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INTRODUCTION |
GTP cyclohydrolase I
(GTPCH1; EC 3.5.4.16) is the
first and rate-limiting enzyme in the biosynthesis of
5,6,7,8-tetrahydrobiopterin (BH4) (1), the required cofactor for
tyrosine, tryptophan, and phenylalanine hydroxylase and the family of
nitric-oxide synthases (2-4). GTPCH is therefore absolutely necessary
for the synthesis of the signaling molecules dopamine (DA),
norepinephrine, epinephrine, serotonin, melatonin, and nitric oxide as
well as the detoxification of the amino acid
L-phenylalanine. GTPCH gene expression is normally restricted to select tissues and cell types and thus determines the BH4
phenotype (1, 5, 6). Moreover, expression of the GTPCH gene is highly
dynamic, can be induced in cell types that do not normally express it,
and is controlled by a growing number of signal transduction pathways
that presumably converge on the GTPCH promoter (5-9). The genes
encoding for human and mouse GTPCH have recently been cloned (10), and
their 5'-flanking regions have been shown to promote transcription of
heterologous reporter genes (11, 12). Nonetheless, virtually nothing is known regarding either the cis-acting elements or
trans-acting factors that control the expression of
GTPCH.
In the rat brain, GTPCH is localized to monoamine-containing neurons
(6, 13, 14). The abundance of GTPCH mRNA (15) and protein (16)
within these cells is very heterogeneous, with particularly low levels
found within nigrostriatal DA neurons. Unlike many other cell types
that contain GTPCH, DA neurons respond to increased levels of cAMP with
a robust increase in GTPCH mRNA and BH4 content that is dependent
upon gene transcription (17). The cellular specificity of this response
suggests that transcription factor(s) other than or in addition to the
relatively ubiquitous CREB (18) are involved in this process. BH4
synthesis within human nigrostriatal DA neurons is known to be
selectively vulnerable to genetic mutations in GTPCH that cause
hereditary progressive dystonia (19). Identification of the gene
promoter elements and their cognate binding proteins that control basal
and cAMP-dependent GTPCH transcription within DA neurons
may thus be crucial to our understanding of this disorder as well as
other diseases that involve this population of neurons. Toward this
end, we have now cloned, sequenced, and begun to identify and
characterize basal and cAMP response elements within 5812 bp of the
5'-flanking region of the rat GTPCH gene. These studies indicate that a
noncanonical CRE and adjacent CCAAT-box that are located within the
142-bp core promoter region and recruit the transcription factors
ATF-4, C/EBP
, and NF-Y in vitro are both necessary and
sufficient to confer sensitivity to cAMP on the GTPCH promoter.
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MATERIALS AND METHODS |
Isolation and Sequencing of the Rat GTPCH 5'-Flanking
Region--
Approximately 106 plaques from a Harlan
Sprague Dawley rat testis genomic library (Lambda DASH II; Strategene,
La Jolla, CA) were screened by hybridization with a random primed
886-bp cDNA containing the entire rat GTPCH coding sequence (20).
Two positive plaques were purified by repeated plating and
hybridization, and their DNA was then used as templates in PCR
screening reactions. Primers for screening PCR (primer 
112/92,
5'-TCGGTGCAGAACTCCTGTCC-3'; primer 293/315,
5'-CTGAGATGGTCTCCTGTTATCCC-3') were designed based upon the rat GTPCH
cDNA sequence and the mouse GTPCH genomic structure to amplify 427 bp within rat exon 1, including a major portion of the 5'-UTR and the
NcoI site at the GTPCH initiation codon. DNA from one of the
positive plaques produced a PCR product of the correct size that when
digested with NcoI yielded fragments of approximately 315 and 110 bp, as would be predicted based upon the rat GTPCH cDNA
sequence. DNA from this plaque was isolated, the insert was freed from
the vector by restriction digestion, and the entire digest was
subjected to Southern blot analysis using as the probe an end-labeled
oligonucleotide corresponding to bases 47 to 23 of the rat GTPCH
5'-UTR (primer 47/23; 5'-AGACACCCGAAGGTGCTACCAAGCG-3'). This
analysis indicated that the insert was approximately 15 kb in size.
Digestion of the 15-kb insert with NcoI yielded fragments of
approximately 6 and 9 kb. Southern blot analysis of this
NcoI digest with the 47/23 5'-UTR probe showed that the
6-kb fragment contained GTPCH 5'-flanking sequence. This 6 kb was
subcloned into the NcoI site of pGEM5Z to produce p6GTPCH5Z.
Both strands of p6GTPCH5Z were sequenced by primer walking using the
dideoxy chain terminator technique. This analysis revealed that the
6-kb insert was actually 5812 bp in size and, as predicted, contained at its 3'-end the entire 127 bp of the rat GTPCH cDNA 5'-UTR
sequence including the initiation codon.
Reporter Plasmid Construction--
p6GTPCH5Z was digested with
NcoI, and the 5.8-kb insert was isolated and cloned in both
directions into the NcoI site of the luciferase-based
reporter vector pGL3basic (pGL3luc; Promega, Madison, WI) to produce
5.8GTPCHluc and the inverted INV5.8GTPCHluc. Based upon the restriction
map of the insert (Fig. 3A), 5.8GTPCHluc was digested with
either ApaI (0.271 kb), NheI (1.768 kb), or HindIII (3.621 kb) to generate the deletion constructs
0.27-, 1.8-, or 3.6GTPCHluc, respectively. The luciferase-based
reporter plasmids TKluc, containing 148 bp of the human Herpesvirus
thymidine kinase minimal promoter (CLONTECH
Laboratories Inc., Palo Alto, CA) or pGL3 enhancer, containing 195 bp
of the SV40 minimal early promoter (Promega, Madison, WI), were
digested with MluI and BglII and ligated with
high pressure liquid chromatography-purified double-stranded
oligonucleotides containing either the GTPCH CRE and CCAAT-box alone
(cre/ccaat; positions 112 to 63; see Fig. 1) or the Sp1/GC box,
CRE, and CCAAT-box (sp1/cre/ccaat; positions 142 to 63) to produce
the minimal promoter constructs GTPCHcre/ccaatTKluc and
GTPCHsp1/cre/ccaatTKluc and the minimal promoter constructs GTPCH-cre/ccaatSV40luc and GTPCHsp1/cre/ccaatSV40luc, respectively. Plasmid DNA was purified either by centrifugation through CsCl or on
QIAGEN columns (QIAGEN, Chatsworth, CA).
Transcription Start Site Analysis--
The transcription start
site was mapped by S1 nuclease protection analysis (21). A
217-nucleotide end-labeled single-stranded cDNA probe was
synthesized by asymmetric PCR using the primer pair 239/218
(5'-GCGCCTTGACGCAAGAGGCTC-3') and 47/23. PCR parameters included 30 1-min cycles at 95, 62, and 72 °C. The primary PCR product was
gel-purified and used as a template for the secondary PCR. The
secondary PCR primer, 47/23, was end-labeled with
[
-32P]ATP (6,000 Ci/mmol; NEN Life Science Products)
using T4 kinase. The conditions for asymmetric PCR were identical to
the primary PCR except that only the 32P-labeled primer was
present. The single-stranded radiolabeled PCR product was gel-purified
and used for S1 nuclease protection analysis. Poly(A+)
mRNA from rat liver (5 µg) and muscle (20 µg) in 20 µl of S1 hybridization buffer (80% deionized formamide, 40 mM
PIPES, pH 6.4, 400 mM NaCl, and 1 mM EDTA) was
hybridized overnight at 56 °C with 50,000 cpm of probe. S1 nuclease
digestion was carried out for 90 min at 37 °C by adding 300 µl of
S1 nuclease buffer (0.28 M NaCl, 50 mM sodium
acetate, pH 4.5, 4.5 mM ZnSO4, 10 µg of
salmon sperm DNA, and 300 units of S1 nuclease). 100 µl of S1 stop
buffer (4 M ammonium acetate, 20 mM EDTA, pH
8.0, and 50 µg/ml tRNA) was added to terminate the reaction. After
ethanol precipitation, samples were run on a 7.5% polyacrylamide, 8 M urea denaturing gel. A dideoxynucleotide sequencing
reaction using 1.7GTPCHluc as template and end-labeled 47/23 as
primer was run simultaneously on the same gel. Gels were dried and
exposed to x-ray film for 1 week at room temperature.
Site-directed Mutagenesis--
Mutations of the CRE and CCAAT
elements were introduced into 0.27GTPCHluc by overlap extension (21).
The primers used for mutagenesis (mutations are underlined) were as
follows: CREmt(+), '-GCGCCTTAATTCAAGAGGCTC-3';
CCAATmt(+),
5'-GCTCGGGACTATGAGAACGCCT-3'; Glprimer2(),
5'-CTTTATGTTTTTGGCGTCTTCCA-3'; Rvprimer3(),
5'-CTAGCAAAATAGGCTGTCCC-3'. Two sets of primary PCR reactions
were run using the CREmt(+) plus Glprimer2 and the
CREmt() plus Rvprimer3 primer pairs. The secondary PCR
reaction was carried out using the combined gel-purified primary PCR
products as templates. PCRs were hot started at 95 °C for 5 min and
then continued for 30 1-min cycles at 95, 60, and 72 °C, with a
final extension at 72 °C for 7 min. The final PCR product was
digested with NcoI and MluI and cloned back into the pGL3 basic vector to produce 0.27GTPCHlucCREmt. The
identical strategy was used for mutation of the CCAAT-box alone or in
combination with the mutated CRE. 0.27GTPCHluc or
0.27GTPCHlucCREmt DNA were used as templates for these
primary PCRs to produce 0.27GTPCHlucCCAATmt and
0.27GTPCHlucCREmtCCAATmt, respectively. All
mutations were confirmed by sequence analysis.
Cell Culture, Transfection, and Enzyme Assays--
Cultures of
PC12 cells were maintained in Dulbecco's modified Eagle's medium,
7.5% fetal calf serum, and 7.5% horse serum. Cultures of C6 glioma
cells were maintained in Dulbecco's modified Eagle's medium, 1 mM sodium pyruvate, and 5% fetal calf serum. Cultures of
Rat2 fibroblast cells were maintained in Dulbecco's modified Eagle's
medium and 10% fetal calf serum. All cultures contained
penicillin/streptomycin and were incubated at 37 °C in a humidified
atmosphere of 10% CO2. Cells were seeded onto poly-D-lysine-coated 16-mm dishes 24 h prior to
transfection and grown to approximately 80% confluency. Cells were
typically transfected with 0.45 µg of experimental DNA and 0.01-0.05
µg of pRSVgal or pCMVgal DNA using LipofectAMINE (Life
Technologies, Inc.). For comparisons across constructs that differed in
size, molar amounts of experimental DNA were equalized by the addition
of pGEM5Z DNA. For co-transfection experiments, 0.3 µg of
0.27GTPCHluc DNA was transfected along with 0.05-0.15 µg of
pCG-hATF4 (cytomegalovirus-driven human ATF-4, a gift from Dr. T. Hai,
Ohio State University) or empty vector DNA along with 0.05-0.15 µg
of pRSVgal DNA. 18 h later, 5 mM 8-Br-cAMP
dissolved in growth-conditioned media was added. Cultures were
harvested 7-18 h later in lysis buffer and assayed for luciferase
(Promega, Madison, WI) and -galactosidase (CLONTECH Laboratories Inc., Palo Alto, CA)
activities as described by the manufacturers. Parallel experiments
using -galactosidase enzyme histochemistry showed that transfection
efficiency was similar for each cell type (15-25%; data not shown).
Luciferase activity was divided by -galactosidase activity to
correct for transfection efficiency and was expressed as relative
luciferase activity.
Nuclear Extracts--
Large scale PC12 nuclear extracts used for
DNase I protection analysis were prepared by the method of Dignam (22).
Small scale nuclear extracts from PC12, C6, and Rat2 cells grown in 100-mm dishes were prepared for use in EMSA analysis by the method of
Andrews and Faller (23). Protein content was determined by the method
of Bradford (24).
DNase I Footprint Analysis--
p0.27GTPCHluc was digested with
NcoI or MluI, and the resulting overhangs were
filled in with Klenow and [
-32P]CTP (3,000 Ci/mmol)
followed by a chase reaction with unlabeled nucleotides. The
NcoI digest was then treated with MluI, and the MluI digest was treated with NcoI in order to
label only the coding or noncoding strands, respectively. Probes were
then purified by acrylamide gel electrophoresis, dialyzed,
phenol-extracted, and concentrated by ethanol precipitation. To 20 µl
of reaction buffer (12.5 mM HEPES-KOH, pH 7.9, 10%
glycerol, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 µg of poly(dI-dC), and 5 µg of acetylated bovine serum albumin) on ice was added 20 µl of nuclear extract containing up to 150 µg of protein. The binding reaction was
initiated by the addition of 10 µl of reaction buffer containing approximately 20,000 cpm of 32P-labeled probe, and the
incubation continued for 20 min at room temperature. Alternatively,
studies using competing oligonucleotides included a 120-min incubation
on ice prior to the addition of probe and then an additional 20 min at
room temperature. Samples were then incubated at room temperature for 1 min with a quantity of RQ1 DNase I sufficient to digest approximately
50% of the labeled DNA probe both in the presence and absence of
binding protein. The RQ1 reaction was terminated by the addition of
stop buffer (400 mM sodium acetate, 0.2% SDS, 10 mM EDTA, 50 µg/ml yeast tRNA, and 20 µg/ml proteinase
K). Samples were then incubated at 55 °C, phenol-extracted, and
ethanol-precipitated. Labeled coding and noncoding strands were
chemically sequenced (25) to yield combined purines (G + A) and
pyrimidines (T + C), which were then run alongside the DNase I-treated
samples on a 6% acrylamide, 8 M urea gel. Gels were dried
and exposed to x-ray film at 80 °C.
Electrophoretic Mobility Shift Assay--
Single-stranded
complementary oligonucleotides were annealed and end-labeled with
[-32P]ATP (3,000 Ci/mmol) and T4 kinase. To 13 µl of
reaction buffer (12.5 mM HEPES-KOH, pH 7.9, 10% glycerol,
100 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 µg of poly(dI-dC), 5 µg of acetylated bovine serum albumin) on ice was added 5 µl of nuclear extract containing up
to 5 µg of protein. The binding reaction was initiated by the addition of 2 µl of reaction buffer containing approximately 50,000 cpm of end-labeled oligonucleotide and continued for 20 min at room
temperature. Antibodies (4 µg; Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) or competing double-stranded oligonucleotides were added to
the reaction buffer before protein and 120 or 30 min, respectively,
prior to the addition of labeled oligonucleotide. Samples were then
returned to ice, and the entire volume was loaded onto a 5% acrylamide
gel containing 1% glycerol and 1× TBE. Running buffer was 1× TBE
containing 1% glycerol. Gels were dried and exposed to x-ray film
overnight at 80 °C.
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RESULTS |
Isolation of the Rat GTPCH 5'-Flanking Sequence and Determination
of the Transcription Start Site--
A combined strategy of Southern
analysis, exon-based PCR, and restriction digestion was used to
identify and clone 5.812 kb of rat GTPCH 5'-flanking sequence up to and
including the translation initiation codon. A three-way alignment of
the rat sequence with GTPCH 5'-flanking sequence currently available
for the mouse and human genes showed a high degree of homology
surrounding the previously established transcription start sites (10,
11) (Fig. 1). Because the human and mouse
start sites are reportedly different, however, the start site for rat
GTPCH was determined using S1 nuclease protection analysis (Fig.
2A). The target mRNAs for
this study were isolated from the liver, which expresses GTPCH
mRNA, and striated muscle, which does not (6). This analysis showed
that only mRNA from liver was able to protect the 217-nucleotide
single-stranded cDNA probe from S1 nuclease digestion (Fig.
2B). Furthermore, only a single band of approximately 100 nucleotides in size was protected, and this was aligned with an A at
position 129 of the sequencing ladder. A transcription start site for
rat liver GTPCH can thus be assigned to the T 129 bp upstream from the
initiation codon and 2 bases upstream from the available cDNA
sequence (20). It should be noted that this position differs from both
the reported mouse and human start sites (Fig. 1). All positions within
the rat 5'-flanking sequence have been indexed relative to this +1 transcription start site.
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Fig. 1.
Nucleotide sequence and organization of the
proximal 5'-flanking region of the rat, mouse, and human GTPCH
genes. Transcription start sites are indicated by
circled and boldface letters. Putative
protein binding sites determined by computer analysis are identified by
boxes and named below the sequence. Actual
protein binding sites determined by DNase I footprint and EMSA analysis
are labeled Domain 1-4 and are drawn as
lines above the sequence. Numbering is based upon
the distance from the rat transcription start site at T+1.
This sequence corresponds to the 142 bp at the 5'-end of the
0.27GTPCHluc construct.
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Fig. 2.
Determination of the rat GTPCH transcription
start site in rat liver. A, a schematic diagram of the
S1 nuclease digestion paradigm. B, 5 or 20 µg of mRNA
isolated from rat liver or striated muscle, respectively, were
hybridized with a single stranded DNA probe complementary to genomic
GTPCH sequence between positions 239 and 23. Following digestion
with S1 nuclease, samples were analyzed alongside a sequencing ladder
generated by the 47/23 primer using 1.7GTPCHluc DNA as template.
Only liver mRNA was able to protect the probe from S1 nuclease
digestion (right arrow). Only a single protected
band was detected, and this band aligned with an A at position 129 of
the sequencing ladder (left arrow). A
transcription start site for rat liver GTPCH can thus be assigned to
the T 129 bp upstream from the initiation codon.
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Functional Characterization of the GTPCH 5'-Flanking
Region--
DNA containing the reporter construct 5.8GTPCHluc was
transiently transfected into PC12 cells, which normally express GTPCH mRNA (6), or C6 or Rat2 cells, which normally do not (9). These
studies showed that transcription from this chimeric gene was roughly
equivalent in each cell line and up to 20-fold greater than produced by
the promoterless pGL3luc or the inverted 5.8GTPCHluc construct (Fig.
3B). This suggests that DNA
elements responsible for restricting expression of the endogenous GTPCH
gene to PC12 cells are either not located within the 5.812 kb of
5'-flanking sequence or cannot be detected using transient transfection
assays. Progressive deletions of the 5.8-kb construct revealed that the minimal promoter sequence necessary to sustain transcription in each
cell line is located within 0.27GTPCHluc, which contains 142 bp
upstream from the cap site and the entire 128 bp of GTPCH mRNA
5'-UTR (+142 to 128; Fig. 1). Deletion analysis also detected a
stepwise increase in transcription as 5'-flanking sequence was eliminated. This suggests that control elements that normally repress
transcription may be found upstream from the core promoter.
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Fig. 3.
Functional analysis of the GTPCH promoter in
PC12, C6, and Rat2 cells. A, a restriction map of the
5.812 kb of GTPCH 5'-flanking sequence showing a number of unique
restriction sites as well as those sites that were used to generate the
luciferase reporter deletion constructs 3.6GTPCHluc
(HindIII), 1.8GTPCHluc (NheI), and 0.27GTPCHluc
(ApaI). B, cells were transfected with GTPCHluc
and pRSVgal DNA. Molar amounts of experimental DNA were equalized by
the addition of pGEM5Z DNA. The next day, 5 mM 8-Br-cAMP
dissolved in growth-conditioned media was added, and the cultures
continued for another 24 h, when cells were lysed and assayed for
luciferase and -galactosidase activities. Luciferase activity was
divided by -galactosidase activity to correct for transfection
efficiency and was expressed as relative luciferase activity. Data are
the mean ± S.E. of three independent experiments each determined
in triplicate.
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PC12 cells transfected with 5.8GTPCHluc responded to 8-Br-cAMP
treatment with a 4-7-fold increase in relative luciferase activity (Fig. 3B). This contrasts with the 1.3-1.9-fold increase
observed for the empty pGL3luc vector or the INV5.8GTPCHluc construct. Deletion of sequence up to the ApaI site (Fig.
3A) did not diminish the response to 8-Br-cAMP. The
cis-acting elements required for the
cAMP-dependent activation of GTPCH transcription are thus located within the 142-bp core promoter contained within 0.27GTPCHluc and presented in Fig. 1. In contrast to PC12 cells, transcription in C6
and Rat2 cells was either unaltered or slightly decreased following
treatment with 8-Br-cAMP (Fig. 3B). The
trans-acting factors(s) required for the
cAMP-dependent enhancement of GTPCH transcription are
therefore found in PC12 cells, which normally express this gene, but
not in two cell lines that do not.
DNase I Footprint Analysis of the GTPCH Core Promoter Region
Reveals Four Protein Binding Domains--
A computerized search of the
142-bp GTPCH core promoter for putative cis-acting elements
that may be responsible for the stimulation of transcription by cAMP
was undertaken using the TFMATRIX transcription factor binding site
profile data base (26). This search revealed an asymmetrical or partial
CRE (TGACGCAA) located between positions 104 and 97 that differed
from the CRE palindrome (TGACGTCA) at positions 6 and 7 of the octomer
(Fig. 1). Computer analysis also revealed a number of other putative
protein binding motifs including three overlapping Sp1/GC box sites
between positions 110 and 137, a CCAAT-box between positions 82
and 86, a TATA-like sequence at positions 45 to 39, and an E-box
located at positions 17 to 12. DNase I footprint analysis of the
core promoter using nuclear extracts prepared from untreated PC12 cells
detected three distinct protein binding domains (Fig.
4). Domain 1 is a 26-bp region that spans
from position +7 to 19 and includes the transcription start site and
the E-box (Fig. 1). Domain 1 also contains two hypersensitive sites; a
C at position 19 of the coding strand and a G at position 7 of the
noncoding strand. Domain 2 is a 29-bp region that spans from position
97 to 69 and is centered upon the CCAAT-box. Domain 4 (numbered out
of order; see below) is a 22-bp region that is 86% GC, spans from
position 132 to 111 and is centered upon the repeating Sp1/GC box
elements. Footprint analysis thus established a general agreement
between putative and actual protein binding sites. Notably missing were
protected sequences associated with the TATA-like ATAAAAA element and
the partial CRE. Incubation of core promoter DNA with 1 µg of
recombinant human CREB-1 bZip domain (amino acids 254-327; Santa Cruz
Biotechnology) produced a weak footprint, referred to as domain 3, that
spanned from position 106 to 97 (Fig.
5). This footprint corresponds to the CRE
predicted by computer analysis and overlaps the 5' boundary of domain
2.
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Fig. 4.
DNase I footprint analysis of the GTPCH core
promoter region reveals three protein binding domains.
32P-Labeled coding and noncoding strands of the 271-bp
0.27GTPCHluc insert were used as probes. Nuclear extracts were prepared
from unstimulated PC12 cells. Maxam-Gilbert sequencing reactions (G+A
and T+C) were performed in parallel to identify the base positions of
protected regions. Three protein binding domains were detected within
this proximal region of the rat GTPCH gene and are referred to as
domains 1, 2, and 4. The locations of these domains and the
cis-acting elements found therein are presented in the
adjacent diagrams and in Fig. 1. Note the presence of two DNase
I-hypersensitive sites within domain 1.
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Fig. 5.
DNase I footprint and competition analysis
detects recombinant CREB-1 binding to domain 3 of the core promoter
region and also demonstrates that the failure of PC12 nuclear extracts
to footprint domain 3 is not due to occupation of domains 2 and 4 by
their cognate binding proteins. A, competing
double-stranded oligonucleotides with consensus sites
underlined and mutations in boldface type.
B, the 32P-labeled noncoding strand of the
271-bp insert from 0.27GTPCHluc was used as the probe. 1 µg of
recombinant human CREB-1 or 35 µg of nuclear extract prepared from
unstimulated PC12 cells was used in these assays. Competing
oligonucleotides were added 120 min prior to the probe. Incubation with
CREB-1 protein resulted in a weak footprint over positions 97 to
106, which corresponds to the CRE and is referred to as domain 3. Elimination of binding to domains 2 and 4 by competing CCAAT and Sp1
oligonucleotides did not result in the appearance of domain 3. The
locations of each domain and the cis-acting elements found
therein are presented in the adjacent diagram and in Fig. 1.
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Nuclear extracts prepared from PC12 cells contain numerous CRE-binding
proteins (see below) yet fail to footprint the GTPCH CRE. DNase I
protection analysis using subsaturating amounts of nuclear extract and
domain-specific competing oligonucleotides was used next to determine
whether this failure could be the result of occupation of the
overlapping domain 2 and adjacent domain 4 by their cognate binding
proteins. Incubation of PC12 nuclear extracts with a 500-fold molar
excess of double-stranded oligonucleotide centered upon the GTPCH
CCAAT-box (Fig. 5A; GTPCHccaat) was able to compete away
domain 2 without affecting domain 1 or 4 or exposing domain 3 to
protein binding (Fig. 5B). This competition was specific in
that a double-stranded oligonucleotide containing a mutated form of the
GTPCH CCAAT-box (Fig. 5A; GTPCHccaatmt) failed
to inhibit the formation of domain 2. The addition of a molar excess of
a double-stranded oligonucleotide centered upon the GTPCH CRE (Fig.
5A; GTPCHcre) or the consensual CRE of the rat tyrosine hydroxylase promoter (Fig. 5A; THcre; Ref. 27), both of
which act to scavenge CRE-binding protein(s) present in the extract (see below), did not enhance the formation of domain 2 or 4 (Fig. 5B). Incubation with a double-stranded oligonucleotide
containing a canonical Sp1 element (Fig. 5A) was able to
compete away domain 4 without affecting domain 1 or 2 or exposing
domain 3 to protein binding (Fig. 5B). The suboptimal amount
of binding protein in these assays also revealed that domain 4 may
contain a low affinity site spanning position 111 to 116. Overall,
these results suggest that occupation of surrounding domains 2 and 4 by
their binding factors is not responsible for the failure of PC12
nuclear extracts to footprint the CRE in domain 3.
Electrophoretic Mobility Shift Analysis Reveals Protein Binding to
the GTPCH CRE--
EMSA using PC12 nuclear extracts and as probe a
radiolabeled double-stranded GTPCHcre oligonucleotide that does not
contain the adjacent CCAAT-box (Fig.
6A) revealed one major and,
depending upon the protein preparation and electrophoresis time, one or two larger but typically minor bands (Fig. 6B). These
complexes were only detected in the presence of nuclear extract,
increased in intensity as the amount of protein in the assay was
increased, and were generally of equal intensity in extracts prepared
from control and 8-Br-cAMP-treated cells. Formation of this complex was
competed away in a concentration-dependent manner by both unlabeled GTPCHcre and THcre oligonucleotides (Fig. 6A), the
latter appearing more potent. The specificity of binding was further established by the failure of a 1000-fold excess of oligonucleotide centered upon either a canonical Sp1 or NF-
B element (Fig.
6A) to compete (Fig. 6B).
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Fig. 6.
Electrophoretic mobility shift assay
demonstrates binding to the GTPCH CRE and competition between the
GTPCHcre and THcre. A, double-stranded oligonucleotides used in
these assays. Consensus sites are underlined. B, binding of
nuclear extracts from control and 8-Br-cAMP-treated PC12 cells to
32P-labeled GTPCHcre oligonucleotide. Protein amounts and
the molar excess of competitors are presented. The arrows
denote the specific binding complex. C, binding of PC12 cell
nuclear extracts to 32P-labeled THcre oligonucleotide. The
molar excess of competitors is presented. The three arrows
denote specific binding complexes.
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Binding to the GTPCH CRE was further characterized by comparison with
the canonical TH CRE. EMSA using PC12 nuclear extracts and the THcre as
probe produced three major complexes, with the smallest being the most
abundant (Fig. 6C). Competition with a 100- and 1000- but
not a 10-fold molar excess of unlabeled GTPCHcre reduced the formation
of each of these complexes. When present at high concentrations,
GTPCHcre thus has the potential to bind the same nuclear proteins
recruited by THcre. Incubation with unlabeled THcre at a 10-fold excess
approximated the competition produced by a 100-fold excess of GTPCHcre.
This observation and the data presented in Fig. 6B indicate
that PC12 nuclear proteins have at least a 10-fold greater affinity for
the canonical TH CRE than for the GTPCH CRE sequence.
Nuclear extracts from control and 8-Br-cAMP-treated PC12, C6, and Rat2
cells were next analyzed by EMSA to determine whether GTPCH CRE binding
activity is correlated with the cellular specificity of the cAMP
response. Fig. 7B shows that
extracts from untreated C6 and Rat2 cells express little GTPCHcre
binding activity but bind avidly to the THcre. GTPCHcre binding
activity was induced, however, in C6 and Rat2 extracts following a 24-h
treatment with 8-Br-cAMP. In contrast, major differences in binding
across treatments were not detected when these same nuclear extracts
were analyzed using the THcre probe (Fig. 7B). These data
further indicate that the GTPCH CRE can recruit a CRE-binding
protein(s) that is normally found in PC12 but not C6 or Rat2 cells.
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Fig. 7.
Electrophoretic mobility shift assay
demonstrates cellular and protein specificity in binding to the GTPCH
CRE. A, double-stranded oligonucleotides used in these
assays. Consensus sites are underlined. B, binding of 5 µg
of nuclear extract from control and 8-Br-cAMP-treated PC12, C6, and
Rat2 cells to 32P-labeled GTPCHcre or THcre
oligonucleotides. The arrow on the left denotes
the specific complexes obtained with the GTPCHcre oligonucleotide. The
three arrows on the right denote the specific
complexes obtained with the THcre oligonucleotide. C,
antibody supershift analysis of control PC12 nuclear extracts using
32P-labeled GTPCHcre, THcre, or GTPCHccaat
oligonucleotides. The arrows denote supershifted binding
complexes.
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Mutagenesis of the CRE Element Inhibits Basal and
cAMP-dependent Transcription--
Three bases within the
CRE were simultaneously mutated to produce the reporter construct
0.27GTPCHlucCREmt (Fig.
8A), a G to A at position
105, a C to T at position 103, and a G to T at position 102 (Fig.
8A). These bases were chosen for mutagenesis because
methylation interference has shown that each is involved in ATF-1
binding to DNA (28). Nonetheless, EMSA using the GTPCHcremt oligonucleotide showed that binding was severely decreased but not
eliminated by these mutations (Fig. 8B). Furthermore, while competition experiments demonstrated a reduced potency, the
GTPCHcremt oligonucleotide still retained the ability to
compete with the wild type oligonucleotide. These results suggest that
the DNA binding specificity of the protein(s) that interacts with the GTPCH CRE is different from that established for CREB and ATF-1. Transient transfection experiments using the
0.27GTPCHlucCREmt construct demonstrated that these
mutations of the CRE decreased basal transcription by 50% (Fig.
9A). The enhancement of
transcription by 8-Br-cAMP was also reduced to 3-fold from the 4.9-fold
observed for the wild type construct (Fig. 9A). While these
results may be explained by the lower affinity of the mutated CRE for
the GTPCH CRE-binding protein(s), it is more likely that some other cis-acting element(s) within the core promoter, such as the
adjacent Sp1/GC- or CCAAT-boxes, may also participate in mediating the cAMP enhancement of GTPCH transcription.
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Fig. 8.
Electrophoretic mobility shift assay of
binding to mutated GTPCH CRE and CCAAT-box elements. A,
double-stranded oligonucleotides used in these assays. Consensus sites
are underlined, and mutations are in boldface
type. B, binding of nuclear extracts from
8-Br-cAMP-treated PC12 cells to 32P-labeled
oligonucleotides containing either the GTPCH wild type or mutated CRE
sequence. The amount of nuclear extract and the molar excess of
competitors is presented. The arrow on the left
denotes the specific complex obtained with the GTPCHcre
oligonucleotide. C, binding of nuclear extracts from control
and 8-Br-cAMP-treated PC12 and C6 cells to 32P-labeled
double-stranded oligonucleotides containing either the GTPCH wild type
or mutated CCAAT box. The molar excess of competitor is presented. The
three arrows denote the specific complexes
obtained with the GTPCHccaat oligonucleotide.
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Fig. 9.
Transient transfection assays of GTPCH
promoter constructs. 18 h after transfection, PC12 cells
cultures were incubated with or without 5 mM 8-Br-cAMP
dissolved in conditioned medium, harvested 7 h later, and assayed
for luciferase and -galactosidase activity. All data are the
mean ± S.E. of at least two independent experiments each
determined in triplicate. A, site-directed mutagenesis of
the GTPCH CRE and CCAAT box decreases basal and
cAMP-dependent transcription. Cells were transfected with
either the wild type 0.27GTPCHluc construct or constructs in which the
CRE (0.27GTPCHlucCREmt), the CCAAT box
(0.27GTPCHlucCCAATmt), or the CRE and CCAAT box
(0.27GTPCHlucCREmtCCAATmt) have been mutated.
The -fold increase in response to 8-Br-cAMP is presented in
parentheses. B, the combined CRE and CCAAT-box
element can enhance basal activity and confer sensitivity to cAMP on a
minimal heterologous promoter. Cells were transfected with either
0.27GTPCHluc or the luciferase-based reporter plasmids TKluc or SV40luc
containing either the GTPCH CRE and CCAAT-box (cre/ccaat; positions
112 to 63; see Fig. 1) or the Sp1/GC box, CRE, and CCAAT-box
(sp1/cre/ccaat; positions 142 to 63) to produce the minimal
promoter constructs GTPCHcre/ccaatTKluc plus GTPCHsp1/cre/ccaatTKluc or
GTPCHcre/ ccaatSV40luc plus GTPCHsp1/cre/ccaatSV40luc, respectively. The
-fold increase in response to 8-Br-cAMP is presented in
parentheses. C, overexpression of ATF-4 enhances
the response to 8-Br-cAMP. 0.3 µg of 0.27GTPCHluc was transfected
along with 0.05-0.15 µg of pCG-hATF4 (ATF-4) or empty vector DNA
along with 0.05 µg of -galactosidase DNA. The -fold increase in
response to 8-Br-cAMP is presented in parentheses.
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Mutagenesis of the CCAAT-box Element Inhibits Basal and
cAMP-dependent Transcription--
EMSA was used first to
establish that nuclear proteins from control and 8-Br-cAMP-treated PC12
are recruited by a double-stranded oligonucleotide that is centered on
the GTPCH CCAAT-box and does not contain the CRE (Fig. 8C;
GTPCHccaat). Depending upon the preparation and electrophoresis time,
these studies detected one large and one or two smaller binding
complexes, the formation of which required nuclear protein, was
effectively competed by unlabeled GTPCHccaat, and generally appeared
unaffected by treatment with 8-Br-cAMP. In addition, unlike binding to
the GTPCH CRE, extracts from control and 8-Br-cAMP-treated C6 cells
bound the CCAAT-box (Fig. 8C). Two bases within the CCAAT
pentamer were mutated to produce the reporter construct
0.27GTPCHlucCCAATmt; a C to A at position 86 and an A to
T at position 84. Methylation interference has shown that either one
or both of these bases are involved in the binding of the transcription
factor NF-Y, CP2, or NF-1 to the CCAAT-box motif (29, 30). This
requirement was corroborated by EMSA using the mutated oligonucleotide
as probe (GTPCHccaatmt; Fig. 8C). Transient
transfection experiments with the 0.27GTPCHlucCCAATmt
construct demonstrated that these mutations decrease basal
transcription by 22% (Fig. 9A). The enhancement of
transcription by 8-Br-cAMP was also reduced to 3.5-fold from the
4.9-fold observed for the wild type construct. The CCAAT-box element
thus contributes to basal and cAMP-dependent GTPCH
transcription, although to a somewhat lesser extent than does the
CRE.
Simultaneous Mutagenesis of Both the CRE and CCAAT-box Elements
Eliminates the Response to cAMP--
A reporter construct was next
prepared in which both the CRE and the CCAAT box were mutated as
described to produce 0.27GTPCHlucCREmtCCAATmt. Transfection with this construct decreased basal levels of
transcription by 66% (Fig. 9A) or approximately the sum of
the declines in basal activity produced by the individual mutations
alone. Furthermore, these combined mutations completely eliminated the
response of the GTPCH promoter to cAMP. The CRE and adjacent CCAAT-box
are thus necessary to confer a major proportion of basal promoter activity as well as the cAMP-dependent enhancement of GTPCH
gene transcription.
The CRE and CCAAT-box Gene Cassette Increases Basal Activity and
Confers Sensitivity to cAMP on a Heterologous Minimal
Promoter--
PC12 cells transfected with the heterologous thymidine
kinase minimal promoter engineered to contain a single copy of the GTPCH CRE and CCAAT-box cassette in normal orientation (112 to 63
bp in Fig. 1; GTPCHcre/ccaatTKluc) exhibited low levels of basal
transcription and did not respond to cAMP with an increase in
luciferase activity (Fig. 9B). Moreover, the addition to
this construct of 30 bp of 5' sequence that includes domain 4 and
contains the Sp1/GC box element (142 to 63 bp;
GTPCHsp1/cre/ccaatTKluc) had little effect on basal or
cAMP-dependent transcription (Fig. 9B). In
contrast, when a single copy of the CRE and CCAAT-box cassette was
placed in the correct orientation upstream of the SV40 minimal early
promoter (GTPCHcre/ccaatSV40luc) a 3-fold stimulation in basal activity
and a 3.6-fold increase in cAMP-dependent transcription were observed (Fig. 9B). The addition of the Sp1/GC box 5'
to this construct substantially reduced basal luciferase activity, although the -fold response to cAMP was maintained and possibly even
enhanced. When combined with the data obtained by mutagenesis, these
observations demonstrate that the CRE and CCAAT-box cassette is
necessary and sufficient to enhance basal and
cAMP-dependent transcription from the rat GTPCH promoter.
Moreover, occupation of the 5' Sp1/GC box was shown to inhibit
CRE-dependent and CCAAT-box-dependent basal
activity without negatively affecting the cAMP response that is
mediated by the same combined element.
Protein Binding to the CRE--
EMSA using PC12 nuclear extracts
and antibodies specific to five members of the CREB/ATF family revealed
that ATF-4 and, to a much lesser degree, CREB-1 are recruited by the
GTPCHcre oligonucleotide (Fig. 7C). Anti-ATF-4 was found to
reproducibly decrease the intensity of both the major and minor binding
complexes and to supershift two distinct bands. When using the THcre
probe, anti-CREB-1 produced a supershift of the middle binding complex,
whereas anti-ATF-4 shifted two faint bands without dramatically
altering overall band intensity. While ATF-4 does not heterodimerize
with CREB, it is known to form heterodimers with the bZip transcription
factors c-Jun, c-Fos, Fra-1, C/EBP, and C/EBP (31, 34). Like
anti-ATF-4, anti-C/EBP was observed here to decrease the intensity
of the GTPCHcre major and minor bands and to supershift the binding
complex (Fig. 7C). In contrast, antibodies specific to
C/EBP or epitopes common to c-Fos, FosB, Fra-1, and Fra-2 or common
to c-Jun, JunB, and JunD were without effect (Fig. 7C). The
GTPCHcre binding complex formed by PC12 nuclear extracts thus includes
the transcription factors ATF-4 and C/EBP.
Overexpression of ATF-4 Transactivates the Response to
8-Br-cAMP--
Expression of endogenous CREB does not appear to
correlate with either the in vivo GTPCH promoter response to
8-Br-cAMP or the in vitro binding of nuclear extracts to the
GTPCHcre. These observations along with the binding of ATF-4 to the
GTPCH CRE suggest that ATF-4 in intact PC12 cells is able to mediate
the response of the GTPCH promoter to cAMP. In order to investigate this, PC12 cells were co-transfected with a fixed amount of
0.27GTPCHluc promoter reporter DNA and varying amounts of DNA
expressing ATF-4 under the control of the cytomegalovirus viral
promoter. Overexpression of ATF-4 was found to increase the
transcriptional response to cAMP in a DNA
concentration-dependent manner, demonstrating that ATF-4 is
able to transactivate the GTPCH promoter in intact cells (Fig.
9C).
Protein Binding to the CCAAT-box--
EMSA using the GTPCHccaat
probe demonstrated that antibodies directed against the C terminus of
the A subunit of the heterotrimeric transcription factor NF-Y partially
shift the large binding complex without altering the mobility of the
smaller band (Fig. 7C). In contrast, antibodies directed
against the C terminus of the NF-Y B subunit completely shift the upper
band, while antibodies to the C terminus of NF-YC were without effect.
Antibodies directed against another CCAAT-binding protein, NF-1, did
not interact with either complex. These results indicate that the
GTPCHccaat oligonucleotide recruits the transcription factor NF-Y but
that only the C terminus of the NF-Y B subunit of the NF-Y trimer is completely accessible to antibody binding.
| |
DISCUSSION |
Progressive deletion analysis of the 5812 bp of the rat GTPCH
5'-flanking sequence revealed that the first 142 bp upstream from the
liver cap site contain the GTPCH core promoter and also mediate the
cAMP-dependent enhancement of gene transcription. This
142-bp region is GC-rich (73%) and exhibits 90% homology with the
mouse and 77% homology with the human GTPCH genes. The human GTPCH
core promoter is also reported to lie within the first 211 bp upstream
from the transcription start site (11). With the exception of the
E-box, which is not present in the human sequence, each of the protein
binding motifs detected in the rat promoter by in vitro
footprinting is found in the mouse and human genes. Moreover, an
additional DNA response element that is found in the human sequence but
is absent from the mouse and rat promoters is a putative Sp1 binding
motif strategically placed between the CRE and CCAAT-box. Whether this
Sp1 site and/or the absence of the E-box are responsible for the
significant differences in GTPCH expression between humans and rodents
awaits determination (1).
The TATA-like ATAAAAA sequence and its position and flanking bases are
also completely conserved within the GTPCH core promoter, where the
sequence may function to position the preinitiation complex (Fig. 1).
Inasmuch as the rat, mouse and human cap sites are all different,
however, it would appear that this sequence serves only a weak
positioning function within the GTPCH promoter. This conclusion is
supported by the failure of PC12 nuclear extracts to footprint the
ATAAAAA sequence and, at least in the case of the mouse cap site, the
lack of consensual distance expected between a bona fide TATA box and a
transcription start site (31). Inasmuch as the recruitment of TFIID to
the promoter is proposed to be an obligatory and probably limiting step
in the formation of the preinitiation complex, the weak positioning of
TBP by the ATAAAAA sequence may actually contribute to GTPCH enhancer
function. Indeed, the capacity of the CRE and CCAAT-box cassette to
enhance cAMP-dependent transcription was only observed
within the context of the GTPCH and SV40 minimal promoters, which,
unlike the thymidine kinase minimal promoter, do not contain canonical
TATA boxes.
The CRE and adjacent CCAAT-box each contribute independently to basal
and cAMP-dependent transcription from the GTPCH promoter, although the contribution of the CRE to both activities is greater than
that of the CCAAT-box. It would also appear that this combined sequence
has the properties of an enhancer element. EMSA and co-transfection studies indicate that the transcription factors ATF-4, C/EBP, and
NF-Y are the likely candidates recruited by the GTPCH CRE and
CCAAT-box. ATF-4 seems to be found in many but not all tissues and cell
lines (32, 33) and, in agreement with the data presented here, is found
to be constitutively expressed along with C/EBP in PC12 cells (34,
35). Although originally characterized as a repressor (32), ATF-4 is
now known to be a strong activator of transcription (34, 36), a role
mediated by physical interactions of the N and C termini with the
general transcription factors TBP, TFIIB, and TFIIF as well as the
co-activator CBP (36). While ATF-4 does not heterodimerize with CREB-1,
it does form heterodimers with a number of other bZip transcription
factors including C/EBP (33, 37). The formation of ATF-4 and
C/EBP heterodimers in vitro is known to modify the DNA
binding properties of this complex so that noncanonical CREs like that
found in the GTPCH promoter are recognized (33). Moreover, ATF-4 and
C/EBP have been shown to interact on the Gadd153
(C/EBP
, CHOP) promoter to enhance transcription in PC12 cells (34).
This observation and the data presented here would seem to establish
that ATF-4 and C/EBP can function as partners in the control of gene
transcription. The other ATF-4 partner on the GTPCH CRE and CCAAT-box
enhancer would appear to be NF-Y, a highly conserved and ubiquitous
heterotrimeric transcription factor composed of A, B, and C subunits,
all of which are required for high affinity DNA binding (29, 38, 39).
Like ATF-4, NF-Y is able to mobilize histone acetyltransferase activity
by interacting directly with CBP as well as p300/CBP-associated factor
(40, 41). Also like ATF-4, the amino-terminal domain of C/EBP has
been established to interact directly with CBP (42). The combination of
ATF-4 and C/EBP arrayed along with NF-Y on the GTPCH core promoter
should thus have enormous potential to affect transcription through the
modification of chromatin structure.
Located immediately 5' to the GTPCH noncanonical CRE is protein binding
domain 4, which is composed of three overlapping Sp1-like elements.
When placed in the context of the SV40 minimal promoter, the addition
of this Sp1-like element to the CRE and CCAAT-box cassette suppressed
basal activity yet maintained and possibly even enhanced
cAMP-dependent transcription. It would thus appear that the
cognate proteins recruited by the Sp1/GC box are somehow capable of
distinguishing between these two modes of CRE-dependent and
CCAAT-box-dependent enhancer activities. Whether the Sp1/GC box acts similarly within the context of the GTPCH promoter awaits determination. Although the protein(s) recruited by this GC-rich sequence have not been identified, an oligonucleotide containing a
canonical Sp1 binding motif was found to compete away domain 4 on the
GTPCH promoter. This suggests that a member(s) of the Sp1 family of
transcription factors, which includes Sp1, Sp3, and Sp4, may bind this
element (43-46). Since Sp4 appears to be exclusively expressed in the
brain (43), the most likely candidates found within PC12 cells, which
are derived from the adrenal medulla, would seem to be Sp1 and/or Sp3.
Unlike Sp1, Sp3 generally acts to repress rather than activate
transcription (47-50) and may thus be responsible for the inhibition
of basal transcription observed here. Alternatively, since Sp1 and
C/EBP are reported to interact physically (51), this interaction may
somehow serve to repress basal GTPCH promoter function without
affecting the cAMP response.
Characterization of a number of promoters has shown that a CRE can act
in concert with a CCAAT-box (52-55) or a CCAAT-box can act alone (40,
56) to enhance gene transcription in response to cAMP. In those cases
where both motifs are involved, the CRE and CCAAT-box can be separated
by as much as 10-20 bp or approximately one or two turns of the double
helix. This spatial geometry is conserved in the rat, mouse, and human
GTPCH promoters and lends support to our contention that both the CRE
and CCAAT-box elements are required for basal and
cAMP-dependent transcription. In promoters where it has
been studied in detail, the phosphorylation of CREB-1 and/or ATF-1 by
protein kinase A has been established to mediate that portion of the
cAMP response contributed by the CRE, while the biological basis for
the NF-Y and CCAAT-box component of the cAMP response remains a
mystery. The case of the GTPCH promoter is further complicated by the
observation that ATF-4 does not appear to be a substrate for protein
kinase A (36). C/EBP is a substrate for protein kinase A, however,
and upon phosphorylation in PC12 cells, it is shuttled from the
cytoplasm to the nucleus (35). How then does cAMP stimulate GTPCH
transcription through the CRE and adjacent CCAAT-box? A model that may
be relevant, at least to the NF-Y and CCAAT-box component of the GTPCH
promoter response, has recently been presented to explain the ability
of protein kinase A to enhance transcription from a CCAAT-box alone (57). This model proposes that the NF-Y B subunit is a substrate for
protein kinase A and that phosphorylation of NF-YB by protein kinase A
enhances its ability to recruit NF-YC, NF-YA, p300/CBP-associated factor, and CBP to the CCAAT-box. The ubiquitous nature of NF-Y, however, and the fact that not all CCAAT-box elements mediate cAMP
responsiveness argue that additional trans-acting factors such as ATF-4 and C/EBP, which can bind noncanonical and therefore cryptic cis-acting elements, may ultimately be found to be
involved in this process.
,
TOP
ABSTRACT
INTRODUCTION
3') a Sp1/GC
box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an
E-box. Transcription from the core promoter in PC12 but not C6 or Rat2
cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis
showed that both the CRE and CCAAT-box independently contribute to
basal and cAMP-dependent activity. The combined CRE and
CCAAT-box cassette was also found to enhance basal transcription and
confer cAMP sensitivity on a heterologous minimal promoter. The
addition of the Sp1/GC box sequence to this minimal promoter construct
inhibited basal transcription without affecting the cAMP response. EMSA showed that nuclear proteins from PC12 but not C6 or Rat2 cells bind
the CRE as a complex containing activating transcription factor (ATF)-4
and CCAAT enhancer-binding protein 
To whom correspondence should be addressed: Dept. of Psychiatry
and Behavioral Neurosciences, 2309 Scott Hall, 540 E. Canfield, Wayne
State University School of Medicine, Detroit, MI 48201. Tel.: 313-577- 5965; Fax: 313-993-4269; E-mail: gkapato@med.wayne.edu.
