRUs), which consist of
constellations of DNA elements and associated transcription factors.
These include the glucocorticoid response unit (GRU), cAMP response
unit (CRU), retinoic acid response unit (RARU), and the insulin
response unit. HRUs are known to have functional overlap. In
particular, the cAMP response element of the CRU is also a component of
the GRU. The purpose of this study was to determine whether known GRU
or RARU elements or transcription factors function as components of the
CRU. We show here that the glucocorticoid accessory factor binding site
1 and glucocorticoid accessory factor binding site 3 elements, which
are components of both the GRU and RARU, are an important part of the
CRU. Furthermore, we find that the transcription factor, chicken
ovalbumin upstream promoter-transcription factor, and two coactivators,
cAMP response element-binding protein-binding protein and steroid
receptor coactivator-1, participate in both the cAMP and glucocorticoid
responses. This provides a further illustration of how the PEPCK gene
promoter integrates different hormone responses through overlapping
HRUs that utilize some of the same transcription factors and coactivators.
Glucose homeostasis is exquisitely controlled by the opposing
actions of a variety of hormonal signals. Much of this control is
achieved through the regulation of hepatic genes that encode the
glycolytic and gluconeogenic enzymes. For example, glucagon (acting via
the cAMP/PKA1 signaling
pathways), glucocorticoids, thyroid hormones, and retinoic acid induce
the phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) gene, which
encodes a rate-determining enzyme in gluconeogenesis. Insulin
dominantly inhibits both the basal and hormone-induced expression of
this gene (1-4). The hormonal regulation of PEPCK gene expression is
mediated by the actions of a diverse array of transcription factors
that bind to several DNA elements in the PEPCK gene promoter. The
complex of factors and DNA elements required for a given hormone
response is known as a hormone response unit (HRU) (5).
The DNA elements that comprise the PEPCK glucocorticoid response unit
(GRU) are illustrated at the top of Fig. 2A. This GRU consists of elements located between Induction of PEPCK gene transcription by glucagon (cAMP) is
mediated by a group of promoter elements and their respective transcription factors, known collectively as the cAMP response unit
(CRU) (see Fig. 2A). The PEPCK gene CRU consists of several known DNA elements located between positions It is important that the action of these various hormones be integrated
into a response that is appropriate for the particular physiologic
situation. How such integration occurs at the molecular level has yet
to be resolved, but the assembly of multiple HRUs in a single promoter
may explain the phenomena of additivity, synergism, and dominant
repression that are observed in the presence of different combinations
of hormones. As the organization of the PEPCK promoter is further
defined, it is also becoming apparent that many of the individual DNA
elements are involved in multiple hormonal responses. As described
above, both gAF1 and gAF3 from the GRU serve as RAREs in the RARU (12,
13). Furthermore, the gAF2 element, which is essential for the
glucocorticoid response, is also part of the insulin response unit
(20).
This study provides a more detailed understanding of the CRU and
identifies elements common to the CRU, GRU, and RARU. We show here that
a deletion of certain elements, specifically gAF1/RARE1 and gAF3/RARE2,
greatly reduces the response of the PEPCK gene to cAMP. Furthermore,
two coactivators, steroid receptor coactivator-1 (SRC-1) and
CREB-binding protein, are utilized by both the CRU and GRU to drive
PEPCK gene transcription.
DNA Constructs--
PEPCK gene promoter truncations were
constructed by PCR amplification of the appropriate regions of the
PEPCK gene promoter in PEPCK/Luc. The primers used to amplify in the 5'
to 3' direction were as follows: 1) the Cell Culture and Transient Transfection--
H4IIE hepatoma
cells were grown to confluence in Dulbecco's modified Eagle's medium
(DMEM) containing 2.5% (v/v) newborn calf serum and 2.5% (v/v) fetal
calf serum. Cells were pelleted and incubated for 30 min at room
temperature with 2 ml of a calcium phosphate:DNA coprecipitate
containing plasmid DNA. Cells were then plated in 10-cm2
culture dishes and incubated at 37 °C. After 4 h, cells were treated with 20% (v/v) dimethyl sulfoxide in serum-containing medium
for 5 min, washed with PBS, and incubated in serum-free media for
18 h. Cells were harvested by trypsin digestion and resuspended in
100 µl of luciferase assay lysis buffer (Promega). Luciferase
activity was measured according to the manufacturer's instructions
(Promega). Luciferase activity was normalized for the protein
concentration in the cell lysate by using the Bio-Rad protein assay reagent.
Primer Extension Assay--
Total RNA for both primer extension
reactions was isolated with Tri-Reagent (Molecular Research Center,
Inc., Cincinnati, OH) using the instructions provided by the
manufacturer. The PC28 and ACT25 oligonucleotides, which are
complimentary to the mRNAs of the rat PEPCK and Chromatin Immunoprecipitation Assay--
The ChIP assay protocol
was adapted from methods described previously (22-26). H4IIE cells
(1 × 108 cells per condition) were washed with PBS
and pre-incubated in serum-free DMEM at 37 °C for CBP and SRC-1 Associate with the Endogenous PEPCK Gene Promoter in
Response to cAMP--
CBP and SRC-1 are recruited to the PEPCK gene
promoter in response to dexamethasone, a synthetic glucocorticoid, as
shown previously
(22).2 cAMP typically
causes a 2- to 3-fold induction of PEPCK mRNA levels in H4IIE
cells, as confirmed in the experiment shown in Fig.
1A. We wanted to determine
whether the same coactivators that associate with the PEPCK gene
promoter after dexamethasone treatment also associate in response to
cAMP. The ChIP assay was therefore used to directly identify
proteins that interact with the PEPCK gene promoter in response to cAMP
treatment of H4IIE cells in vivo (Fig. 1B). We
found that CBP interacts with the PEPCK promoter after cAMP treatment.
This result was expected, because CBP action is regulated by cAMP
signaling pathways (27, 28). It was somewhat surprising to observe
recruitment of SRC-1 to the PEPCK gene promoter in response to cAMP,
however, because relatively few studies exist showing the involvement
of this coactivator in cAMP action. SRC-1 is a coactivator with
COUP-TF, which is an important mediator of the glucocorticoid response
in the PEPCK gene, and CBP interacts with CREB on a variety of
promoters. ChIP assays were therefore performed using a COUP-TF or CREB
antibody. We found that COUP-TF is constitutively bound to the
promoter, in agreement with previous electrophoretic gel shift mobility analysis of the PEPCK gene promoter (10). CREB is also bound to the
promoter under basal conditions, and binding was not increased when
cells were treated with cAMP, as unchanging bands were detected under
these conditions (Fig. 1B). These data suggest that CREB and
COUP-TF are constitutively bound to the PEPCK gene promoter, whereas
CBP and SRC-1 are recruited in response to cAMP treatment. The next set
of experiments were performed to specifically locate the region of the
PEPCK promoter targeted by COUP-TF, CREB, CBP, and SRC-1.
CREB and CBP Function at the CRE to Mediate the PKA
Response--
The CRE is necessary for both cAMP- and
glucocorticoid-mediated PEPCK gene expression, and CBP is likely to act
through the proteins bound to this element. H4IIE rat hepatoma cells
were transiently transfected with a construct that contains the region from GRU/RARU Elements Are Necessary for the PKA
Response--
A G4CBP construct was cotransfected with
(C/G)-PEPCK/Luc to further explore the role of CBP in PKA-mediated gene
expression (Fig.
3A). G4CBP, in
the absence of cotransfected PKA, increased reporter gene activity
7-fold (Fig. 3A, lanes 1 and 2).
Cotransfection of PKA further increased the response to nearly 20-fold
over the basal value (Fig. 3A, lane 3); thus CBP
has substantial activity at the CRE, even in the absence of CREB
binding at the CRE. This activity is much greater than the 3- to 4-fold
response observed in the experiment illustrated in Fig.
2B.
CBP interacts with several coactivators to stimulate gene
transcription. It is thus possible that CBP interacts with upstream coactivators to mediate the PKA response. A series of 5' deletions (to
positions
To further investigate the involvement of the GRU/RARU DNA binding
elements, in the context of the wild-type promoter, 5' deletions (to
gAF1 and gAF3 of the GRU/RARU Mediate the PKA
Response--
In addition to the CRE, the GRU consists of the elements
gAF1, gAF2, and gAF3 that bind to HNF-4/COUP-TF, HNF-3
The gAF3/RARE2 element and an immediately adjacent E box bind COUP-TF
and USF, respectively (Fig. 4B, top) (10). RARE2
is also a DR5-type RARE composed of three half-sites ( COUP-TF and SRC-1 Rescue the PKA Response--
COUP-TF, which was
originally identified as a stimulatory transcription factor required
for the expression of the chicken ovalbumin gene (31), also acts as a
repressive factor in that it counteracts the positive transcriptional
effects mediated by factors such as RAR/RXR, thyroid hormone
receptor/RXR, 1,25-dihydroxyvitamin D3 receptor/RXR, peroxisome
proliferator-activated receptor/RXR, HNF-4, and estrogen receptor
(32-35). In the context of the PEPCK gene, COUP-TF acts as a
stimulatory factor at gAF3, and it is required for a complete
glucocorticoid response. A chimeric protein in which the G4DBD is
ligated to COUP-TF was used to determine whether COUP-TF can rescue the
PKA response of PEPCK/Luc when gAF3/RARE2 is replaced by a Gal4 DNA
binding element. Although the G4DBD did not mediate a PKA response
when bound to gAF3/RARE2, G4COUP-TF rescued this response (Fig.
5A, compare lanes 3 and 4 with lanes 5 and 6).
SRC-1 is a coactivator with COUP-TF and is an important regulator of
the glucocorticoid response of the PEPCK gene promoter (36-38).
Furthermore, SRC-1 is regulated by cAMP, as 8-bromo-cAMP treatment
increases the phosphorylation of SRC-1 on threonine 1179 and serine
1185, an effect that is inhibited by the PKA inhibitor (38).
Phosphorylation of these sites is necessary for a functional cooperation between SRC-1 and CBP for coactivation of the progesterone receptor (39). A chimeric protein in which the G4DBD is ligated to
wild-type SRC-1 was therefore tested at gAF3/RARE2 to determine whether
SRC-1 can also rescue the PKA response. Although G4DBD did not mediate
a PKA response when bound to gAF3/RARE2, G4SRC-1 rescued the PKA
response (Fig. 5B, compare lanes 3 and
4 with lanes 5 and 6).
Experiments were also performed to determine whether the threonine 1179 and serine 1185 phosphorylation sites of SRC-1 are essential for the
PKA response. H4IIE cells were cotransfected with a PEPCK/Luc reporter
gene in which gAF3 is replaced by a Gal4 DNA binding element and with a
G4SRC-1 construct in which residues 1179 and 1185 are mutated to
alanine (referred to at G4SRC-1 Ala/Ala). A mutation of these two amino
acids completely prevented responsiveness of the PEPCK gene to PKA
(Fig. 5B, compare lanes 5 and 6 with
lanes 7 and 8), suggesting that either the phosphorylation of these residues and/or the interaction of this region
of SRC-1 with other factors is important. The expression of G4SRC-1
Ala/Ala and wild-type G4SRC-1 could not be tested in H4IIE because of
the extremely low transfection efficiency of these cells. Instead,
H4IIE cells were transfected with the 5× Gal4/E1b-Luc construct that
has five Gal4 DNA binding elements upstream of the E1b promoter and
luciferase reporter gene. Cells were cotransfected with G4DBD, G4SRC-1,
or G4Ala/Ala. As shown in Fig. 5C, G4SRC-1 and G4SRC-1
Ala/Ala had comparable activity at this promoter, suggesting that these
two chimeric proteins are expressed at comparable levels. The data also
show that residues 1179 and 1185 of SRC-1 are specifically important
for expression of the PEPCK gene promoter but not the E1b gene.
SRC-1 at gAF3/RARE2 Does Not Effectively Mediate the
Retinoic Acid or Glucocorticoid Response--
Because this is the
first time that SRC-1 was shown to function at gAF3/RARE2 (Fig.
5B), we wanted to determine whether SRC-1 also plays a role
at this element for the retinoic acid (RA) and glucocorticoid responses
of the PEPCK gene. SRC-1 supports the glucocorticoid response when
tethered to gAF1/RARE1 and gAF2 (36), so it is possible that it also
functions at gAF3/RARE2. The role of SRC-1 for the RA response of the
PEPCK gene has yet to be studied. Experiments were performed,
therefore, to determine whether G4SRC-1 rescues either the RA or
glucocorticoid responses when bound at gAF3/RARE2. G4SRC-1 Ala/Ala was
also used to determine the role of residues 1179 and 1185 of SRC-1 for
these responses. As expected, both the RA and glucocorticoid responses
were markedly reduced when gAF3/RARE is mutated to a Gal4 DNA binding
site (Fig. 6, compare lanes 2 and 5 and lanes 3 and 6). Unlike the
PKA response, however, G4SRC-1 was unable to recover the RA response,
which was somewhat surprising, because SRC-1 can coactivate with
RAR/RXR heterodimers (40, 41) (Fig. 6, compare lane 8 with
lane 2). G4SRC-1 caused a small increase of the
glucocorticoid response when tethered to gAF3/RARE2 but did not recover
the response to the full extent, as observed at gAF1/RARE1 and gAF2
(36) (Fig. 6, compare lane 9 with lane 3).
Although not significant, mutation of residues 1179 and 1185 partially
blunted the small recovery of the glucocorticoid response mediated by
SRC-1 (Fig. 6, compare lanes 9 and 12),
suggesting that these residues may play a role in the glucocorticoid
response.
Transcription of the PEPCK gene is regulated by a number of
hormones, including glucagon (acting via PKA), glucocorticoids, retinoic acid, and insulin (5, 42-44). Each hormone response is
mediated by a set of cis-elements and associated
transcription factors that are collectively termed a hormone response
unit or HRU. In this study, we further describe the components of the PEPCK CRU. The CRU was originally defined as extending from Although a simple hormone response element may provide "on or off"
control, a HRU provides a more flexible and versatile means of
regulating gene transcription and thus may allow for a more precise
control of a specific metabolic process. The sharing of components
between several HRUs provides additional flexibility and regulatory
control. Several genes, in addition to PEPCK, encode metabolic enzymes
that integrate multiple hormone responses. For example, the tyrosine
aminotransferase and insulin-like growth factor-binding protein-1 genes
possess GRUs that contain an insulin response sequence that
overlaps with the HNF-3 binding site, allowing insulin to repress these
genes in a dominant manner over glucocorticoid-mediated activation
(45-47). Mutation of two insulin response elements that bind to HNF-3
and NF1-like proteins prevents insulin-mediated repression of the
cytosolic aspartate aminotransferase gene. Mutation of these
elements also partially decreases the response of this gene to
glucocorticoids (48). Furthermore, the hepatic glucose 6-phosphatase
(G6Pase) gene is composed of different overlapping HRUs that mediate
its activation by cAMP and glucocorticoids and dominant repression by
insulin (49-53). A CRE is an accessory factor element for both the CRU
and GRU of hepatic G6Pase, whereas an HNF-1 With regard to the PEPCK gene, gAF1 and gAF3 of the GRU are also RAREs
that comprise a functional RARU. It is unlikely that RAR/RXR is
involved in the PKA response, because retinoic acid is not present in
our culture media. Furthermore, we have found that RAR is recruited to
the PEPCK promoter only in the presence of retinoic
acid.4 We find here that
COUP-TF binding to gAF3/RARE2, which is essential for a full
glucocorticoid response, is also required for a full PKA response. In
addition, the coactivators SRC-1 and CBP are shared by both the PEPCK
CRU and GRU. This overlapping structure of the PEPCK gene promoter is
termed a metabolic control domain (5, 13, 56). Our current thinking is
that the arrangement of several functionally interacting HRUs may
explain the phenomena of additivity, synergism, and dominant repression
observed in the presence of different combinations of hormones. The
metabolic control domain may thus integrate the action of a variety of
hormonal signals to provide a transcriptional response that is
appropriate for a particular physiological process, which is
gluconeogenesis in the case of the PEPCK gene.
Different hormonal inputs have combinatorial effects on PEPCK gene
expression. For instance, glucocorticoids and cAMP have additive
effects, whereas glucocorticoids and retinoic acid have additive to
synergistic effects on PEPCK gene
transcription.5 The fact that
both gAF1/RARE1 and gAF3/RARE2 are components of each of these three
hormone responses suggests that the factors that bind to these sites,
and their associated coregulators, play a central role in these
combined stimulatory effects. Several mechanisms may explain how these
hormones act together to enhance PEPCK gene expression. For example, it
is possible that a greater number of CBP and SRC-1 molecules are
recruited to the PEPCK gene promoter under combined treatment with
dexamethasone and cAMP, although we have not observed an increase in
the association of these coactivators with the PEPCK promoter under
these conditions in the ChIP assay (data not shown). Alternatively,
these hormones may cause a modification of the coactivators recruited
to the PEPCK gene promoter. PKA signaling causes phosphorylation of
SRC-1 at residues 1179 and 1185 (39), and both of these sites are important for the component of the PKA response that is mediated through gAF3/RARE2. Furthermore, SRC-1 potentiates the activity of
COUP-TF when COUP-TF is bound to the DR4 element of the CYP1A gene
promoter, and COUP-TF associates with SRC-1 in vivo (37). We
are currently trying to determine whether a direct interaction between
COUP-TF and SRC-1 is important for the PKA response of the PEPCK gene.
Perhaps phosphorylation of SRC-1 is necessary for such an interaction.
Furthermore, the phosphorylation of the coactivators that associate
with gAF1/RARE1 and gAF3/RARE2 may lead to their interaction with the
basal transcription machinery, thereby increasing the rate of transcription.
CBP is also recruited to the PEPCK gene promoter in response to
glucocorticoid or cAMP treatment. We show here that CBP works at the
CRE to mediate the PKA response. CBP most likely interacts with CREB at
the CRE, although it is possible that other factors, such as C/EBP Organization of a gene promoter into multiple, interacting HRUs may
allow for a more precise regulation of gene transcription in the face
of fluctuating environmental conditions. The PEPCK gene is used as a
model system to study the integration of multiple hormones into a
transcriptional response. The organization of its promoter into
multiple HRUs, and the inherent redundancy found therein, may help
prevent the dysregulation of this gene. For instance, deletion of gAF1,
gAF2, gAF3, or the CRE still allows for 50% of the PEPCK
glucocorticoid response. Thus, if one of the transcription factors that
binds to these elements is defective in its DNA binding or
transactivation capabilities, a partial increase in PEPCK gene
expression and gluconeogenesis can still be achieved. Furthermore,
several different transcription factors are capable of binding to
individual elements such as gAF3, which can bind to COUP-TF and
RAR/RXR. Perhaps these factors possess redundant roles and can serve in
place of each other if one is disabled, which would allow for the
maintenance of PEPCK gene expression, gluconeogenesis, and normal blood
glucose levels.
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ABSTRACT
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467 and
300, including two
tandem glucocorticoid receptor binding elements and several flanking
accessory factor elements that are essential for a full response. These
elements include gAF1, gAF2, and gAF3, which bind HNF-4/COUP-TF,
HNF-3
, and COUP-TF, respectively (6-10). C/EBP
also serves as a
glucocorticoid response accessory factor at the CRE (6, 11). The PEPCK
promoter also contains a retinoic acid response unit (RARU) that
consists of two retinoic acid response elements (RARE1 and RARE2) that
bind retinoic acid receptor heterodimers. RARE coincides with gAF1 of
the GRU, whereas RARE2 coincides with gAF3 (12, 13).
300 and
86 relative to
the transcriptional start site, including the CRE and the P3 and P4
elements. P3(I) and P4(II) are essential for a full cAMP/PKA response
(2, 4, 14-16). These elements are located between
300 and
200 and
contain binding sites for the AP-1 and C/EBP family members. The
CRE is located between
93 and
86 and binds to members of the
CREB/cAMP response element modulator, C/EBP, and AP-1 families
of transcription factors in vitro (11, 17, 18). The CRE has
three functions: 1) it binds CREB as part of the basal promoter; 2) it
is essential for the cAMP response, in which case CREB is
phosphorylated on serine 133 by PKA; and 3) it is necessary for a full
glucocorticoid response (2, 6, 14, 19).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
467 primer 5',
CGGGGTACCCCGTGAATTCCCTTCTCATGACC 3'; 2) the
299 primer, 5'
CGGGGTACCCCGAGCCTATAGTTTGCATCAGC 3'; and 3) the
200 primer, 5'
CGGGGTACCCCGCAACATTCATTAACAACAGCAA 3'. The primers were paired with the
reverse primer, 5' TCCCCCCGGGGGGAACCTGGAGGCTCGCCTC 3'. The PCR
amplification was carried out for 30 cycles. PCR products were digested
with KpnI and BglII and inserted into pGL3b at
the corresponding restriction sites. The
120 truncation of the PEPCK gene promoter was constructed by excising the PEPCK gene promoter from
PEPCK/Luc with NcoI and inserting the resulting fragment into pGL3b at the corresponding restriction site. The QuikChange site-directed mutagenesis kit (Stratagene) was used to construct the
120, (GRU/RARU)/
200, AF3
m, and AF3Em plasmids. The sequence of
all subcloned fragments was verified by DNA sequencing. The construct
with G4DBD ligated to SRC-1 with residues 1179 and 1185 mutated to an
alanine (referred to as G4SRC-1 Ala/Ala) was kindly provided by Dr.
Bert O'Malley, Baylor College of Medicine.
-actin genes at
positions 102-129 and 42-67, were used in primer extension assays, as
described previously (21).
48 h. Following
hormone treatments, cells were cross-linked with 1% formaldehyde
(Fisher Scientific) in serum-free DMEM at 37 °C for 5 min. To arrest
cross-linking, glycine was added directly to the media at a final
concentration of 125 mM, and the cells were rinsed with
ice-cold PBS. Cells were harvested with cell scraping buffer (1 ml per
plate of ice-cold PBS with protease and phosphatase inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 20 mM NaF, 1 mM Na3VO4, 10 mM
Na4P2O7, pH 8, 0.4 mM
Na2MoO4, 125 nM okadaic acid).
Cells were then pelleted by centrifugation at 700 × g
for 4 min at 4 °C, resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8, with protease
and phosphatase inhibitors), and incubated on ice for 10 min. The
lysates were transferred to pre-chilled Eppendorf tubes containing
25-µm-diameter glass beads (ratio of lysate to bead volume was 3:1),
pre-washed with cell scraping buffer. To shear chromatin, the
lysate/bead mixture was sonicated (VirSonic; 2.5-mm tip) on ice for
12 × 10-s pulses at a setting of 3 (output of 4-5 watts),
yielding chromatin fragments of 100-600 bp in size. Samples were
centrifuged at 14,000 rpm for 10 min at 4 °C to remove detritus, and
the supernatant was divided into aliquots for subsequent 10-fold
dilution in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, pH 8, 16.7 mM Tris-HCl, pH 8, with
protease and phosphatase inhibitors). To provide a positive control for
each condition, one undiluted aliquot was retained for further
processing in parallel with all other samples at the reversal of
cross-linking step. To reduce nonspecific background, each 1-ml
chromatin sample was pre-cleared with 10 µl of protein A/G
PLUS-agarose slurry (Santa Cruz Biotechnology, Inc.), supplemented with
100 µg/ml sonicated salmon sperm DNA (Stratagene), for 1 h at
4 °C on a rotating wheel, after which the beads were pelleted, and
the supernatant was transferred to a new tube. Chromatin complexes were
immunoprecipitated for 12-18 h at 4 °C while rotating with amounts
(5-10 µg) of primary antibody optimized for selective
immunoprecipitation of signal or without antibody to provide negative
controls. Immune complexes were collected with 40 µl of protein
A/G-agarose + 100 µg/ml salmon sperm DNA, while rotating for 3 h
at 4 °C, followed by centrifugation at 1000 × g for
1 min at 4 °C. The beads were washed for 5 min at 4 °C with 1 ml
of each of the following buffers in succession: low salt wash buffer
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, pH 8, 20 mM Tris-HCl, pH 8, 150 mM NaCl), high salt wash
buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, pH 8, 20 mM Tris-HCl, pH 8, 500 mM NaCl), LiCl wash
buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, pH 8, 10 mM Tris-HCl,
pH 8), and twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Chromatin complexes were eluted from the
beads in three consecutive 30-min rotating incubations with 200 µl of
elution buffer (1% SDS, 0.1 M NaHCO3) at
22 °C. To reverse cross-linking and digest RNA present in the samples, NaCl (200 mM final concentration) and RNase
mixture (Ambion) were added, and the samples were incubated at 65 °C
for
6 h. To digest proteins, samples were incubated at 45 °C for
90 min after the addition of the following at their final
concentrations: 10 mM EDTA, 40 mM Tris-HCl, pH
6.5, and 50 µg/ml proteinase K. Samples were extracted twice with
phenol:chloroform:isoamyl alcohol (25:24:1) and once with
chloroform:isoamyl alcohol (24:1). DNA was precipitated with 5 µg of
glycogen azure (Sigma) and 2× volumes of 95% ethanol, and
pellets were collected by microcentrifugation for 30 min at 4 °C.
Samples were resuspended in 100 µl of nuclease-free water (Promega)
and stored at
80 °C for subsequent PCR analysis.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
CBP and SRC-1 are recruited to the PEPCK gene
promoter in response to cAMP. As shown in panel A,
PEPCK mRNA is induced after treatment of H4IIE cells with 100 µM 8-(4-chlorophenylthio)-cAMP for 2 h.
N, no hormone; C, 100 µM
8-(4-chlorophenylthio)-cAMP. -Actin mRNA was also measured as a
control. The association of COUP-TF (the antibody was provided by Dr.
Ming Tsai), CREB (Santa Cruz antibody sc-186), CBP (Santa Cruz antibody
sc-369), and SRC-1 (Santa Cruz antibody sc-6098) with the endogenous
PEPCK gene promoter was measured by ChIP assay on H4IIE cells treated
for 2 h under the following hormone conditions: N, no
hormone; C, 100 µM 8-(4-chlorophenylthio)-cAMP
(panel B). Control lanes show the results of
immunoprecipitations performed in parallel without the application of
primary antibodies. Input lanes show the results from samples not
subjected to immunoprecipitation. All results depicted are
representative of at least three independent experiments.
467 to +69 of the PEPCK gene promoter positioned upstream of the
luciferase reporter gene (referred to as PEPCK/Luc). This construct
typically gives a 2- to 4-fold induction when cotransfected with the
catalytic subunit of PKA in H4IIE cells (Fig.
2A, compare lanes 1 and 3). For convenience, we refer to cAMP-mediated PEPCK gene expression in transient transfection assays as the PKA response, because this is the effector employed. The role of the CRE in PKA-stimulated PEPCK gene expression was examined next. Replacement of
the CRE with a Gal4 DNA binding element (referred to as
(C/G)-PEPCK/Luc) resulted in a 50% reduction of the PKA response (Fig.
2B, compare lanes 2 and 4). The same
result was observed for the glucocorticoid response in earlier
experiments, as either deletion of the CRE or its replacement with a
Gal4 DNA binding element also reduces this response by 50% (6, 11).
Others have shown that G4CREB (a chimeric construct in which the Gal4
DNA binding domain (G4DBD) is ligated to full-length CREB) is effective
at restoring the PKA response when it binds to a Gal4 element that has
replaced the CRE (29). This result is confirmed here, as G4CREB
increased the PKA response ~3-fold over that mediated by the G4DBD
alone (Fig. 2B, lanes 4 and 7). The
activity mediated by G4CREB is higher than that mediated by PKA alone.
Wilson et al. (30) showed recently that, besides G4CREB,
G4C/EBP
and G4C/EBP
also mediate this response (30). It is
therefore possible that different combinations of these factors bind to
the CRE to mediate the PKA response of the endogenous PEPCK
gene. CBP is an important coactivator for glucocorticoid-mediated PEPCK
gene expression, and it has been shown to drive PEPCK gene
transcription by interacting with proteins bound to the CRE (22). The
role of CBP at the CRE was therefore examined for the PKA response. CBP
enhanced the PKA response of wild-type PEPCK/Luc by ~2-fold (Fig.
2A, lanes 2 and 4) and of G4CREB by 3- to 4-fold (Fig. 2B, compare lanes 6 and
8).
View larger version (14K):
[in a new window]
Fig. 2.
CBP enhances the PKA response. The DNA
elements essential for the GRU, RARU, and CRU are illustrated at the
top of panel A. H4IIE cells were transiently
transfected with PEPCK/Luc (5 µg) in the presence or absence of
expression vectors encoding the catalytic subunit of PKA (2.5 µg) or
CBP (10 µg) or an equivalent amount of plasmid DNA as a control.
Cells were treated for 18 h in serum-free DMEM, and cell lysates
were prepared for luciferase assays as described under "Experimental
Procedures." The results illustrated in panel A are
normalized to the basal activity of PEPCK/Luc. Data represent the mean
of four experiments ± S.E. The H4IIE cell transfection
experiments were repeated using (C/G)-PEPCK/Luc, in which the CRE is
replaced by a Gal4 binding site, in the presence or absence of 1 µg
of transfected G4DBD or G4CREB expression vectors or an equivalent
amount of plasmid DNA (panel B). The data are normalized to
the basal activity of (C/G)-PEPCK/Luc in the presence of G4DBD alone,
and the data represent the mean of four experiments ± S.E.
View larger version (19K):
[in a new window]
Fig. 3.
GRU and RARU elements are necessary for the
PKA response. H4IIE cells were transiently transfected with
(C/G)-PEPCK/Luc (5 µg) or constructs in which (C/G)-PEPCK/Luc was
truncated to 299,
200,
120, or
100. These various reporter constructs
were transfected in the presence or absence of 1 µg of expression
vectors encoding G4DBD or G4CBP or an equivalent amount of plasmid DNA
(panel A). The data are normalized to the basal activity of
(C/G)-PEPCK/Luc in the presence of G4DBD and represent the mean of four
experiments ± S.E. Deletion constructs of PEPCK/Luc were also
made and are illustrated at the top of panel B.
The GRU/RARU elements were added 5' of the
200 deletion construct
(referred to as (GRU/RARU)/
200). These reporter constructs (5 µg)
were transfected into H4IIE cells in the presence or absence of an
expression vector encoding the catalytic subunit of PKA (2.5 µg) or
an equivalent amount of plasmid DNA (panel B). The data are
normalized to the basal activity of PEPCK/Luc, and the data represent
the mean of four experiments ± S.E.
299,
200,
120, and
100 relative to the transcription start site) of (C/G)-PEPCK/Luc was constructed to examine the role of
upstream promoter elements/factors in the PKA response. Truncation of
the promoter to
299, which removes the GRU elements gAF1 (RARE1 of
the RARU), gAF2, and gAF3 (RARE2 of the RARU), nearly abolished the PKA
response when G4CBP is bound to a Gal4 element at the CRE (Fig.
3A, lanes 4 and 5). Removal of the P3 and P4 regions by truncation to
200, or further deletion to
120, provides reporter gene constructs that exhibit no PKA response (Fig.
3A, lanes 6-9). Further truncation of the
promoter to
100, which removes the CAAT box, an element that contains
a binding site for the transcription factor NF1, resulted in a reporter gene with greatly decreased basal activity and no PKA responsiveness, even in the presence of G4CBP (Fig. 3A, lanes 10 and 11).
299,
200, and
120) were made in PEPCK/Luc (Fig. 3B).
In this circumstance, the PKA response was also inhibited by truncation
of the PEPCK promoter to
299 (Fig. 3B, compare lanes
1 and 2 with lanes 3 and 4).
Truncation of the promoter to
200 and
120 also resulted in a lack
of stimulation by PKA (Fig. 3B, lanes 5-8). In
fact, there was a trend toward repression in the presence of the
catalytic subunit of PKA (Fig. 3B). The role of the GRU/RARU
elements was further verified by adding these elements (bases
467 to
300 relative to the transcription start site) immediately upstream of
the
200 construct, referred to as (GRU/RARU)/
200. H4IIE hepatoma
cells were transiently transfected with this construct, and a
restoration of the PKA response was observed (Fig. 3B,
lanes 9 and 10). These results confirm that some
component of the GRU/RARU is necessary for full stimulation of PEPCK
gene transcription by PKA.
, and COUP-TF, respectively (8-10). The RARU consists of two RAREs that are
coincident with gAF1 and gAF3 and bind retinoic acid receptor
(RAR)/retinoid X receptor (RXR) heterodimers (12, 13). The gAF elements
were initially identified by deletion and mutational analysis, and the
identity of the factors that bind these elements was later confirmed
using a electrophoretic mobility gel shift assay, the Gal4 system, and
the ChIP assay. A similar approach was used to evaluate the role of the
GRU/RARU in the PKA response, starting with the Gal4 system. Each of
the GRU or RARU elements was replaced by a Gal4 DNA binding site, and
these constructs were tested for PKA responsiveness. Replacement of
gAF2 had no effect on the PKA response (Fig.
4A, compare lanes 1 and 2 with lanes 3 and 4). Mutation of
either gAF1/RARE1 or gAF3/RARE2, however, significantly reduced this
response (Fig. 4A, compare lanes 1 and
2 with lanes 5 and 6 or with
lanes 7 and 8). Because replacement of gAF3/RARE2 with the Gal4 DNA binding site was particularly detrimental to the PKA
response, we focused our attention on this element.
View larger version (16K):
[in a new window]
Fig. 4.
gAF1 and gAF3 are essential for the PKA
response. H4IIE cells were transiently transfected with 5 µg of
PEPCK/Luc or PEPCK/Luc in which the gAF1, gAF2, or gAF3 elements were
replaced by a Gal4 DNA binding element. These constructs were
cotransfected with 2.5 µg of an expression vector encoding the
catalytic subunit of PKA or an equivalent amount of plasmid DNA
(panel A). The data are normalized to the basal activity of
PEPCK/Luc and represent the mean of eight experiments ± S.E. The
PKA responses of gAF1/Gal4 and gAF3/Gal4 were significantly reduced (*,
p < 0.05; **, p < 0.01 by Student's
t test). The sequence of the region containing the gAF3
element, with the sites for COUP-TF and USF binding as revealed by DNA
methylation studies, is illustrated at the top of
panel B. The half-site or the E box was mutated in
PEPCK/Luc, and 5 µg of each construct was transiently transfected
into H4IIE cells with or without 2.5 µg of the catalytic subunit of
PKA (panel B). The data are normalized to the basal activity
of PEPCK/Luc and represent the mean of eight experiments ± S.E.
,
, and
) (Fig. 4B, top). RARE2 binds an RAR/RXR
heterodimer but only in the presence of retinoic acid
(13).3 Because retinoic acid
is not necessary for the PKA response, it is unlikely that RAR/RXR play
a role in this response. We therefore wanted to determine whether
COUP-TF or USF mediate the PKA response. Methylation interference
studies show that COUP-TF makes contact with the
half-site, and USF
binds to the E box. Furthermore, mutations that prevent COUP-TF or USF
binding to the PEPCK gene promoter show that the
half-site is
necessary for the glucocorticoid response, whereas the E box is not
involved (10, 13). The mutations shown to prevent binding of COUP-TF or
USF to the PEPCK gene promoter were therefore introduced into PEPCK/Luc
to examine for a possible role of these sites in the PKA response.
Mutation of the
half-site is referred to as AF3
m and prevents
COUP-TF binding, whereas the E box mutation is referred to as AF3Em and prevents USF binding. In transient transfection assays of H4IIE hepatoma cells, a mutation of the E box had no effect on the PKA response (Fig. 4B, compare lanes 1 and
2 with lanes 3 and 4), but a mutation
of the
half-site completely abolished this response (Fig.
4B, lanes 5 and 6). Treatment of H4IIE
cells with cAMP did not cause additional protein complexes to bind to
gAF3/RARE2 in electrophoretic mobility shift assays (data not shown).
These data strongly suggest that COUP-TF, which is a member of the GRU, is involved in the PKA response. These observations are compatible with
the ChIP data presented in Fig. 1.
View larger version (21K):
[in a new window]
Fig. 5.
COUP-TF and SRC-1 rescue the PKA
response. H4IIE cells were transiently transfected with 5 µg of
PEPCK/Luc or PEPCK/Luc with gAF3 mutated to the Gal4 DNA binding
element (referred to as gAF3/G4) in the presence or absence of 2.5 µg
of an expression vector encoding the catalytic subunit of PKA. In
addition, 1 µg of an expression vector encoding either G4DBD or
G4COUP-TF was cotransfected in these experiments (panel A).
The data are normalized to the basal activity of PEPCK/Luc and
represent the mean of four experiments ± S.E. Experiments
identical to those performed in panel A were performed in
panel B except that G4SRC-1 or G4SRC-1 Ala/Ala (a construct
with serine residues 1179 and 1185 mutated to an alanine) was employed.
The data are normalized to the basal activity of PEPCK/Luc and
represent the mean of eight experiments ± S.E. The activity of
G4DBD, G4SRC-1, and G4SRC-1 Ala/Ala on the E1b promoter was also
examined by transfecting H4IIE cells with 1 µg of each construct and
5 µg of the 5× Gal4/E1b-Luc reporter gene construct. The data are
normalized to the activity of 5× Gal4/E1b-Luc in the presence of G4DBD
and represent the mean of four experiments ± S.E. (panel
C).
View larger version (24K):
[in a new window]
Fig. 6.
SRC-1 partially rescues the glucocorticoid
response but not the RA response. H4IIE cells were transiently
transfected with 5 µg of PEPCK/Luc or PEPCK/Luc with gAF3 mutated to
the Gal4 DNA binding element. In addition, 1 µg of an expression
vector encoding G4DBD, G4SRC-1, or G4SRC-1 Ala/Ala was cotransfected in
these experiments. Cells were treated with 5 µM RA or 500 nM dexamethasone (Dex), a synthetic
glucocorticoid, for 18 h. The data are normalized to the basal
activity of PEPCK/Luc and represent the mean of four experiments ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
300 to
86 relative to the transcriptional start site (2). This region
encompasses the CRE, which binds the CREB/cAMP response element
modulator, C/EBP, and AP-1 families of transcription factors, and the
P3(I) and P4(II) elements, which bind to members of the C/EBP
and
AP-1 families of transcription factors (2, 4, 11, 15-18). We find here
that the CRU requires the participation of additional upstream
elements, as the deletion or mutation of components of the GRU/RARU
severely restricts the PKA response. A mutation of the gAF1/RARE1 or
gAF3/RARE2 elements, which are shared by both the GRU and RARU,
significantly reduces the PKA response. On the other hand, a mutation
of gAF2, which disables its ability to function in the GRU, has no
effect on the PKA response.
DNA binding element is
an accessory factor site for the hepatic G6Pase GRU and is also an
accessory factor element for the G6Pase CRU in the kidney (49, 54,
55).
,
may mediate this response, as well. C/EBP
, which also interacts with
CBP, binds to the CRE to mediate the glucocorticoid response. This
response is enhanced by CBP, which also suggests a role for this
coregulator at the CRE (22). Little is known about phosphorylation of
CBP, although it contains several PKA consensus phosphorylation sites.
It is noteworthy that insulin, a repressor of PEPCK gene transcription,
causes the dissociation of CBP from the PEPCK promoter, underscoring
the importance of the interaction of this coactivator with the promoter
for induction of PEPCK gene expression (11, 22).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Cathy Caldwell for excellent technical assistance and Deborah Brown for manuscript preparation.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK02887 (to M. W.-L.), DK35107 and DK07061 (to D. K. G.), and DK20593, the Vanderbilt Diabetes Research and Training Center, the Veterans Administration Research Service, and the Vanderbilt University School of Medicine Medical Scientist Training Program GM07347 (to D. T. D.).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 & Biophysics, 707 Light Hall, Vanderbilt University School
of Medicine, Nashville, TN 37232-0615. Tel.: 615-322-7004; Fax:
615-322-7236; E-mail: daryl.granner@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M211846200
2 D. T. Duong, unpublished data.
3 X. L. Wang, unpublished data.
4 X. L. Wang, unpublished observation.
5 X. L. Wang, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, protein kinase A; PEPCK, phosphoenolpyruvate carboxykinase; HRU, hormone response unit; GRU, glucocorticoid response unit; CRU, cAMP response unit; RARU, retinoic acid response unit; RA, retinoic acid; gAF, glucocorticoid accessory factor binding site; SRC, steroid receptor coactivator; DMEM, Dulbecco's modified Eagle's medium; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; HNF, hepatic nuclear factor; CRE, cAMP response element; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; G4, Gal4; USF, upstream stimulatory factor; C/EBP, CAAT enhancer-binding protein; RAR, retinoic acid receptor; RARE, retinoic acid response element; Luc, luciferase; PBS, phosphate-buffered saline; RXR, retinoid X receptor; G6Pase, glucose 6-phosphatase; AP-1, activator protein-1.
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