From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935 and the ¶ Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44106
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ABSTRACT |
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Nuclear factor I (NFI) binds to a region of the
phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent
to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of
protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by
transcription factors, regardless of the presence of factors that
stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin)
transcription from the PEPCK gene promoter. The NFI effects on
transcription from the PEPCK gene promoter were observed even in the
absence of the NFI binding site, suggesting the possibility of other
weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to
demonstrate direct interaction between the transactivation domain of
NFI-C and the CREB binding domain of the CREB-binding protein (CBP).
Overexpression of a gene fragment encoding the CREB binding domain of
CBP stimulates transcription from the PEPCK gene promoter. The
inhibitory effect of NFI on transcription of the PEPCK gene induced by
the catalytic subunit of protein kinase A appears to be the result of
an interaction between NFI and the CREB-binding protein in which NFI
competes with CREB for binding to the CREB-binding site on CBP. In
contrast, glucocorticoids and thyroid hormone use the steroid hormone
receptor binding domain of CBP to stimulate transcription from the
PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid
hormone in an additive manner to stimulate PEPCK gene transcription. We
conclude that CBP coordinates the action of the multiple factors known
to control transcription of the PEPCK gene.
The regulation of phosphoenolpyruvate carboxykinase (GTP) (EC
4.1.1.32) (PEPCK)1 gene
transcription is coordinated by the action of a number of transcription
factors that bind to specific cis-elements on the promoter and mediate
the effect of various hormones and other regulatory molecules (1, 2).
Most of the cis-acting elements on the PEPCK gene promoter are located
within the first 550 base pairs immediately 5' to the transcriptional
start site (3-6). The cAMP response element (CRE) is critically
important for mediating both basal and cAMP-induced transcription (7,
8) and is located between In general transcription factor activation domains fall into four main
groups based on recurring motifs (12). NFI has a proline-rich
transactivation domain and a DNA binding domain that is highly
conserved among the various NFI isoforms but shows no homology to other
well characterized domains (13). Bandyopadhyay et al. (13)
showed that four of five cysteine residues located in the N-terminal
DNA binding domain of NFI are crucial for DNA binding. Crawford
et al. (14) grouped the four isoforms of NFI proteins into
two categories based on their ability to alter the level of basal
transcription from the PEPCK gene promoter. NFI-A and -B stimulate
basal transcription, whereas NFI-C and -X have mild inhibitory effects.
All four isoforms of NFI markedly inhibited the induction of
transcription from the PEPCK gene promoter caused by cAMP or by
transfection of the gene for the catalytic subunit of protein kinase A
(PKA-c) (14). Chaudhry et al. (15) used domain-swap
experiments to show that these different transactivation capabilities
of the NFI isoforms resided wholly in their C-terminal transactivation domains.
Many transcription factors are capable of interacting cooperatively.
The NFI cis-element on the PEPCK promoter is located immediately
adjacent to the CRE. Altering the relative positions of the NFI site
and CRE on the PEPCK gene promoter did not affect the ability of NFI to
abrogate CRE-mediated PKA-c induction of transcription in hepatoma
cells (14). Thus, the proximity of P1 and CRE sites in the PEPCK gene
promoter was not critical for the NFI effect. The ability of NFI to
block CRE-mediated stimuli such as that achieved by the co-transfection
of PKA-c, apparently without direct interaction between the proteins,
suggested that another signal transduction pathway must exist.
Recently we have studied the effects of the various isoforms of NFI on
cAMP induction of transcription of the PEPCK gene (14). The NFI site in
the PEPCK gene promoter contains the consensus NFI binding sequence
TTGGC(N)5GCCAA (16-18). There are four genes encoding NFI
proteins, all of which are expressed in the liver (19). Mutating the
NFI-binding site (P1) of the PEPCK gene promoter results in a marked
increase in the level of basal expression of a linked structural gene
(bGH) in the livers of transgenic mice and the premature expression of
bGH mRNA in the liver in utero, as compared with control
mice carrying a transgene with an intact PEPCK promoter (20). We
speculate that NFI serves the important function of maintaining a low
basal level of transcription of the PEPCK gene in the liver until
stimulation by hormones such as glucagon, glucocorticoids, and thyroid
hormone occurs. In the current study we confirm the function of NFI in
the control of PEPCK gene transcription, and we describe the key role
of CBP in coordinating not only the effects of NFI but other
transcription factors involved in determining the level of hepatic
PEPCK gene transcription.
Materials
[3H]Chloramphenicol (30-60 Ci/mmol),
[ Methods
In Vivo UV Photo-footprinting--
In vivo
photo-footprinting was carried out essentially as described by Becker
and Grossmann (21). Minimal deviation H4 hepatoma cells were grown to
70% confluence in 100-mm plates in a 1:1 mixture of Dulbecco's
modified Eagle's and Ham's F-12 media supplemented with 10% fetal
calf serum. Before hormone treatment, cells were grown for 20 h in
serum-free medium. Four hours prior to UV irradiation, hormones were
added to modulate endogenous PEPCK expression as follows: basal
(serum-free), stimulatory (0.5 mM Bt2cAMP, 1 mM theophylline and 1.0 µM dexamethasone), or
inhibitory (0.5 mM Bt2cAMP, 1 mM
theophylline, 1.0 µM dexamethasone, and 50 nM
insulin). The medium was then removed from the cells, and the cell
monolayer was irradiated with 254 nm UV light at 1200J/m2.
Genomic DNA was then harvested from the irradiated cells. An oligonucleotide primer specific to bases Construction of Chimeric Genes--
The details of the
construction of the chimeric PEPCK-CAT and the P1-CAT genes used in the
transfection experiments were outlined previously (22). Block mutations
were introduced into the PEPCK gene promoter at the P1 (NFI-binding
site) as described by Liu et al. (23). These mutations
eliminate binding of the respective cognate binding proteins to the
PEPCK gene promoter. NFI expression vectors were as described
previously (14).
Cell Culture--
HepG2 human hepatoma cells were purchased from
the American Type Culture Collection and grown in 100-mm tissue culture
plates in Dulbecco's minimal essential medium, supplemented with 50% F-12 nutrient mixture (Dulbecco's modified Eagle's medium/F-12). Cells were passaged at least four times prior to use and were allowed
to grow to 30-40% confluence prior to DNA transfection. The H4
hepatoma cells used for the in vivo footprinting study were
grown as described previously (24).
Assay of Transcription from the PEPCK Gene
Promoter--
Chimeric genes containing the PEPCK gene promoter,
modified as described above, were transfected into HepG2 cells using
calcium phosphate as described previously (7). In practice, three
replicate 100-mm plates of 30-40% confluent HepG2 cells were
transfected with 2.5 µg of 490-CAT. Other plasmids were
co-transfected into HepG2 cells as indicated under "Results."
Control cells were transfected with buffer alone. The cells were given
fresh medium at 24 h after transfection, harvested at 48 h,
and cell extracts prepared for the determination of CAT activity.
Preparation of Cell Extracts for the Determination of CAT
Activity--
Cells were harvested 48 h after transfection.
Plates were drained and cells were rinsed in 1× PBS buffer. Ice-cold
1× PBS (1 ml) was added to each plate, and cells were scraped free of the surface and transferred to a 1.5-ml microcentrifuge tube kept on
ice. Samples were centrifuged for 20 s at 10,000 rpm; the
supernatant was removed and the pellet resuspended in 250 mM Tris-Cl, at pH 7.8. Samples were subjected to three
freeze-thaw vortex cycles using ethanol/dry ice and a 37 °C water
bath and then centrifuged for 10 min at 10,000 rpm. The supernatant was
transferred to a new tube and placed in a 58 °C water bath for 10 min to denature inhibitors of CAT (25, 26). The samples were
centrifuged again for 10 min at 10,000 rpm, and the supernatant was
used to assay CAT activity using [3H] chloramphenicol,
butyryl-CoA, and the xylene phase extraction method (27). All
transfection studies were performed in duplicate or triplicate.
Differences between means of each treatment were tested for statistical
significance using Student's t test (one-tailed) (28).
Experiments were repeated at least twice with three values per
experiment. Values are expressed as the mean ± S.E. of the mean
for the number of determinations noted in the legends to the figures.
Mammalian Two-hybrid Assay for Direct Protein-Protein
Interaction--
Plasmids encoding the CREB and E1A binding domains of
CBP (amino acid residues 451-682 and 1678-2441, respectively) linked to amino acids 1-147 of Gal4 (the DNA binding domain) were gifts from
Dr. Richard Goodman, Vollum Institute, Portland, OR. For brevity these
plasmids have been renamed pBCBP451 and pBCBP1678, the initial "B"
indicating its role of "Binding" the luciferase promoter. Two other
regions of CBP encompassing the steroid binding domain (amino acids
1-299) and a more restricted E1A binding domain (amino acids
1678-1843) were also subcloned into the pBIND vector supplied as a
part of the mammalian two-hybrid kit (Promega). The primer pairs
used for these were
5'-GCTAGGATCCATGGCCGAGAACTTGCTGC-3'/5'-GCTAGCTAGCGGCCGCTTTGCTGGCTAACTGGGG-3' for the steroid binding domain and
5'-GCTAGGATCCGAATTCTCTTCCTTACGCCGC-3'/5'-GCTAGCGGCCGCGAGGCAGAAGGGCACAGGGC-3' for the restricted E1A binding domain. The region of the pHANFI-C plasmid encoding the C-terminal transactivation domain (amino acids
220-499) was amplified using these polymerase chain reaction primers,
5'-GCTAGGATCCTTTGTCACGTCAGGTGTGTTC-3', in which the added BamHI site is underlined and immediately precedes the codon
for amino acid 220 of NFI-C. The downstream primer
5'-GCTAGCGGCCGCCTAATCCCACAAAGGGACTGTCTG-3' has an
introduced NotI site (underlined) immediately following the
stop codon of NFI-C. The amplified fragment was subcloned into the
NotI/BamHI-digested pACT vector supplied as part
of the Checkmate® mammalian two-hybrid kit (Promega). The resultant
plasmid was named pAC220, the initial "A" indicating its putative
"Activator" role in the two-hybrid system. The luciferase reporter
gene contained five tandem repeats of the Gal4-binding site. The
plasmids pBCBP451, pBCBP1678, and pAC200 were then used in
co-transfection experiments to test for the presence of an interaction
with Gal4-CBP fragment fusion proteins, along with the pertinent
controls. A base line was generated by co-transfecting HepG2 cells with
a plasmid, pBind, that contains the Gal4 DNA binding domain together
with pACT containing the VP16 transactivation domain and a luciferase
reporter gene (pG5Luc).
UV Photo-footprinting of the PEPCK Gene Promoter in Vivo--
DNA
structural changes and the binding of sequence-specific binding
proteins to DNA may be detected by comparing the rate of UV
photoproduct formation in naked DNA irradiated in vitro with
the rate of UV photoproduct formation in DNA irradiated in vivo. In order to determine the physiological circumstances under which the NFI-binding sites, CRE and TATA box on the PEPCK gene promoter (see Fig. 1A for
organization of the promoter) were occupied by transcription factors,
we carried out in vivo irradiation. UV photo-footprinting of
the endogenous PEPCK gene was carried out using H4 cells treated in a
manner calculated to provide widely divergent transcriptional responses
(Fig. 1B). These cells were maintained for 3 h in a
serum-free (basal conditions) medium or were treated with a combination
of Bt2cAMP and dexamethasone to stimulate PEPCK gene
transcription or with a combination of three hormones
(Bt2cAMP, dexamethasone, and insulin). Insulin inhibits PEPCK gene transcription (29). Cells treated as above were subjected to
UV irradiation for the time indicated in Fig. 1B in order to introduce covalent bonding between consecutive pyrimidine moieties in
the genomic DNA. Proteins bound to the DNA can act in a manner that
either magnifies the effect of UV irradiation or deflects and thereby
decreases the rate at which inter-pyrimidine covalent bonds are formed.
Taq DNA polymerases used in subsequent primer extension
analyses have difficulty reading through the modified pyrimidines
leading to pausing and banding patterns that correlate with the
location of pyrimidines (see Ref. 21 for a detailed description of the
method and its limitations). Proteins bound to the DNA increase or
decrease the banding patterns and thereby provide a unique footprint.
Several significant differences in UV photoproduct signal intensity are
evident between H4 DNA irradiated in vivo under different
PEPCK transcription states (Fig. 1B, lanes 1-3) and H4
genomic DNA irradiated in vitro (lanes 4 and
5). The banding patterns in the P1, CRE, and TATA box sites
in the PEPCK gene promoter, under a variety of hormonal treatment
conditions, are different from the patterns seen in the control cells
(lanes 4 and 5). Four changes of footprint map to
the TATA box and early RNA coding regions. These have been designated
with plus symbols next to the vertical bar and
are seen only in the presence of the Bt2cAMP and
dexamethasone. These bands are consistent with initiation site
occupancy by the TATA box binding factor and RNA polymerase,
respectively. The rate of UV photoproduct formation at the CRE and
NFI-binding site (bands e and f in lanes
1-3) is markedly and reproducibly different in vivo
from the corresponding DNA sites irradiated in vitro
(bands e and f in lanes 4 and 5). We
interpret the differences in the rate of UV photoproduct formation within the NFI-binding site/CRE in vivo, relative to the
naked DNA, to signify that these promoter elements are continuously occupied regardless of the transcriptional state of the PEPCK gene.
Faber et al. (30) have reported a similar finding using hepatoma cells treated with Bt2cAMP. Since the NFI-binding
site appears to be continuously occupied by proteins, modulation of transcription caused by NFI may reflect the binding of different NFI
isoforms during different physiological stimuli.
A Co-activator Molecule Is Involved in the Effect of NFI on PEPCK
Transcription--
The extent to which the P1 site on the PEPCK
promoter is necessary for the NFI effects was determined by
transfecting a PEPCK-CAT reporter gene containing a block mutation in
the P1 site ("P1-CAT") into HepG2 cells in the presence and absence
of the various NFI-A and NFI-C isoforms (Fig.
2). NFI-A induced transcription from the
intact PEPCK promoter 6-fold but increased transcription from the PEPCK
gene promoter with a mutation in the P1 site only 2.5-fold (Fig.
2A). Similar results were observed for NFI-C (Fig.
2B). NFI-C blocked PKA-c-induced transcription from the
intact PEPCK gene promoter by 60%, whereas PKA induction of the
chimeric PEPCK-CAT gene with a mutation in the P1 site was inhibited by
only 25%. Thus, removal of the NFI-binding site clearly reduced the
effectiveness of NFI in altering PEPCK gene. There are two possible
explanations for the partial effects observed in the absence of the NFI
site: (i) NFI may be promiscuous in its binding and have other weaker binding sites on the promoter, and (ii) NFI may be capable of direct
interaction with components of the initiation complex. Mutant
transcription factors that cannot dimerize or bind DNA have been shown
to be capable of activating or repressing transcription (31, 32). One
candidate for mediating these site-independent effects is a
co-activator molecule such as the CBP/p300 family of proteins. Members
of this family have multiple binding domains and have been implicated
as co-activators in the signal transduction of members of the steroid
receptor superfamily as well as members of the leucine zipper group of
transcription factor such as c-Fos and c-Jun (33). Although the
transcriptional regulation of the PEPCK gene promoter has never been
linked directly to CBP/p300, such an interaction is likely since PEPCK
gene expression is induced by thyroid hormone (34), glucocorticoids
(5), cAMP (9, 7, 8), and c-Jun (35), all of which are known to bind to
CBP. In addition, the adenovirus early protein, E1A, which binds to
CBP, markedly inhibits cAMP-induced transcription from the PEPCK gene
promoter in hepatoma cells (36). However there has been no report of a
direct interaction of members of the NFI family of transcription
factors with CBP.
Co-transfection of increasing amounts of an expression vector for CBP
caused a linear, 7-fold increase in expression of the PEPCK-CAT gene
(Fig. 3A). Further increasing
the amount of transfected CBP plasmid caused a dramatic decrease in CAT
activity. Another indication that CBP coordinates the transduction of a
stimulus to the initiation complex on the PEPCK gene promoter is its
susceptibility to the action of E1A, which binds CBP at the E1A binding
domain located near the C-terminal of CBP (37). E1A has only a marginal effect on the low level of basal transcription from the PEPCK gene
promoter but can markedly inhibit the induction of transcription by
PKA-c (Fig. 3B). An E1A mutant ( Protein-Protein Interaction Assays Show That the Transactivation
Domain of NFI-C Can Interact with the CREB-binding Domain of
CBP--
Chaudhry et al. (15) used domain swap experiments
to show that the transactivation domain of NFI isoforms resides wholly in the C-terminal regions. The transactivation domain of NFI-C in
particular appears to be the most active in transfection studies using
the promoters for both the PEPCK gene (14) and the mouse mammary tumor
virus.2 For these reasons we
used a mammalian two-hybrid assay to test whether the NFI-C
transactivation domain could interact with different regions of CBP
protein. We used plasmids encoding fusion proteins comprising the Gal4
DNA binding domain linked to four different CBP domains representing
amino acids 1-299 (pBCBP299), 451-682 (pBCBP451), 1678-1843
(pBCBP1678), and 1678-2441 (pCBP2441). In addition, the
transactivation domain of NFI-C comprising amino acids 220-499 was
subcloned into the pACT vector containing the VP16 transactivation
domain (pAC220). The two plasmids were then used in standard two-hybrid
assays, the results of which may be seen in Fig.
4.
Basal activity (1st bar) was that seen when the reporter
gene was co-transfected with the pACT and pBIND vectors without
inserts. The levels of activity observed for the two fusion genes in
combination were corrected for increases in background caused by
individual components, and the results are presented as fold induction
over basal. The 2nd bar indicates the result when positive
control fusion genes supplied by the manufacturer of the kit (pACTMyo and pBindId) were used. There was a robust interaction between pAC220
and pBCBP451 which codes for the CREB binding domain of CBP (100-fold
over background), with virtually no interaction occurring with the E1A
domain of CBP. These experiments were repeated in NIH 3T3 cells with
very similar results and suggest that the effects of NFI may be
mediated by competition for the CREB binding domain of CBP.
CBP Fragments Containing Binding Sites for Specific Transcription
Factors Prevent Modulation of Transcription from the PEPCK Gene
Promoter--
Expression vectors coding for fragments of CBP were used
to examine whether they alter the ability of well characterized
transcription factors to modulate transcription from the PEPCK gene
promoter. The effect of CREB and E1A was determined because the CBP
domains involved in the signal transduction of these two transcription factors are well characterized. CREB binds to a region between amino
acid residues 451 and 562, whereas E1A binds to a region between amino
acids 1805 and 1854 (37), and both proteins are known to alter
transcription from the PEPCK gene promoter. Hep G2 cells were
co-transfected with plasmids for PEPCK-CAT and E1A in the presence and
absence of expression vector CBP451, which corresponds to the CREB
binding domain of CBP (amino acid residues 451-682) or an expression
vector CBP1678, which encompasses the E1A binding domain of CBP (amino
acid residues 1678-2441). CBP451 and CBP1678 stimulated expression of
the PEPCK-CAT gene 5- and 12-fold, respectively (Fig.
5A, lanes 2 and 5).
When E1A was co-transfected along with the individual CBP fragments,
the results differed. E1A caused a substantial inhibition of
transcription from the PEPCK gene promoter in the presence of CBP451
(compare Fig. 5A, lanes 2 and 3) but had no
effect on transcription in the presence of CBP1678 (the CBP fragment
that contains the E1A binding domain; compare Fig. 5A, lanes
5 and 6). This effect was observed even when the amount
of transfected E1A plasmid used in the transfection was increased
4-fold (Fig. 5A, lanes 4 and 7).
Complementary results were observed when a plasmid coding for CREB was
transfected into HepG2 cells along with vectors encoding the different
binding domains of CBP. CREB is a transcription factor that binds to
the CRE of the PEPCK gene promoter and interacts with the CREB binding
of CBP (Fig. 5B). When co-transfected with the PEPCK-CAT
gene, CBP451 and CBP1678 caused a similar, approximately 8-fold
increase in CAT activity. When CREB was co-transfected with CBP451 (the
fragment that contains the CREB-binding domain of CBP), no effect was
seen (compare Fig. 5B, lanes 3 and 4). However,
when CREB was co-transfected with CBP1678, the CAT activity dropped
from that seen with CBP1678 alone to a level almost identical to that
observed with CREB alone (compare Fig. 5B, lanes 5, 6, and
2). CREB alone caused a modest increase in PEPCK-CAT
expression (lane 2). CREB produces approximately this same
level of activity in the presence of CBP1678 but not in the presence of
the CBP fragment which contains the CREB-binding site (CBP451). Thus, these two fragments of CBP may be used to distinguish which region of
the CBP molecule is involved in the transduction of signal from these
two well characterized transcriptional regulatory factors. One model
consistent with all these data is that the protein products of the
co-transfected CBP fragment expression vectors sequester transcription
factors that normally bind to endogenous CBP. When CBP451 was
co-transfected alone, we observed a strong increase in transcription.
The two simplest explanations for this is that CBP451 transactivated
the PEPCK-CAT reporter gene or that it removed endogenous inhibitors by
sequestration. Since CBP451 lacks a transactivation domain, we suggest
the latter the more likely to be the case. Hence, the protein products
of overexpressed CBP451 most likely sequestered CREB and prevented the
modulation of the PEPCK-CAT transcription. The protein products of
CBP451 do not bind E1A and so, in the presence of overexpressed CBP451,
E1A still functioned to block transcription from the PEPCK gene promoter.
NFI Isoforms Are Prevented from Acting on Transcription from the
PEPCK Gene Promoter by the Protein Product of Overexpressed CBP451 That
Codes for the CREB Binding Domain of CBP--
In parallel experiments,
NFI-A or NFI-C were co-transfected with PEPCK-CAT into HepG2 cells in
the presence and absence of plasmids CBP451 and CBP1678. NFI-A is a
known stimulator of PEPCK-CAT and when co-transfected along with
PEPCK-CAT produced a robust 7-fold stimulation of transcription (Fig.
6, lane 2). Co-transfection of
NFI-A together with the CREB binding domain of CBP (CBP451) did not
increase the level of CAT expression beyond that found with CBP451
alone (compare Fig. 6, lanes 3 and 4), whereas
co-transfection of NFI-A with the E1A binding domain of CBP (CBP1678)
resulted in a marked increase in expression (compare Fig. 6,
lanes 5 and 6). Clearly NFI-A could function in
the presence of CBP1678 but not in the presence of CBP451. Similar
results were observed for NFI-C that is an inhibitor of PEPCK-CAT.
Transcription of the PEPCK-CAT gene was inhibited by NFI-C in the
presence of the overexpressed protein products of CBP1678 (compare Fig.
6, lanes 11 and 12), but no inhibitory effect was
observed in the presence of CBP451 (compare Fig. 6, lanes 9 and 10). These results are consistent with our model that
the CBP451 fragment of CBP sequesters NFI and suggest that the CREB
binding region of CBP may be responsible for relaying the NFI signal to
the initiation complex to alter transcription from the PEPCK gene
promoter.
Transcription Factors Compete for Binding Domains on CBP--
If
the effect of NFI is mediated through the CREB binding domain of CBP,
it would predict that the isoforms of NFI should interact cooperatively
with some stimuli and not with others, based on which CBP domains relay
their signal to the transcription initiation complex. To examine this
issue, co-transfection experiments were designed to test whether CBP
could coordinate two diverse stimuli simultaneously to regulate
transcription from the PEPCK promoter. We examined the interaction
between NFI-A, which we assume also binds to the CREB binding domain of
CBP, and members of the superfamily of steroid receptors that are known
to operate via the N-terminal 117 residues of CBP. In two separate
series of experiments we co-transfected HepG2 cells with NFI-A along with expression vectors for the ligand-activated human glucocorticoid or thyroid hormone receptors. Dexamethasone (10 nM) and
triiodothyronine (1.0 µM) were added as ligands for their
respective receptors, and the level of CAT activity in the HepG2 cells
was determined 48 h later. The results were similar for both
steroid and thyroid hormone stimulation of transcription from the PEPCK
gene promoter. Thyroid hormone stimulated transcription from the PEPCK
promoter approximately 8-fold (Fig. 7,
lane 3) whereas NFI-A alone produced a 6-fold stimulation
(lane 2). NFI-A in combination with thyroid hormone produced
an additive effect (approximately 20-fold; compare Fig. 7, lanes
2-4). Similar results were observed in experiments in which NFI-A
and dexamethasone were combined. Individually they stimulated PEPCK-CAT
transcription by 6- and 3-fold respectively; in combination their
effect on PEPCK-CAT activity in the HepG2 was additive (approximately
10-fold; compare Fig. 7, lanes 2, 5, and 6).
In a second series of experiments, NFI-A was co-transfected with
PEPCK-CAT into HEP G2 cells in the presence or absence of expression
vectors for CREB or PKA-c. Individually, NFI-A and CREB increased
transcription from the PEPCK-CAT gene 8- and 3-fold, respectively (Fig.
8, lanes 2 and 3).
When NFI-A and CREB were co-transfected together, the increase in
transcription was not additive but was not significantly different from
that found with NFI-A alone (Fig. 8, lane 4). A similar
pattern was observed when NFI-A was co-transfected along with an
expression vector PKA-c, a potent stimulator of PEPCK-CAT transcription
whose actions are mediated through the CREB binding domain of CBP.
PKA-c alone produced a 10-fold induction (Fig. 8, lane 5).
When PKA-c was co-transfected with NFI-A, we observed again that the
resulting induction was not significantly different from that produced
by NFI-A alone (Fig. 8, lane 6). It thus appears that NFI-A
blocks PKA-c and CREB-mediated activation of transcription from the
PEPCK gene promoter by successfully competing for the CREB binding
domain of CBP. These studies demonstrate that CBP has the capability of
coordinating the transcriptional response of the PEPCK-CAT gene to
multiple stimuli simultaneously.
The Role of NFI in Regulating PEPCK Gene Transcription--
The
family of four NFI genes give rise to four distinct isoforms (NFI-A,-B,
-C, and -X (38, 39), each of which may give rise to splice
variants by post-transcriptional processing (16, 38, 40). NFI isoforms
bind as dimers (41), raising the possibility of subtle and complex
interactions with DNA through heterodimerization. NFI was initially
studied for its role in the replication of adenovirus (42). Later it
was reported to be important for transcription of mouse mammary tumor
virus (43). More recently, NFI has been implicated in transcriptional
control of genes of physiological importance. For example, NFI is
involved in glucocorticoid receptor-mediated transcriptional response
of the gene for mouse mammary tumor virus (44) and aspartate
aminotransferase (45). It is also a negative modulator of transcription
of the gene for L-type pyruvate kinase (46).
The PEPCK gene promoter contains an NFI-binding element that maps
immediately adjacent to the CRE (3). Mutating this element resulted in
a marked increase in basal gene expression in the livers of transgenic
mice that contained a chimeric PEPCK-bovine growth hormone (PEPCK-bGH)
gene (20). In addition, there was premature expression of the transgene
in the livers of 19-day-old fetal mice in utero (20).
Recently, Crawford et al. (14) demonstrated that expressing
any of the four isoforms of NFI in hepatoma cells inhibits the normally
robust induction of transcription from the PEPCK gene promoter caused
by cAMP. Understanding the role of NFI in the regulation of PEPCK gene
transcription is complicated by the fact that the major isoforms of NFI
exert different effects on the level of basal transcription (NFI-A and
NFI-B increase basal transcription and NFI-C and NFI-X decrease this
process). The results of the present study show that NFI does not alter PEPCK gene transcription by binding to an unoccupied site on the PEPCK
gene promoter. This suggests that NFI exerts its effect by differential
expression of one of the four NFI genes in response to different
stimuli, and differences in modulation of PEPCK expression would result
from an exchange of NFI occupying the P1 site. Alternatively, the NFI
bound to the P1 site could be covalently modified by phosphorylation or
glycosylation (47, 48) which would alter its ability to affect gene
transcription. Toohey et al. (49) showed that although NFI
binding to the mouse mammary tumor virus promoter sequences could be
enhanced by mutating specific bases, the effect of NFI on
steroid-induced transcription from the mouse mammary tumor virus gene
promoter did not change. This suggests that affinity to the NFI-binding
site is not the principal determinant of transcriptional activation by
NFI protein.
From the results of Faber et al. (30) and those published in
this paper (see Fig. 1B), it seems likely that both the CRE and NFI sites of the PEPCK gene promoter are occupied by transcription factors even in the absence of cAMP or glucocorticoid stimulation of
transcription. Christ et al. (50) reported similar results that showed complex formation between protein nuclear extracts and an
oligonucleotide containing an NFI-binding site was not altered when
cells were pretreated with glucagon, which stimulates PEPCK gene
transcription. From the available data it seems reasonable to assume
that NFI acts to maintain a low level of basal transcription from the
PEPCK promoter and that it interacts with protein(s) binding to the CRE
to regulate PEPCK gene transcription. The proximity of the NFI-binding
site and the CRE on the PEPCK gene promoter suggests that there is a
functional interaction between NFI and the protein(s) that bind to the
CRE. These include CREB, C/EBP, and Jun/Jun homodimers, all of which
have been shown to bind to the CRE of the PEPCK gene promoter. However,
Crawford et al. (14) demonstrated that the proximity of the
CRE to the NFI binding domain was not required for the negative effect
of NFI on transcription from the PEPCK gene promoter. In the present
paper we show that the NFI-binding site is not required for NFI-C to
block cAMP induction of PEPCK gene transcription in transfected HepG2
cells. This suggests that NFI interacts with a co-activator protein,
such as CBP/p300, to block the normal induction of PEPCK gene
transcription caused by cAMP.
The Role of CBP in the Regulation of PEPCK Gene
Transcription--
A role for CBP/p300 in the regulation of PEPCK gene
transcription was suggested by a previous report by Kalvakolanu
et al. (36) which demonstrated that the adenoviral early
protein, E1A, totally inhibited the cAMP induction of PEPCK gene
transcription in hepatoma cells, even though E1A itself does not bind
to the PEPCK promoter. Subsequently it was established that the
mechanism of E1A action involves its binding to CBP/p300. CBP/p300 has
a number of discrete functions in regulating gene transcription. It is
considered by some to be a subunit of RNA polymerase II and can
integrate regulatory signals from transcription factors and chromatin
by virtue of its intrinsic acetyltransferase activity (51). CBP/p300
can itself be phosphorylated on serine and threonine residues
during retinoic acid-induced differentiation of F9 embryonal carcinoma cells (52), suggesting that phosphorylation of the protein by a cyclin-dependent kinase can control its
transcriptional activity. At the present time there are more than 30 proteins known to bind to CBP/p300, and the list is growing (33).
Nakajima et al. (53) have suggested a model in which the
cAMP regulatory element-binding protein (CREB) is phosphorylated by PKA
and then binds to CBP resulting in an association of CBP/p300 with
TFIIB and RNA polymerase II.
The PEPCK gene promoter has binding domains for a large variety of
transcription factors so that it is critical that the effect of these
factors on transcription be integrated and coordinated with the
transcriptional machinery at the TATA box. Based on the results of the
current paper, we propose that CBP controls the interaction of the
various effector molecules and acts to regulate the rate of PEPCK gene
transcription. CBP has specific binding sites for many of the proteins
that are known to be involved in the control of PEPCK gene
transcription, including thyroid hormone and glucocorticoid receptors,
CREB, Jun, and C/EBP (33). There are several lines of evidence to
support a role for CBP in the regulation of PEPCK gene transcription.
First, CBP in the absence of any other extrinsic factors can stimulate
transcription of a chimeric PEPCK-CAT gene (Fig. 5). Second, E1A
strongly inhibits cAMP-induced transcription from the PEPCK gene
promoter by binding to CBP/p300. In support of the effects of E1A on
PEPCK gene transcription is the report of Arany et al. (37)
demonstrating a similar effect of E1A on cAMP-induced transcription
from the somatostatin gene promoter. Third, transcription factors such
as C/EBP, which play a key role in the response of the PEPCK gene
promoter to cAMP (54-56), also bind to CBP/p300. Recently, Mink
et al. (57) reported that the N terminus of C/EBP interacts
with the E1A binding domain of CBP, suggesting that the cAMP-induced
effect of C/EBP isoforms on PEPCK gene transcription may be exerted at
the E1A domain of CBP. A direct competition between C/EBP and E1A for
binding to the same domain on CBP could explain the mechanism of the
dominant negative effect of E1A on cAMP-induced PEPCK gene
transcription (36). This possibility is reinforced by the fact that E1A
totally inhibits the stimulatory effects of Jun/Jun homodimers on PEPCK gene transcription (36); Jun also binds to the E1A domain of CBP,
suggesting a common mechanism.
A complicating factor in this interpretation of the action of E1A on
PEPCK gene expression is a paper by Klemm et al. (58), which
reported that infection of HepG2 cells with an adenovirus expressing
E1A resulted in an increase rather than a decrease in transcription
from the PEPCK gene promoter. It is not clear why there is such a
marked difference in the response of PEPCK gene transcription in the
two studies. One notable difference is that Klemm et al.
(58) used whole virus as a source of E1A and reported viral titers of
100-200 infectious particles per cell. Their cells were maintained for
20 h after adenoviral infection, so that viral-induced
transcription factors could participate in the regulation of PEPCK gene
transcription. Kalvakalanu et al. (36) infected HepG2 cells
with adenovirus but isolated nuclei for transcription measurements
within 2.5 h. In addition Kalvakalanu et al. (36, 58)
determined the direct effect of E1A, introduced into the HepG2 cells
via an expression plasmid, on transcription from the PEPCK gene
promoter. The most likely explanation for the differences between the
two studies is that the long term expression of the adenoviral genes in
hepatoma cells could alter PEPCK gene expression in an indirect manner
due to the expression of viral genes in the late stage of infection.
Control of PEPCK Gene Transcription Requires Coordination of the
Activity of Transcription Factors through Their Interaction with
CBP--
The integration of the numerous signals that control
transcription of the gene for PEPCK is critical to ensure the
appropriate expression of the gene in response to physiological
stimuli. CBP is an ideal co-activator, since it contains binding sites
for multiple transcription factors, has intrinsic histone acetylase activity (51), and interacts with RNA polymerase (53). Shikama et
al. (33) have suggested that "CBP/p300 may provide a
transcriptional scaffold that enables regulatory molecules, such as
kinases, acetyltransferases, and perhaps other enzymatic activities, to
be recruited to and assembled at relevant sites for transcriptional
activity." Fig. 9 presents our model
for the proposed interaction of CBP and the transcription factors that
control the expression of the gene for PEPCK. NFI binds to the
CREB-binding site of CBP (Figs. 4 and 5), whereas C/EBP, which is
critical for the cAMP regulation of PEPCK gene transcription, binds to
the E1A site of CBP (57). The transcriptional activation domain of CBP
is brought into contact with the TATA binding factors of the PEPCK gene
promoter by first binding to transcription factors such as C/EBP; this
in turn aligns CBP properly on the promoter to activate gene
transcription. There are a number of lines of evidence that indicate
that C/EBP rather than CREB is involved in the hepatic cAMP-induced
transcription of the PEPCK gene (these are reviewed in detail in Ref.
59). However, since both CREB and C/EBP bind to the CRE of the PEPCK gene promoter, it is also possible that CREB mediates the cAMP effect
on PEPCK gene transcription.
Since the CRE and NFI sites of the PEPCK gene promoter are always
occupied by transcription factors (30), it is likely that CBP is
recruited to bind to C/EBP, most likely through the E1A or CREB binding
domains of CBP. Cyclic AMP control of PEPCK gene transcription would
involve PKA-c phosphorylation of C/EBP (or CREB), resulting in an
interaction of these transcription factors and CBP, aligning it
appropriately to allow for interaction with RNA polymerase II and
increased gene transcription. According to this model (Fig. 9), NFI
blocks cAMP-induced transcription from the PEPCK gene promoter by
binding to the CREB site on CBP and preventing the appropriate
alignment of CBP or by blocking the binding of C/EBP (or CREB) to CBP.
The strong negative effect of E1A on PEPCK gene transcription is a
prototype of the negative control exerted by proteins that bind to
critical sites of CBP. Our model predicts that intermediate molecules
activated in the insulin signaling cascade will act by blocking the
interaction of CBP with critical proteins bound to the PEPCK promoter,
thus interfering with the appropriate alignment of CBP with the
transcriptional machinery. The finding that the mitogen-regulated S6
kinase pp90Rsk, which binds to the E1A domain of CBP, is
responsible for inactivating the cAMP-inducible gene transcription
caused by CREB (53) supports a model in which an intermediate in a
signaling cascade is able to interact with CBP to control gene
transcription. CBP can also coordinate the effects of more than one
transcription factor simultaneously and can distinguish between a
positive and a negative effector molecule. As an example, NFI-A, which
inhibits cAMP-induced transcription from the PEPCK gene promoter,
causes an additive increase in PEPCK gene transcription in the presence
of glucocorticoids (Fig. 7). This indicates that CBP can coordinate a
response to two signals simultaneously and has a high degree of
selectivity in orchestrating the response of the PEPCK gene promoter to
various signals. Further work is required to more fully delineate the
role played by CBP in coordinating the dietary and hormonal control of
PEPCK gene transcription.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
90 and
83 (9, 10) on the PEPCK gene
promoter. A glucocorticoid regulatory unit, which contains an insulin
response element and two glucocorticoid response elements, is also
located from
415 to
440 in the promoter (5, 11). In this study we
examine how nuclear factor I (NFI) transduces its signal to the
initiation complex and how it interacts with other transcription factors, either competitively or cooperatively.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]ATP (3,000 Ci/mmol), and [32P]dCTP
(3000Ci/mmol) were purchased from NEN Life Science Products. Bt2cAMP and butyryl-coenzyme A were bought from Sigma.
HepG2 cells were originally purchased from ATCC (Rockville, MD). The
Checkmate® mammalian two-hybrid assay kit was purchased from the
Promega Corp. (Madison, WI). The expression vector for catalytic
subunit of protein kinase A was a gift from Dr. Masa-Aki Muramatsu.
Dulbecco's modified Eagle's medium/Ham's F-12 cell culture medium
and fetal calf serum were from Life Technologies Inc. All other
reagents used in this study were of the highest quality obtainable. All expression vectors for the NFI proteins were the same as those used in
an earlier study (19) and express murine proteins homologous to chicken
NFI-A1.1, NFI-B2, NFI-C2, and NFI-X2 (39). The expression vectors for
the full-length CBP and the fragments of CBP was generous gifts from
Dr. Richard Goodman, Vollum Institute, Portland, OR.
399 to
341 of the PEPCK gene promoter was labeled to high specific activity with
32P, hybridized to the endogenous PEPCK gene, and extended
using Taq DNA polymerase at 74 °C. Synthesis of the
complementary DNA by Taq DNA polymerase is terminated one
nucleotide before sites of photo-damage on the template DNA. The
extended products were subjected to electrophoresis on an 8%
polyacrylamide sequencing gel. Sites of photoproduct formation were
mapped against Sanger di-deoxy sequence patterns in parallel lanes.
Non-irradiated genomic DNA from H4 hepatoma cells was analyzed as a
control to display sequence-dependent extension artifacts.
Signals were visualized using a PhosphorImager®. Enhanced bands
(relative to those found in the control DNA) are indicative of an
increased rate of photoproduct formation, whereas a weaker band is
indicative of a decreased rate of photoproduct formation. Proteins
bound to DNA may lead to either increased or decreased rates of
photoproduct formation depending upon the properties of the protein
binding to the DNA. Changes in the rate of UV photoproduct formation in
the genomic DNA of the treated cells are taken as evidence of
protein-DNA interaction. Phenylalanine, tyrosine, and tryptophan absorb
relatively weakly at 254 nm. The changes in band intensity is likely to
be caused by a DNA structural distortion brought about by the binding of a protein at that locale, which forces photosensitive bases into a
geometry less favorable to the formation of photoproduct. As a negative
control, genomic DNA was recovered from untreated H4 cells, purified,
and irradiated for similar periods with UV light. The banding pattern
on the control DNA is used for comparison with the banding pattern on
the stimulated DNA.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
In vivo footprinting of the PEPCK
gene promoter. A, the arrangement of selected
regulatory elements in the PEPCK gene promoter ( 490 to +73).
B, UV photoreactivity of the proximal region (
100 to +73)
of the PEPCK gene promoter. H4 hepatoma cells were preconditioned in
serum-free (SF) medium for 20 h before treatment for
4 h with the indicated hormones in serum-free medium
(CD, 0.5 mM Bt2cAMP, 1 mM theophylline, 1 µM dexamethasone;
CDI, CD + 50 nM insulin). Plates were drained,
and the cells were irradiated with a dose of 254 nm of UV light at
1,200 watts/m2 254 nm of UV light. Genomic DNA was
isolated, heat-denatured, and annealed with a
[
-32P]ATP]labeled 59-base oligonucleotide primer
specific to the PEPCK gene promoter. Primers were extended using
Taq DNA polymerase, and primer extension analysis was
performed by electrophoresis of the extended products on an 8%
polyacrylamide gel electrophoresis sequencing gel. The gel was dried
and placed in a PhosphorImager screen overnight. Abbreviations used are
as follows: GRE, glucocorticoid response element;
TRE, thyroid hormone response element; NFI,
nuclear factor I; CRE, cyclic AMP response element; plus and
minus denote an increase or decrease of band intensity.
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Fig. 2.
The effects of NFI-A and NFI-C on basal and
PKA-c-induced transcription from the PEPCK gene promoter in the
presence and absence of the NFI-binding site. A, HepG2
cells were grown to between 45 and 60% confluence on 100-mm tissue
culture plates and were co-transfected with 2.5 µg of a chimeric
PEPCK-CAT gene which contained either the intact PEPCK gene promoter
(490-CAT) or the promoter with a mutation in the NFI-binding site
(P1-CAT) together with 0.5 µg of NFI-A. Cells were harvested 48 h later, lysed, and assayed for CAT activity as described under
"Experimental Procedures." B, HepG2 cells were
co-transfected with 2.5 µg of either 490-CAT or P1-CAT along with 2.5 µg of an expression vector for PKA-c and/or NFI-C (0.25 µg per
plate). All values for CAT activity were normalized for protein content
and were expressed as the mean ± S.E. for 2-3 replicates.
2-36), defective in its
ability to bind CBP by virtue of a mutation in its N-terminal region, did not block the expected stimulation of expression of the PEPCK-CAT gene by PKA-c (Fig. 3B, inset).
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Fig. 3.
CBP is involved in coordination transcription
from the PEPCK promoter. A, increasing amounts of a CBP
expression vector (0, 2.5, 7.5, 12.5, 25, 37.5, and 50 µg per 100-mm
plate) were transfected into HepG2 cells grown under basal conditions
along with 2.5 µg of PEPCK-CAT. Cells were harvested 48 h later
and assayed for CAT activity. Relative CAT activity is expressed as the
fold induction caused by CBP over the basal CAT activity in the absence
of CBP. B, HepG2 cells were co-transfected with 2.5 µg of
PEPCK-CAT along with 2.5 µg of an expression vector for the catalytic
subunit of PKA-c. An E1A expression vector was also co-transfected at
1.0 µg per plate Inset, the effect of a mutation in the
CBP-binding site of E1A on its ability to alter PKA-c induction from
the PEPCK promoter. All values for CAT activity were normalized for
protein content and were expressed as the mean ± S.E. for 2-3
replicates.
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Fig. 4.
Determination of the direct interaction of
the NFI-C with specific domains of CBP using the mammalian two-hybrid
system. Basal activity was established by co-transfecting pACT and
pBIND vectors (without inserts) with the luciferase reporter gene
(pG5Luc). Four domains of CBP were linked to the Gal4 DNA binding
domain to generate plasmids pBCBP299, pBCBP451, pBCBP1678, and
pBCBP2441. The transactivation region of the expression vector for
NFI-C encoding amino acids 220-499 was subcloned into pACT to generate
pAC220. HepG2 cells were grown to approximately 40% confluence in
6-well plates and transfected with a total of 1.8 µg of DNA using
Lipofectamine Plus®. Equal amounts (0.6 µg) of reporter, activator,
and binder plasmids were used for transfection. The plasmids containing
the various regions of CBP fused to the Gal4 DNA binding domain were
co-transfected individually with pACT to assess the effect of the
individual CBP fusion genes on background activity. Similarly, pBIND
was co-transfected with pAC220 to assess the effect of the NFI-C fusion
on the basal level of luciferase activity. These background activities
were used to correct the activity levels observed when each CBP fusion
was co-transfected with pAC220. The results are presented as fold
activation of transcription of the pG5Luc gene over base
line.
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Fig. 5.
The ability of E1A to repress PEPCK
transcription in the presence of specific fragments of CBP.
A, HepG2 cells were co-transfected with PEPCK-CAT (2.5 µg)
together with 20 µg per plate of expression vector CBP451 (containing
the CREB binding domain of CBP) or CBP1678 (containing the E1A binding
domain of CBP) in the presence or absence of an expression vector for
E1A (0.05 and 0.2 µg per plate). Cells were later assayed for CAT
activity as described under "Experimental Procedures."
B, the effect of CREB on the rate of transcription of
PEPCK-CAT in the presence of various fragments of CBP was determined.
The experimental design was the same as shown in A except
that a plasmid expression vector for CREB (2.5 µg per plate) was
substituted for that of E1A. All values for CAT activity were
normalized for protein content and expressed as the mean ± S.E.
for 3 replicates. Results were analyzed using a one-tailed t
test and were significant at p 0.05.
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Fig. 6.
The effects of NFI-A and NFI-C on PEPCK-CAT
transcription in the presence of specific fragments of CBP.
A, an expression vector for NFI-A (2.5 µg per plate) was
tested for its ability to stimulate transcription of the PEPCK-CAT gene
(2.5 µg) in the presence of 20 µg per plate of expression vector
CBP451 (containing the CREB binding domain of CBP) or CBP1678
(containing the E1A binding domain of CBP). Cells were later assayed
for CAT activity as described under "Experimental Procedures."
B, an expression vector for NFI-C was tested for its ability
to inhibit PEPCK-CAT transcription in the presence of CBP protein
fragments. The amount of plasmids transfected were the same as in
A with NFI-C substituted for NFI-A. Values for CAT activity
were normalized for protein content and expressed as the mean ± S.E. for 3 replicates. Results were analyzed using a one-tailed
t test and were significant at p 0.05.
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Fig. 7.
The effects of NFI-A, dexamethasone, and
thyroid hormone on transcription from the PEPCK gene promoter.
A, HepG2 cells were co-transfected with 2.5 µg of
PEPCK-CAT, along with a similar amounts of expression vectors for
NFI-A, the human glucocorticoid receptor, and human thyroid hormone
receptor, individually and in combination, as indicated in the figure.
After 24 h, the cells were treated overnight with 10 nM dexamethasone (DEX) or 1.0 µM
triiodothyronine (T3) and assayed for CAT activity as
described under "Experimental Procedures." All values for CAT
activity were normalized for protein content and expressed as the
mean ± S.E. for 3 replicates. Results were analyzed using a
one-tailed t test and were significant at p 0.05.
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Fig. 8.
The combined effects of NFI-A with CREB or
PKA-c on transcription from the PEPCK gene promoter. A,
HepG2 cells were co-transfected with 2.5 µg of PEPCK-CAT together
with a similar amount of expression vector for either NFI-A or the
catalytic subunit of PKA-c individually and in combination. Cells were
assayed 2 days later for CAT activity. B, as in A
except that an expression vector for CREB was substituted for that of
PKA-c. All values for CAT activity were normalized for protein content
and expressed as the mean ± S.E. for 2 to 3 replicates. Results
were analyzed using a one-tailed t test and were significant
at p 0.05.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
The role of CBP in the coordinating the
interaction of multiple transcriptional stimuli of the PEPCK gene
promoter. NFI is shown bound to the P1 (NFI-binding) site on the
PEPCK promoter and successfully competing with CREB (bound to the
adjacent CRE) for the CREB binding domain on CBP. Note that the binding
of NFI to CBP does not preclude the binding of either thyroid hormone
receptor or glucocorticoid receptors from binding to the N-terminal
steroid receptor binding region of CBP. pol., polymerase;
GRU, glucocorticoid regulatory unit; GR,
glucocorticoid receptor; TRE, thyroid hormone response
element; TR, thyroid hormone receptor.
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ACKNOWLEDGEMENT |
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We are indebted to Dr. David Samols for helpful discussions during the course of this work.
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FOOTNOTES |
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* This research was supported in part by Grants DK-25541 (to R. W. H.) and HD-34908 (to R. G.) from the National Institutes of Health.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 Biochemistry,
Case Western Reserve University School of Medicine, Cleveland, OH
44106-4935. Tel.: 216-368-5302; Fax: 216-368-454.
§ Supported by Metabolism Training Grant DK 07319 from the National Institutes of Health.
2 R. M. Gronostajski, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; CBP, CREB binding protein; CREB, cAMP regulatory element-binding protein; NFI, nuclear factor I; CRE, cAMP regulatory element; PKA-c, catalytic subunit of protein kinase A; Bt2cAMP, dibutyryl cAMP; CAT, chloramphenicol acetyltransferase; bGH, bovine growth hormone.
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