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INTRODUCTION |
Neurotensin (NT)1 is a
13-amino acid peptide (1) that is expressed in a complex pattern in the
limbic regions of the brain and in the gastrointestinal tract (2). NT
and the related peptide neuromedin N are generated from a common
precursor protein and are thought to have overlapping signaling
functions (3, 4). There is considerable anatomical and functional
evidence indicating that NT functions as a neuromodulator in the
dopamine (DA) pathways in the central nervous system. The majority of
midbrain DA neurons express the cloned high affinity NT receptor
(NTR-1), and there is evidence that at least some DA neurons are
directly contacted by NT-positive axons (5, 6). NT appears to excite
midbrain DA neurons both directly (7, 8) and through inhibition of DA
D2 autoreceptors (9) resulting in locomotor activation (10, 11). However, NT also attenuates D-amphetamine locomotor
activation after intracerebroventricular administration or after
direct application in the ventral striatum possibly through the
inhibition of post-synaptic D2 signaling (12-14). These
results suggest that the expression of NTR-1 in midbrain DA neurons is
important for appropriate regulation of DA-mediated behaviors.
NTR-1 was cloned using an expression assay in frog oocytes, and
sequence analysis revealed that it is a member of the G protein-coupled receptor superfamily (15). A lower affinity levocobastine-sensitive receptor (NTR-2) was subsequently cloned by low stringency
hybridization using an NTR-1 cDNA probe (16, 17). Recent evidence
suggests that NT does not stimulate signaling through NTR-2, suggesting that NT acts mainly through NTR-1 (18). NTR-1 is expressed at high
levels in midbrain DA neurons, and dopamine regulates NTR expression in
corticolimbic structures in the rat brain (19-21). The ability of DA
to modulate NTR expression suggests that changes in DA signaling result
in plastic changes in NT signaling. This hypothesis is further
supported by the observations that the indirect DA agonists cocaine and
methamphetamine and D2 antagonists stimulate NT gene
expression in the dorsal and ventral striatum (22, 23). Long term NTR-1
blockade and continuous infusion of NT also result in alterations in
NTR-1 expression (24, 25). These results collectively indicate that DA,
NT, and perhaps other signals can result in plastic changes in NTR-1
expression; however, the underlying mechanisms controlling NTR-1
expression are poorly understood.
Tissue culture cell lines that express NTR-1 in a regulated or
constitutive manner potentially provide model systems for understanding the mechanisms controlling Ntr-1 gene expression. The
N1E-115 neuroblastoma cell line was isolated as part of a screen for
catecholamine-producing neuronal cell lines from a mouse brain
neuroblastoma (26), and Me2SO-induced neuronal
differentiation of these cells (27) is accompanied by increased
expression of high affinity NT-binding sites (28). The transition of
these cells to a post-mitotic stationary phase also results in the
induction of NTR expression (29). A neuroblastoma × glioma cell
line has also been described that expresses high constitutive levels of
high affinity NT-binding sites (30). The N1E-115 cells are a
particularly attractive model to probe Ntr-1 gene regulation
since the Ntr-1 gene is expressed in catecholaminergic
neurons in vivo (31). They should also be useful for
identifying signaling mechanisms that activate the Ntr-1
gene during neuronal differentiation (28).
To investigate the pathways controlling Ntr-1 gene
expression in these and other cell types, we have cloned the mouse
Ntr-1 gene and sequenced the promoter region. Detailed
mutational analysis of the Ntr-1 promoter has revealed
sequence elements that are crucial for Me2SO induction and
basal expression in N1E-115 cells that are conserved in the rat and
human promoters. A CACCC sequence appears to be the most critical
sequence element for Me2SO responsiveness, and gel shift
and DNase I footprinting experiments indicate that an
Me2SO-inducible complex binds to this site. Several
sequence elements contribute to basal expression, including an
Sp1-related site and a sequence that is similar to the initiator
element first identified in the terminal deoxynucleotidyltransferase
gene (32). We also present evidence that a transcriptional silencer
controls the activity of this positive regulatory region. These results provide evidence that the Ntr-1 gene is transcriptionally
activated during Me2SO-induced neuronal differentiation of
N1E-115 cells most likely through a mechanism involving the induction
of a complex that binds to a sequence that includes a CACCC motif.
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EXPERIMENTAL PROCEDURES |
Isolation of Mouse Ntr-1 Genomic Clones--
A mouse genomic
library was constructed by ligating partially digested high molecular
weight DNA isolated from D3 embryonic stem cells (15-20-kb fragments)
into BamHI-digested 
EMBL4 using standard methods (33).
The library was screened using a 32P-labeled
SmaI fragment of the rat NTR-1 cDNA (15) that contains the 5' 1.3 kb of the cloned sequences and standard filter lift procedures (33). Positives were plaque-purified and used to prepare
phage DNA. Two positives were further characterized by restriction
mapping (mNTR2-2 and mNTR10-1). Promoter fragments from
mNTR2-2 were subcloned into pGEM4 (Promega), and the promoter region was sequenced using the chain terminator method.
Cell Culture--
N1E-115 cells were passaged in Dulbecco's
modified Eagle's medium containing 4.5 g of glucose/liter and
supplemented with 10% fetal bovine serum (Sigma) and 2 mM
L-glutamine. NG108 cells were passaged in the medium
described above for N1E-115 cells except supplemented with
hypoxanthine, aminopterin, and thymidine (Life Technologies, Inc.) as
described (30). Mouse embryonic fibroblast (MEF) cells were passaged in
Dulbecco's modified Eagle's medium containing 1.0 g of
glucose/liter and supplemented with 10% fetal bovine serum and 2 mM L-glutamine.
Transfections--
N1E-115 and MEF cells were subcultured from
confluent dishes by diluting the cells 1:4 with fresh medium 3 days
prior to transfection. All lines were subcultured the day before
transfection at a density that resulted in 1 × 106
cells per 10-cm dish at the time of transfection. CsCl-purified plasmid
DNA was transfected by calcium phosphate precipitation using 10-cm
dishes. Briefly, for each reporter plasmid, two 10-cm dishes were fed
with 8 ml of fresh medium just prior to transfection, and 25 µg of
plasmid DNA as a calcium phosphate precipitate were added to each dish.
DNA precipitates were prepared by diluting 50 µg of plasmid DNA
(luciferase reporter and pPGK
-gal standardization plasmids) into 700 µl of NTE (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA), 100 µl of 2 M
CaCl2, and 800 µl of transfection buffer (50 mM HEPES, pH 7.1, 180 mM NaCl, 2 mM
NaPO4), and the mixtures were incubated at room temperature
for 20-30 min. The calcium phosphate precipitate (800 µl) was added
dropwise to the cells, and after 16 h the medium was replaced, and
the cells from one 10-cm dish were resuspended by trituration and
plated into two 60-mm dishes. The cells were allowed to adhere to the
dish for 1 h; 1.5% Me2SO was added to one dish of
each pair, and the cells were incubated for an additional 72 h.
Cells were incubated for 72 h, and cell extracts were prepared
after washing the cells with ice-cold phosphate-buffered saline by
lysis in buffer containing 1% Triton X-100 as described (34).
Luciferase and -galactosidase activities were determined as
described previously (35).
Luciferase Reporter Constructs--
The initial NTR luciferase
reporter construct was created by ligating a mouse Ntr-1
gene 1.2-kb BamHI fragment, containing sequences 
1425 to
185 relative to the NTR methionine initiator codon, into a
BglII site just upstream of the luciferase gene in the pXP2
reporter plasmid (36). Convenient restriction sites (SmaI
and SacI) and exonuclease III digestion were used to create a series of 5' deletion mutants in pXP-2. Additional 5' and 3' deletion
constructs were created by PCR using appropriate primers. The PCR
primers contained 17 nucleotide regions that were complementary to
deletion end points and resulted in the introduction of a
BamHI site at the 5' end and a SacI site at the
3' end. The PCR fragments were digested with BamHI and
SacI and cloned into the SacI 5' deletion
construct described above digested with BamHI and
SacI. This minimal promoter fragment was initially selected
based on the positions of the transcriptional start sites that had been determined in rat and human (37, 38); however, the start point in the
mouse actually lies 5' to the SacI site used to create the
deletion clones. Clustered point mutations were created using the PCR
overlap extension protocol (39). Briefly, two overlapping PCR fragments
were generated using either a 5' primer that ends at the promoter
SmaI site and introduces a BamHI site and a 3' primer containing the clustered point mutations flanked by 10 and 6 nucleotide stretches of complementary sequence on the 3' and 5' sides,
respectively, or a 5' primer containing clustered point mutations as
described above and a 3' primer with the promoter SacI site
as an end point. The two fragments were isolated by electrophoresis on
a low melting temperature gel; the gel slices were melted, mixed, and a
third PCR reaction was performed using the outer 5' and 3' primers
described above. The resulting fragment was digested with
BamHI and SacI, gel-isolated, and ligated into the SacI deletion clone in pXP-2 described above digested
with BamHI and SacI. The promoter regions of the
mutant constructs were completely sequenced to verify that only the
intended changes had been introduced.
RNase Protection Assay--
Riboprobes were synthesized using
either mouse Ntr-1 gene promoter fragments or a fragment of
the rat NTR-1 cDNA subcloned into pGEM4 (Promega Biotech). The
mouse Ntr-1 gene plasmids were constructed by ligation of
either a 293-bp SacI fragment (SacII93, derived
from a 2.4-kb PstI fragment subclone) or a 497-bp
EcoRI/BamHI fragment (B/E 497, derived from a
1.2-kb BamHI subclone) into either SacI- or
BamHI- and EcoRI-digested pGEM4. A 300-bp
PstI fragment derived from pBSNTR2-2 (15) was subcloned
into pGEM4 digested with PstI and treated with calf
intestinal phosphatase to generate a probe for the quantitation of
NTR-1 mRNA (PstI 300). To synthesize
32P-labeled riboprobes, plasmids were linearized with
either EcoRI (B/E 497) or HindIII
(SacI 293 and PstI 300) and transcribed with either T7 (B/E 497) or SP6 (SacI 293, PstI 300)
RNA polymerase as described (40).
RNase protection assays were performed as described (40). Briefly, 10 µg of total RNA was mixed with 5 × 105 cpm of
32P-labeled riboprobe, dried, and dissolved in 30 µl of
hybridization buffer (80% formamide, 40 mM PIPES, pH 6.7, 0.4 NaCl, 1 mM EDTA). Reactions were denatured by heating
to 95 °C for 5 min and hybridized overnight (~16 h) at 45 °C.
Following hybridization, 300 µl of RNase digestion buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 300 mM NaCl, 20 µg/ml nuclease P1, 2 µg/ml RNase T1) was
added, and the reactions were incubated at room temperature for 1 h. Reactions were terminated by the addition of 20 µl of 10% SDS, 5 µl of proteinase K (10 mg/ml), incubation at 37 °C for 15 min,
followed by phenol extraction and ethanol precipitation. Protection
products were analyzed on a sequencing gel and visualized either by
autoradiography or on a PhosphorImager (Molecular Dynamics).
32P-Labeled RNA size markers were synthesized from a pGEM4
rat NT/N gene subclone (pGEM4-NT1.8) using a mixture of templates that were linearized with different restriction enzymes to yield transcripts of 113 (RsaI), 158 (HaeIII), 196 (PvuII), 242 (AvaII), 305 (HinfI), and
496 (DraI) nucleotides.
RT-PCR--
Mouse brain poly(A)+ RNA was treated
with DNase I to remove trace amounts of contaminating genomic DNA and
RT-PCR reactions using avian myeloblastosis virus reverse transcriptase
(Promega) and Taq polymerase (Roche Molecular Biochemicals)
using the conditions specified by the manufacturers, except that RT
reactions were performed at 50 °C and betaine was added to the PCR
reactions. The reverse transcription reaction was initiated after heat
inactivation of the DNase I (70 °C for 5 min) by the addition of 5 pmol of a gene-specific primer (PEX-411c
5'-TGGCGCAGAGAGGAGCGCACGCACGGCTGCCCAGC-3') complementary to nucleotides
411 to 376 of the mouse Ntr-1 gene and 25 units of avian
myeloblastosis virus reverse transcriptase (Roche Molecular
Biochemicals). PCR reactions were performed using either mouse brain
cDNA or embryonic stem cell genomic DNA templates mixed with 10 pmol of the PEX-411c primer and 10 pmol of one of three different 5'
primers (513, 5'-GTGGAAGCGCGAGGAGCCCG-3'; 548,
5'-TTTTGGATCCACTGCTGGGCGCGCC-3'; 566,
5'-CTCCAACACCCACCCTCCTCCACTG-3', the numbers correspond to the
position of the 5' end of the oligonucleotide on the mouse NTR-1
promoter). PCR reactions were supplemented with 2.5 M
betaine (Sigma) due to the high GC content of the promoter region, and
30 amplification cycles (94 °C, 15 s; 62 °C, 1 min; 72 °C, 1 min; the extension time was increased to 4 min on the last
cycle) were performed after an initial 1-min denaturation step at
94 °C (41).
DNase I Footprinting and Methylation Interference
Assays--
32P-End-labeled probes spanning the promoter
region were prepared by digestion of a 1.2-kb BamHI gene
fragment subcloned into pGEM4 with either EcoRI or
BamHI followed by Klenow fill-in with [32P]dATP. The plasmids were subsequently digested with
either BamHI or EcoRI, respectively; the
resulting fragments were separated on a 1% agarose gel, and the 496-bp
labeled EcoRI/BamHI fragments labeled at either
the EcoRI or BamHI sites were recovered by
centrifugation through a Gen Elute column (Supelco) and ethanol
precipitation. Nuclear extracts were prepared from N1E-115 cells that
had either been treated with 1.5% Me2SO for 48 h or
grown under control conditions as described previously (42) with minor
modifications (43) using approximately 20 dishes of cells grown to
confluency for each preparation. Protein concentrations were determined
using the Bradford method. DNase I footprint reactions were performed as described (35) by mixing 20,000 cpm probe, 160 µg of nuclear extract, and treatment with various amounts of DNase I (DNase I, pure,
ribonuclease-free, Worthington). Reactions were also performed without
the addition of nuclear extract to identify protected regions. The
DNase I-treated reactions were phenol/chloroform-extracted, ethanol-precipitated, analyzed on sequencing gels, and visualized by
autoradiography with an intensifying screen. Chemical sequencing reactions (G, G + A) were also run so that the footprinted regions could be precisely aligned with the DNA sequence.
Methylation interference reactions were performed as described (44)
using a 32P-labeled probe spanning nucleotides 589 to
507 generated by PCR and labeled by Klenow fill-in of a
BamHI site at the 5' end. The probe (10 µl, 5 × 105 cpm) was partially methylated by incubation in 200 µl
of dimethyl sulfate reaction buffer (sodium cacodylate, 1 mM EDTA, pH 8.0) containing 1 µl of dimethyl sulfate for
5 min at room temperature followed by the addition of dimethyl sulfate
stop buffer (1.5 M sodium acetate, 1 M
-mercaptoethanol, pH 7.0) and ethanol precipitation. The methylated
probe was then reacted with 50 µg of nuclear extract from N1E-115
cells that had been induced with 1.5% Me2SO for 72 h,
and bound complexes were separated from free probe by electrophoresis on a non-denaturing gel. The complexes were visualized by
autoradiography, and the region of the gel containing complexes 1 and 2 and unbound probe were excised, the DNA was electroeluted,
phenol/chloroform-extracted, and recovered by ethanol precipitation.
The DNA was resuspended in 1 M piperidine, incubated at
95 °C for 30 min, lyophilized several times, and analyzed on a
sequencing gel.
Gel Shift Analysis--
N1E-115 cells were propagated as
described for transfection analysis and treated with 1.5%
Me2SO or grown under control conditions in 10-cm dishes.
The cells were washed twice with ice-cold phosphate-buffered saline,
resuspended in 400 µl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 2 mM aprotinin, 2 mM pepstatin, and 2 mM leupeptin), and
incubated on ice for 15 min. The cells were lysed by the addition of 25 µl of 10% Nonidet P-40 and vigorous vortexing for 10 s followed
by centrifugation for 30 s. The supernatant was discarded; the
pellet was washed once with buffer A on ice, centrifuged, and
resuspended in 30 µl of buffer C (20 mM HEPES, 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 2 mM aprotinin, 2 mM pepstatin, and 2 mM leupeptin) on ice by vortexing for 10 s every 5 min
for 30 min. Nuclear extracts were stored at 80 °C. Binding
reactions were performed by mixing (10 µg, 1-2 µl) N1E-115 nuclear
extract in binding buffer (12.5 mM HEPES, pH 7.9, 100 mM KCl, 10% glycerol, 0.1 mM EDTA, pH 8.0, 1.5 mM dithiothreitol) supplemented with 3 µg of poly(dI-dC),
1 µg of acetylated bovine serum albumin, and in some cases unlabeled
competitor DNA (16-µl final volume). The reactions were incubated on
ice for 10 min, and 1 × 105 cpm probe was added
followed by incubation for 15-20 min at room temperature. The probe
was the same as that used for methylation interference. Binding
reactions were analyzed on low ionic strength Tris acetate acrylamide
gels as described (44).
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RESULTS |
Cloning and Characterization of the Mouse Ntr-1 Gene--
Mouse
NTR-1 clones were isolated by screening a genomic library constructed
from D3 mouse embryonic stem cell DNA with a 32P-labeled
1.3-kb SmaI 5' fragment of NTR-1 cDNA (15), and two were
further analyzed (2-2 and 10-1) by restriction mapping and Southern
blotting. Exon one was localized to an ~7-kb HindIII fragment that contained sequences extending ~2.0 kb 5' to the initiator methionine codon. A portion of the first exon and the 5'-flanking region were sequenced (Fig.
1A), and comparison with the
corresponding regions of the rat (37) and human (38) NTR-1 genes revealed a region that is highly conserved between all three species (Fig. 1B). This region contains Sp1-, CRE-, CACCC
box-, and initiator element-related sequences as well as a large 40-bp palindromic sequence (mPAL) that includes the initiator element-related sequences (Fig. 1B). This conserved sequence is located
within a 200-bp region that is required for expression.
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Fig. 1.
Sequence of the promoter region of the mouse
Ntr-1 gene and comparison with other NTR-1 sequences
and the dopamine D2 receptor
gene. A, a HindIII restriction
fragment of the mouse Ntr-1 gene was subcloned, and the
sequence of the 5'-flanking region and a portion of exon one was
determined on both strands using the chain terminator method. The
positions of the transcriptional start site (+) and the 5' end of a
mouse NTR-1 cDNA clone (*) are shown. B, comparison of
the mouse NTR-1 sequence underlined in A with the
corresponding regions of the rat and human NTR-1 promoters (sequence
differences are indicated). Potential transcriptional regulatory
sequences (overlined) in the conserved region and the
transcriptional start site (+) are indicated. C, sequence
comparison between the mouse NTR-1 and rat dopamine D2
receptor promoter (67) regions. Sequences that are identical are
boxed.
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RNase protection experiments were performed to map the approximate
location of the transcriptional start site. Two 32P-labeled
antisense riboprobes (depicted schematically in Fig. 2A) were hybridized to total
brain, kidney, spleen, or yeast RNA, followed by digestion with a
mixture of nuclease P1 and ribonuclease T1, and the resulting products
were analyzed on a sequencing gel. An antisense probe transcribed from
a subcloned SacI fragment (Sac293, 448 to 149
relative to the AUG start codon) was fully protected after
hybridization with mouse brain RNA but not the control RNAs, indicating
that the start site is located upstream of the region encompassed by
this probe (Fig. 2B). Hybridization of mouse brain RNA with
an antisense probe that extends 233 nucleotides further in the 5'
direction (B/E 497) resulted in three closely spaced protected
fragments (Fig. 2C). The estimated sizes of these protection
products indicate that transcription starts within the mPAL region (in
the vicinity of position 523). Identical protection results were
obtained with RNA isolated from Me2SO-induced N1E-115 cells
(data not shown). We attempted to confirm this result using several
different oligonucleotides for primer extension experiments using
poly(A)+ brain RNA and control RNAs isolated from several
different tissues, but no specific products were detected. Therefore,
we employed RT-PCR to delineate the 5' boundary of the gene using a
common 3' primer and several closely spaced 5' primers located at
successively further 5' positions. Control PCR reactions using the same
primer sets to amplify mouse embryonic stem cell genomic DNA were also performed (Fig. 2D). Specific products were detected by
electroblotting the RT-PCR products and hybridizing with a
32P-labeled rat Ntr-1 gene probe from within the
amplified region. Primers with 5' end points at 513 and 548
produced products of the expected size; however, much less product (at
least 10-fold less) was detected after RT-PCR with the 566 primer
(Fig. 2D). These results indicate that the major
transcriptional start site is close to position 548 (most likely at
545) near the 5' end of the mPAL within a sequence that is strikingly
similar to the consensus initiator element (32, 45).
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Fig. 2.
Mapping the transcriptional start site by
RNase protection and RT-PCR. RNase protection experiments were
performed by hybridizing either 32P-labeled
Sac293 (B) or B/E 497 (C) antisense
riboprobes with total RNA (10 µg) from the indicated tissues,
subsequent digestion with nucleases P1 and T1, and analysis of the
products on a sequencing gel. 32P-Labeled RNA markers were
prepared by transcription of a mixture of pGEM4-NT1.8 templates
linearized at different sites to produce transcripts of the indicated
lengths as described under "Experimental Procedures." A,
schematic diagram of the antisense riboprobes used. Thick black
lines represent Ntr-1 gene sequences, and open
lines represent pGEM4 vector sequences. The sizes of the regions
of the transcript derived from NTR-1 sequences and vector sequences are
indicated. The Sac293 probe encompasses a 293-nucleotide
region located between the SacI sites at 448 and 155.
The B/E 497 probe spans 497 nucleotides of the Ntr-1 gene
between the EcoRI site at 682 and the BamHI
site at 185. B and C, RNase protection products
resulting from hybridization to the Sac293 (B) or
B/E 497 (C) probes were analyzed on sequencing gels and
visualized by autoradiography. Specific protection products were
detected only in reactions containing brain RNA and are indicated by
arrows. D, RT-PCR reactions were performed as
described under "Experimental Procedures" using a common 3' primer
and three different 5' primers with 5' end points at the indicated
positions (566, 548, 513) to map the transcriptional start site
more precisely. RT-PCR reactions in which reverse transcriptase was
omitted from the initial reaction (no RT) were performed to ensure that
PCR-amplified products were derived from reverse-transcribed mRNA
templates. Control PCR reactions were also performed with the same
primers using mouse embryonic stem cell DNA as template. The reaction
products were analyzed on a 5% polyacrylamide gel, electroblotted, and
probed with a 32P-labeled fragment of the mouse
Ntr-1 gene (514 to 448, see A, Southern
probe). The gel was stained with ethidium bromide and photographed
prior to electroblotting (top panel), and hybridization
signals were detected by film autoradiography (bottom
panel). The results indicate that the major transcriptional start
site is in the vicinity of 548, since much less RT-PCR product was
generated with the 566 primer than with the 548 and 513
primers.
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Sequence analysis revealed that an approximately 500-bp region
surrounding the transcriptional start site has the structural features
of a CpG island (Fig. 3). CpG islands
have been identified in the promoter regions of a variety of
housekeeping and tissue-specific genes from higher eukaryotes (46, 47).
The CpG island in the mouse NTR-1 promoter region has close to the
expected frequency of CpG dinucleotides (CpG/GpC 
0.6) in a
region that has greater than 50% G + C content and encompasses at
least 500 bp (Fig. 3). Although the function of CpG islands remains
uncertain, they are commonly found in promoter regions, including a
large number of promoters that lack TATA elements like the NTR-1
promoter (46, 47).
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Fig. 3.
The transcriptional start site of the
Ntr-1 gene is located in a CpG island. The
sequence of the promoter region was analyzed in 100 nucleotide blocks
for G/C content and the frequency of CpG and GpC dinucleotides. The
ratio of CpG/GpC was calculated, and values of 0.6 in regions that
had 50% G + C content were defined as meeting the criteria for CpG
islands as described (43). A 500-bp region extending from approximately
140 bp upstream of the transcriptional start site to 350 bp into exon
one has the characteristics of a CpG island. A graph of the ratio of
CpG/GpC is shown, and the positions of the GpC and CpG dinucleotides
are depicted schematically below along with a schematic of the mouse
NTR-1 promoter region. The transcriptional start site is denoted by an
arrow, and the coding region is depicted as a black
box.
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Cell Density and Me2SO Treatment Increase Ntr-1 Gene
Expression in N1E-115 Cells--
N1E-115 cells plated at low density
are devoid of high affinity NT-binding sites; however, large increases
in binding activity are observed when the cells are grown to high
density (29) or treated with Me2SO (28). To examine whether
these culture conditions result in increased Ntr-1 gene
expression, NTR-1 mRNA levels were quantitated by nuclease
protection assay using a 32P-labeled antisense riboprobe
corresponding to nucleotides 296-632 of the rat NTR-1 cDNA
(PstI 300). N1E-115 cells were plated at low density (5 × 105 cells/15-cm dish) and propagated without subculture
but with frequent medium changes in the first set of experiments as
described (29). Cells were harvested from duplicate cultures at the
indicated times; RNA was prepared, and NTR-1 mRNA levels were
quantified using an RNase protection assay (Fig.
4A). Culturing the cells at
low density resulted in an initial decline in Ntr-1 gene
expression, followed by a gradual increase during growth to stationary
phase. NTR-1 mRNA levels increased >20-fold compared with day 3 levels after 17 days of culture but declined precipitously thereafter as the cultures deteriorated (Fig. 4). To examine Ntr-1 gene
expression following Me2SO treatment, cells were plated in
duplicate in medium containing reduced serum and treated with 1.5%
Me2SO for the indicated times (Fig. 4B).
Me2SO treatment resulted in a 4-fold increase in
Ntr-1 gene expression over the course of 72 h. These
results indicate that increased Ntr-1 gene expression
underlies the previously observed increases in NT-binding sites in
these cells (28, 29).
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Fig. 4.
Cell density and Me2SO induce
Ntr-1 gene expression in N1E-115 cells.
A, cell density experiments were performed in duplicate by
plating N1E-115 cells at low density and incubation without further
subculturing but with frequent medium changes as described under
"Experimental Procedures." Cells were harvested at the indicated
times for RNA preparation. Total RNA isolated from each duplicate
culture dish (10 µg) was hybridized with 32P-labeled
PstI 300 riboprobe and nuclease-treated, and the protected
products were analyzed on sequencing gels. The relative levels of NTR-1
mRNA were quantitated using a PhosphorImager, and autoradiographs
of the gels are shown below the graphs. The two
lanes under each time point represent the protection products
obtained with RNA preparations from each of the duplicate culture
dishes. B, Me2SO induction was analyzed by
treating N1E-115 cells with 1.5% Me2SO in duplicate for
the indicated times and quantitation of NTR-1 mRNA as described in
A.
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Promoter Sequences Required for Me2SO Induction in
N1E-115 Neuroblastoma Cells--
To determine what promoter sequences
are required for the response to Me2SO, a series of
promoter deletion constructs in which up to 1.4 kb of 5'-flanking
sequences were fused to a luciferase reporter gene were transfected
into N1E-115 cells, and the cells were either grown under control
conditions or treated with 1.5% Me2SO for 72 h. To
control for variations in transfection efficiency, a PGK-gal plasmid
was co-transfected, and -galactosidase activity was used to
standardize luciferase activity. The initial series of constructs
revealed two interesting features of the regulatory region. First,
sequences upstream of 640 (we have used the numbering system
previously used for the rat and human NTR-1 genes (37, 38)
where +1 is the A of the initiator methionine codon) appear to suppress
basal promoter activity (Fig.
5A). Second, promoter elements
required for Me2SO induction are located between a
SmaI site at 640 and a SacI site at 448 (Fig.
5A). The 640 deletion construct is induced 3-5-fold upon
Me2SO treatment; however, deletion to the SacI
site at 448 completely abolishes Me2SO induction, and
this construct is actually repressed after Me2SO treatment (Fig. 5A). The effect of Me2SO was selective,
since the expression of a reporter gene controlled by the Rous sarcoma
virus-long terminal repeat was not affected by Me2SO
treatment (data not shown). These experiments define an
Me2SO-responsive region between 640 and 448 and
indicate that sequences upstream of 640 suppress basal promoter
activity.
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Fig. 5.
Transfection analysis of Ntr-1 gene promoter region deletion constructs in N1E-115 cells.
A, an initial series of deletion constructs was constructed
by cloning the indicated restriction fragments upstream of the
luciferase reporter gene in pXP-2 (33). The plasmids were
co-transfected with a PGK-gal standardization plasmid into N1E-115
cells using calcium phosphate precipitation, and the cells were either
grown under control conditions (open bars) or in the
presence of 1.5% Me2SO (black bars) for 72 h. The cells were harvested, extracts were prepared, luciferase and
-galactosidase activities were determined, and -galactosidase
activity was used to correct for variations in transfection efficiency.
The constructs are depicted schematically, and their activity in light
units is plotted. B, an extensive series of 5' and 3'
deletion constructs was generated using PCR methods and tested as
described above. The constructs are depicted schematically and activity
is plotted as either fold induction or relative basal expression.
Transfections were performed in duplicate, and the luciferase and
-galactosidase activities were determined in duplicate for each
transfection. To correct for variations in transfection efficiency,
luciferase activity was divided by -galactosidase activity. Fold
induction was calculated by dividing the corrected induced value by the
corrected control value. Relative basal expression was calculated by
dividing the corrected mutant control value by the corrected wild type
control value. The mean values are plotted and the error
bars indicate the S.E. (n = 3-8). Promoter
fragments generated by PCR were completely sequenced to verify that
only the intended changes had been introduced.
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To determine more accurately the boundaries of the regulatory region,
additional 5' and 3' deletion mutants were constructed and tested in
N1E-115 cells (Fig. 5B). The response to Me2SO
was maintained through deletion to 589; however, deletion to 569 nearly abrogated the response. In contrast, basal activity decreased in
a graded fashion as sequences were deleted from the 5' end, except for
a small increase when sequences between 589 and 569 were deleted,
perhaps indicating that a weak repressor element is located in this
region. Deletion in the 3' direction from the SacI site at
448 to 526 had a severe impact on both basal activity and
Me2SO induction. Further 3' deletions had no effect until removal of an Sp1-like sequence (compare constructs 3'-590 and 3'-603
that have deletion end points on either side of the Sp1-like site),
which essentially eliminated expression, indicating that this Sp1-like
site is important for basal expression. These results indicate that the
Me2SO regulatory region lies in the 83-bp region located
between 589 and 507 and that the region between 640 and 507 is
required for full basal expression. This regulatory region is highly
conserved in the rat and human NTR-1 promoters (Fig. 1B),
indicating that it is functionally important in vivo.
The mPAL was examined in more detail by evaluating the effects of
clustered mutations along the length of the palindrome (Fig. 6A, mPal mut-1 to -6). Several
mutations reduced basal activity; however, mPal mut-6 had the largest
effect, reducing activity by ~70%. Removal of this sequence in the
3'-526 deletion construct greatly reduced both basal activity and
Me2SO induction (Fig. 5, A and B).
Mutations near the 5' end of the mPal all had similar effects, reducing
basal activity by about half (Fig. 6A, mPal mut-1, -3, and
-5). The transcriptional start site lies within the region affected by
mPal mut-3. Deletion of the entire mPAL (Fig. 6A,
dmPal) reduced basal expression by ~60%, consistent with
this sequence being important for basal expression; however, the
residual basal expression of this construct indicates that transcription can also initiate outside this region. A mutation near
the center of the mPAL (mPal mut-2) had little effect on basal activity
but severely curtailed Me2SO induction (Fig.
6A). Two other mutants that reduced basal expression had no
effect on Me2SO induction (mPal mut-4 and-5), whereas the
remaining mutations (mPal mut-1, -3, -6) reduced both basal and
Me2SO-inducible expression. These results indicate that the
mPAL is composed of multiple cis-regulatory sequences that
contribute to basal and Me2SO-induced expression. The mPAL
core (affected by mPal mut-2) appears to be most critical region
involved in Me2SO induction, although sequences at the 5'
border of the mPAL are also important.
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Fig. 6.
Sequences required for Me2SO
induction and basal expression of the Ntr-1 gene.
Clustered point mutations were introduced into the regulatory region
using the PCR overlap extension technique (36) and cloned upstream of
the luciferase reporter gene. All mutant promoters were completely
sequenced to verify the mutation and to confirm that no other
substitutions had occurred. The mutant promoters were analyzed as
described in Fig. 5. The mean values are plotted with the S.E.
indicated by error bars (n = 5-8).
Mutational analysis of the mPAL region (A) and the region
immediately upstream (B) are shown. Schematic diagrams of
the promoter regions analyzed and the specific substitutions are shown
below the graphs.
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To examine the regulatory properties of the mPAL in more detail, one
(mP12-4) or two (mP10-1) copies of an oligonucleotide spanning the
mPAL were cloned upstream of the SacI deletion construct (5'-448) that displays very low basal expression and is actually inhibited by Me2SO treatment (Fig. 6A). Both
constructs conferred some Me2SO responsiveness to the
deleted promoter, but two copies were required to increase basal
expression, although neither construct had full activity. To determine
whether inclusion of sequences just upstream of the mPal could restore
full Me2SO responsiveness, a construct containing sequences
between 589 and 507 cloned upstream of the SacI promoter
deletion was tested (Fig. 6A, URmP-1). Inclusion
of these more 5' sequences restored Me2SO induction nearly
completely, although the basal activity of this construct was increased
only marginally (similar to the mP10-1 construct). These results
provide additional evidence that sequences immediately upstream of the
mPAL and the mPAL itself are required for full Me2SO responsiveness.
To define the additional sequences located in the upstream region that
are required for full Me2SO induction, a series of mutations in this region were analyzed by transfection in N1E-115 cells
(Fig. 6B, mut-1 to -4). Mutants 2-4 had little effect on Me2SO induction (Fig. 6B), and none of the
mutants had a large effect on basal expression (data not shown). In
contrast, mut-1, which specifically alters the conserved CACCC box,
nearly abolished Me2SO inducibility (Fig. 6B).
These results indicate that the conserved CACCC box that lies
immediately upstream of the mPAL is required for Me2SO
induction. This result was somewhat surprising in view of the fact that
5' deletion to 569 results in a large drop in Me2SO
responsiveness (Fig. 5, 5'-569), and this deletion removes the
sequences mutated in mut-2 to -4. This apparent discrepancy could
result from either functional redundancy within the region mutated in
mut-2 to -4 (e.g. the sequence GTGGC is directly repeated), the presence of a cis-regulatory element within the deleted
region that is not inactivated by any of the individual mutations, or the creation of junction sequences during cloning that have an adventitious effect on activity. Thus, although it remains possible that one or more sequences in the region affected by mut-2 to mut-4
contribute to Me2SO responsiveness, the CACCC box is
clearly an important cis-regulatory element required for
Me2SO induction.
Similar Promoter Elements Are Required for Expression in NG108
Neuroblastoma Cells--
NG108 neuroblastoma × glioma cells
display high constitutive levels of high affinity NT-binding sites
(30). To determine whether similar promoter elements are required for
constitutive expression and to examine possible regulation by
Me2SO, these cells were transfected with a series of
promoter constructs using the same methods as were used for N1E-115
cells. Me2SO treatment reproducibly increased expression of
the wild type promoter construct (5'-640) by 2-3-fold (Fig.
7A). The results obtained in
these cells were similar to those obtained in N1E-115 cells. The 5'- and 3'-deletion mutants define a region between 589 and 507 that is
required for full induction, and the analysis of selected clustered
point mutations indicates that the CACCC box (URmut-1) and the center
of the mPAL (mPalmut-2) are critical sequence elements required for
induction (Fig. 7A). The only real difference was that mPal
mut-6 had a modest negative effect on Me2SO induction in
N1E-115 cells but slightly enhanced the response in NG108 cells. The
region required for constitutive expression (622 to 507) was also
similar to that found in N1E-115 cells. The major differences were that
5' deletion to 622 had no effect on constitutive expression in NG108
cells but decreased basal expression in N1E-115 cells by about
one-half, and several mPal mutants that reduce basal expression in
N1E-115 cells had no effect on expression in NG108 cells (mPalmut-1,
-3, and -5). These results indicate that similar promoter elements are
required for expression in these two neuroblastoma cell lines and
provide further evidence that the CACCC box and the core of the mPAL
are critical elements required for Me2SO induction.
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Fig. 7.
Transfection analysis of Ntr-1 gene promoter constructs in NG108 and MEF cells. A subset of
the reporter constructs diagrammed in Figs. 5 and 6 were transfected
into either NG108 cells or MEF cells together with a PGK--gal
standardization plasmid as described for N1E-115 cells. Initial
experiments indicated that the full-length Ntr-1 gene
promoter construct was induced by Me2SO in NG108 cells but
not in fibroblasts. Therefore, reporter constructs were transfected
into NG108 cells and assayed exactly as described for N1E-115 cells in
the presence or absence of 1.5% Me2SO, but
Me2SO induction was not further analyzed in MEF cells.
A, NG108 cells were transfected with the indicated
constructs and grown either under control conditions or in the presence
of 1.5% Me2SO for 72 h. The data are plotted as fold
induction as described for N1E-115 cells (n = 3-4).
B, the basal activity of the indicated NTR-1 promoter
constructs in either NG108 (solid bars) or MEF
(diagonal fill bars) cells is plotted relative to the
activity of the 640 wild type promoter construct (n = 3-4). C, the relative activity of the 640 wild type
Ntr-1 gene promoter construct in the different cell lines
was calculated by dividing luciferase activity by -galactosidase
activity. In each case, cells were transfected with 10 µg of the
640 NTR-1 promoter luciferase and 2.5 µg of the PGK--gal
reporter constructs. The data are plotted as light
units/-galactosidase units, and the standard error is indicated
(n = 3).
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Distinct Promoter Requirements for Expression in Mouse Embryo
Fibroblasts--
To determine whether the NTR-1 promoter is expressed
in a cell type-specific manner, the same series of constructs that were tested in NG108 cells were also transfected into mouse embryo fibroblasts that do not express the endogenous Ntr-1 gene
(data not shown). Preliminary experiments revealed that
Me2SO treatment of these cells had no effect on the
expression of the wild type promoter, indicating that Me2SO
induction is cell type-specific. The wild type promoter (5'-640) was
active in these cells; however, this basal expression was dependent on
largely different promoter elements compared with the neuroblastoma
cell lines. There were several major differences. First, the Sp1-like
sequence that is removed in the 5'-589 promoter construct appears to be
unimportant for expression in MEF cells (Fig. 7B). Second,
deletion of the region between the Sp1-like site and the mPAL (5'-548)
essentially abolished expression in MEF cells but had only a modest
effect in NG108 cells (Fig. 7B) and essentially no effect in
N1E-115 cells (Fig. 5B). The CACCC box appears to be
critical for basal expression in these cells (Fig. 7B), in
contrast to the situation in the neuroblastoma cell lines where the
CACCC box is required for Me2SO induction and makes at most
a modest contribution to basal expression (data not shown). The mPAL
was also found to be important for basal expression in MEF cells;
however, the core of the mPAL appears to be extremely important for
basal expression in these cells (Fig. 7B, mPal mut-2) but
again is mainly involved in Me2SO induction in the
neuroblastoma cell lines. These results indicate that substantially
different sequence elements are responsible for basal expression in
MEFs as compared with neuroblastoma cells. The endogenous
Ntr-1 gene must normally be repressed in fibroblasts so that
the sequence elements identified in transient transfection experiments
are masked in the endogenous gene.
Factors Binding to the Regulatory Region--
DNase I footprint
analysis was used to identify protein-binding sites in the
Ntr-1 gene regulatory region. Nuclear extracts were prepared
from N1E-115 cells that were either grown under control conditions or
treated with 1.5% Me2SO for 48 h.
32P-End-labeled promoter fragments were mixed with protein
from either control or induced cells, incubated on ice, treated with different concentrations of DNase I, phenol-extracted, and analyzed on
a sequencing gel (Fig. 8A). A
probe labeled at the BamHI site at 175 that extends to an
EcoRI site at 680 revealed that the functional promoter,
defined by the mutational analysis described above, is extensively
protected by nuclear extracts from Me2SO-induced cells
(Fig. 8A). The specific protections are indicated by
circles and fall into six footprinted regions
(FP1-6). Inducible or partially inducible protections are
represented by open and gray circles, respectively, and constitutively protected sites are represented by
black circles. Comparison of the patterns obtained with
Me2SO-induced and control N1E-115 nuclear extracts
indicates that FP1-3 and FP6 are induced by Me2SO; FP4 is
better protected by Me2SO-induced cell extracts, and FP5 is
constitutive. PC12 cell nuclear extract resulted in a protection
pattern that was nearly identical to that obtained with control N1E-115
cell extracts (data not shown), indicating that the pattern obtained
with uninduced control cell extracts is not cell type-specific. PC12
cells express no detectable NTR-1 mRNA (data not shown). A cluster
of constitutive hypersensitive sites (indicated by arrows in
Fig. 8A) was observed just downstream of a putative Sp1
site, indicating that this region is occupied in both
Me2SO-induced and control cells. The significance of FP5 and FP6 is uncertain since deletion of this region has no effect on
either basal expression or Me2SO induction. However, the
Me2SO-inducible footprints FP3 and FP4 lie over
functionally important sequences.
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Fig. 8.
DNase I footprint analysis of the mouse
Ntr-1 gene regulatory region. DNase I
footprinting assays were performed using an
EcoRI/BamHI mouse NTR-1 promoter fragment
32P-end-labeled at either the BamHI
(A) or EcoRI (B) site and nuclear
extracts (160 µg) from either control () or
Me2SO-induced (48 h, +) N1E-115 cells. A series of
reactions containing increasing amounts of DNase I were performed, and
equal counts (2,000 cpm) were loaded on a sequencing gel for analysis.
The products of a reaction in which the probe was treated with DNase I
in the absence of protein (NP) were also analyzed. Lanes in
which the extent of probe digestion was similar were compared and are
depicted. Chemical sequencing reactions (lanes G/A, G) were
used to generate markers for the alignment of footprinted regions on
the promoter sequence. DNase I protections are indicated by either
open circles (Me2SO-inducible), gray
circles (partially inducible), or black circles
(constitutive). Footprinted regions are indicated schematically by
boxes shaded like the circles and are
numbered 1-6. The mouse NTR-1 promoter sequence in the
region analyzed is depicted to the right of the
autoradiographs and promoter schematics, and the positions of the mPAL
and CACCC box are indicated. B, the antisense strand was
labeled, but the coding sequence is shown for clarity.
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To examine FP-1 in more detail, the EcoRI/BamHI
promoter fragment was labeled at the EcoRI site and
subjected to footprint analysis using Me2SO-induced and
control N1E-115 cell nuclear extracts (Fig. 8B). The
footprint was found to consist of a region of Me2SO-induced
protections (FP1A) and an adjacent region that was constitutively
protected (FP1B). The constitutive footprint at least partially
overlaps a half-CRE site (CGTCA), and many CREs bind cAMP-response
element family proteins constitutively (48). The
Me2SO-inducible FP1A is within a region that the 5'-622 deletion construct indicates is required for full basal expression in
N1E-115 cells but does not affect Me2SO induction (Fig.
5B); thus, the significance of this site is uncertain. Since
there is a relative paucity of strong DNase I cleavages 5' of FP1A, it
remains possible that additional proteins may bind to this region.
To characterize further the factors binding to the promoter region, gel
shift experiments were performed using a probe that spans the mPAL and
CACCC element (589 to 507). Nuclear extracts were prepared from
N1E-115 cells that were either treated with 1.5% Me2SO for
72 h or grown under control conditions and incubated with the
32P-labeled promoter fragment, and the resulting complexes
were analyzed on non-denaturing acrylamide gels (Fig.
9A). At least four complexes
(Fig. 9A, complexes 1-4) were identified that
were specifically competed by cold wild type probe fragment (longer exposure times were required to detect complexes 3 and
4, Fig. 9A, bottom panel). To examine
whether the functional sequence elements defined in the transfection
experiments described above were required for the formation of these
complexes, competition experiments were performed with wild type and
mutant promoter fragments. An oligonucleotide corresponding to the
collagenase TRE (49) was used as a nonspecific competitor. Competition
with a cold mutant promoter fragment containing clustered point
mutations in the CACCC element (mut-1 in Fig. 6B)
identified two complexes that require this sequence for binding (Fig.
9A, 4th lane, complexes 1 and 2). Comparison between the induced and control lanes
indicates that Me2SO treatment results in the specific
induction of complex 1 (Fig. 9A, compare 2nd and
6th lanes). Close DNA contacts in the inducible
complex were analyzed using a methylation interference assay (Fig.
9B). Complexes were formed by mixing partially methylated 32P-end-labeled probe (the same probe used in the
experiment depicted in Fig. 8A) with nuclear extract from
Me2SO-induced N1E-115 cells, separated on a native
acrylamide gel, and the regions corresponding to complex 1/2 and free
probe were excised for analysis of methylated G residues on sequencing
gels. Several bands were underrepresented in complex 1/2 compared with
free probe, specifically the Gs that are complementary to last three Cs
in the CACCC sequence (Fig. 9B). Analysis of the other
strand was not informative most likely due to the absence of G residues
in the region containing the CACCC sequence. These results indicate
that the Me2SO-inducible complex makes specific DNA
contacts in the CACCC element, since complex 1 is the major component
of the complex 1/2 band in Me2SO-induced cell extracts (see
Fig. 9A).
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Fig. 9.
An Me2SO-inducible complex binds
to the CACCC element in the NTR-1 regulatory region. A,
a 32P-labeled probe spanning the mPAL and immediate
5'-flanking sequences (589 to 507) was incubated with nuclear
extracts prepared from N1E-115 cells that had either been treated with
1.5% Me2SO for 72 h or grown under control conditions
as indicated, and the resulting complexes were analyzed on a
non-denaturing acrylamide gel and visualized by autoradiography with an
intensifying screen for 16 h. A longer exposure (72 h) of the
upper part of the gel is shown below to visualize complexes 3 and 4. Binding reactions were performed either in the absence of unlabeled
competitor DNA (2nd and 6th lanes) or
in the presence of a 100-fold molar excess of either wild type
(WT), CACCC mutant (Mut 1), or collagenase TRE
(Col TRE) DNA. Complexes 1 and 2 require an intact CACCC
element for binding. B, nucleotide contacts required for
binding of complexes 1 and 2 were analyzed by methylation interference.
The probe described above was partially methylated with dimethyl
sulfate, mixed with nuclear extract from Me2SO-induced
cells, and the resulting complexes were separated on a low ionic
strength polyacrylamide gel. The regions of the gel containing
complexes 1 and 2 (lane B, bound) and free probe (lane
F, free) were excised, the DNA was purified, cleaved with
piperidine, and the products were analyzed on a sequencing gel. The
input probe was also analyzed (lane P, probe). C,
complex 2 is closely related to Sp1. The probe described above was
incubated with nuclear extracts of cells treated with 1.5%
Me2SO for 72 h, and the resulting complexes were
analyzed on a non-denaturing acrylamide gel. Binding reactions were
performed either in the absence of competitor (No comp) or
in the presence of a 100-fold molar excess of oligonucleotides
containing either a consensus Sp1-binding site (Sp1) or the
collagenase TRE (Col TRE). To examine whether complexes 1 and 2 were immunologically related to Sp1, two different amounts (1 and
4 µl) of either an Sp1-specific or control JunB-specific antibody
were added to the binding reactions as indicated. The free probe was
also loaded (Probe).
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The CACCC box is a binding site for a number of zinc finger
transcription factors, including Sp1-related and Krüppel-like proteins (50-52). To examine whether complexes 1 and 2 were
Sp1-related factors, competition and antibody detection experiments
were performed using the same probe (Fig. 9C). A competitor
oligonucleotide containing a consensus Sp1 site effectively competed
for binding of complex 2 but not complex 1, indicating that complex 2 has a binding specificity similar to Sp1. To determine whether complex
2 contains Sp1, a specific Sp1 antibody was added to the binding
reaction at two different concentrations (1-4 µl), and for
comparison a Jun B-specific antiserum was added to control reactions.
Complex 2 was abolished by addition of 4 µl of Sp1 antiserum but was
not affected by the same amount of JunB antiserum, indicating that
complex 2 contains Sp1 (Fig. 9C). These results provide
evidence that complex 2 is due to binding of Sp1 to the CACCC box;
however, complex 1 appears to be due to a distinct factor(s), perhaps a
Krüppel-like protein, that is specifically induced during
Me2SO-induced neuronal differentiation of N1E-115 cells.
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DISCUSSION |
Several Sequences, Including a CACCC Box, Are Required for
Me2SO Induction of Ntr-1 Gene Expression--
DNA
transfection experiments were used to define promoter elements required
for Ntr-1 gene induction in neuroblastoma cells. Previous
work has shown that Me2SO-stimulated neuronal
differentiation of N1E-115 neuroblastoma cells is accompanied by
increased expression of high affinity neurotensin-binding sites (28).
We demonstrate here that Me2SO treatment induces the
expression of the endogenous Ntr-1 gene from 3- to 5-fold
and has a similar effect on the expression of a luciferase reporter
gene driven by NTR-1 promoter sequences. Me2SO also
increased Ntr-1 gene expression in NG108 neuroblastoma × glioma hybrid cells that have previously been shown to express high
affinity NT-binding sites (30). This was a selective effect since the
expression of a luciferase reporter gene controlled by the Rous sarcoma
virus-long terminal repeat was not affected by Me2SO
treatment of either neuroblastoma cell line. In contrast, Me2SO had no effect on NTR-1 reporter constructs
transfected into fibroblasts. Mutational analysis of the NTR-1 promoter
has defined an 80-bp region encompassing a CACCC box and large GC-rich
palindrome as critical for Me2SO induction. The CACCC box
is bound by an Me2SO-induced complex in N1E-115 cells.
Several regions within the mPAL are required for full Me2SO
induction; however, the central core appears to be the most critical
Me2SO-responsive sequence. The transcriptional start site
was mapped to the 5' end of the mPAL to a region that closely matches
the initiator element that was first identified in the terminal
deoxynucleotidyltransferase gene (32). The regulatory sequences
identified through transfection analysis here are highly conserved
between mouse, rat, and human (Fig. 1B), indicating that
they are functionally important in vivo. These results
provide evidence that a conserved region surrounding the
transcriptional start site is critical for Ntr-1 gene
activation during a program of neuronal differentiation initiated by
Me2SO treatment in N1E-115 cells.
The CACCC box was first identified through sequence comparisons of
-globin genes and has been shown to be important for expression in
erythroid cells. This site binds a number of related zinc finger transcription factors, including Sp1- and Krüppel-related
proteins (50, 53). The majority of the characterized
Krüppel-related genes are expressed in tissue-restricted
patterns. Gene targeting approaches have revealed that the erythroid
Krüppel-like factor is essential for high level
erythroid-specific -globin gene expression (54, 55) and that lung
Krüppel-like factor is essential for T-cell development (56),
indicating that specific Krüppel-like proteins play key roles in
transcriptional control and terminal differentiation. The -globin
CACCC boxes are required for activation during
Me2SO-induced differentiation of mouse erythroleukemia cells (57), possibly through the phosphorylation of erythroid Krüppel-like factor (58). The CACCC box in the NTR-1 promoter is
clearly required for Me2SO induction and is bound by both
constitutive and Me2SO-inducible complexes in N1E-115
neuroblastoma cells. The major constitutively expressed complex is
closely related to Sp1 (Fig. 9C, complex 2). The
inducible complex clearly has a binding specificity different from that
of the Sp1-related proteins and makes close contacts within the CACCC
box, similar to Krüppel-like proteins (see for example Ref. 53).
These data suggest a model in which the induction of one or more
Krüppel-like factors is required for Ntr-1 gene
activation during N1E-115 cell differentiation. Intriguingly, both
gut-enriched (52) and neuron-enriched (59-61) Krüppel-like
factors have been described, and these are the major sites of
Ntr-1 gene expression in vivo (15, 31).
The mutational analysis of the mPAL revealed that it most likely
consists of several independent cis-active elements that influence Me2SO-induced and basal expression. Several
mutations along the length of the mPAL reduced Me2SO
inducibility in both N1E-115 and NG108 cells (Figs. 6A and
7A). Alteration of sequences near the center of the mPAL
greatly reduced Me2SO responsiveness (Figs. 6A
and 7A, mPal mut-2). This sequence is a perfect
palindrome consisting of alternating G and C residues and is conserved
in both the rat and human genes; however, comparisons with known transcription factor-binding sites did not reveal any close
similarities, indicating that either it is a novel binding site or
functions as a DNA structural element. Mutation of an additional
sequence located at the 5' end of the palindrome (mPal mut-3) also
decreased Me2SO activation in both N1E-115 and NG108 cells
but appears to affect a distinct functional element. This sequence is
nearly identical to the consensus initiator element, and mPal mut-3
also reduced basal activity in N1E-115 and MEF cells but surprisingly not NG108 cells (Figs. 6B and 7B). The major
transcriptional start site was also mapped to this location making it
likely that this is a functional initiator. DNase I footprint analysis
indicates that factor binding to this site is regulated by
Me2SO in N1E-115 cells (Fig. 8A). This could be
due to either increased expression of a factor that binds to this site
or cooperative interactions with the Me2SO-inducible CACCC
box-binding factor. Thus, the mutational analysis indicates that there
are at least two distinct cis-regulatory elements within the
mPAL that are required for full Me2SO responsiveness, and
DNase I footprint analysis indicates that Me2SO-inducible complexes bind in the mPAL region (Fig. 8, A and
B). The central portion of the palindrome contains few DNase
I-sensitive sites; thus, further analysis will be required to determine
whether Me2SO-inducible complexes bind to the mPAL core
region. These results coupled with the results discussed above for the
CACCC box indicate that Me2SO induction of Ntr-1
gene expression requires cooperative interactions between multiple
sites, including the CACCC box, the mPAL core, and the initiator element.
Different Promoter Sequences Required for Basal Expression in
Neuroblastoma and MEF Cells--
There were several differences in the
sequences required for basal expression in the three cell lines,
although the results obtained in N1E-115 and NG108 cells were similar.
A notable difference was that the CACCC box and the central region of
the mPAL are required for high level basal expression in MEF cells but
are principally involved in Me2SO responsiveness in the
neuroblastoma cell lines. The contribution of the CACCC box to basal
expression in MEFs is most likely due to the expression of one or more
CACCC box-binding proteins in these cells, for instance BKLF is
expressed in fibroblasts (62). A CACCC box has been shown to be
important for -globin gene expression in transient expression assays
in non-erythroid cell lines (63), although this element mediates cell-specific expression through the binding of erythroid
Krüppel-like factor in erythroid cells (54, 55, 57). The
endogenous Ntr-1 gene is most likely repressed in most cell
types and only accessible to Krüppel-like proteins and perhaps
other CACCC box-binding proteins in neurons and a restricted set of
other cell types. The induction of these proteins during neuronal
differentiation or in response to environmental cues could underlie
Ntr-1 gene activation in specific neuronal populations.
There were also differences in the requirement for the Sp1-like
sequence located between 590 and 603 and a sequence near the 5' end
of the positive regulatory region for basal expression. The Sp1-related
sequence was clearly important for basal activity in the neuroblastoma
cell lines but not in MEF cells (compare 3'-590 and 3'-603 in Figs.
5B and 7B). Sp1 is constitutively expressed in
N1E-115 cells (see Fig. 9A, complex 2), and
nuclear extracts from both control and Me2SO-induced cells
create DNase I-hypersensitive sites just downstream of the Sp1-related
sequence indicating that this site is constitutively occupied.
Sequences near the 5' end of the positive control region were found to
be important for basal expression in N1E-115 cells (Fig. 5B,
compare 5'-640 and 5'-622) but not in NG108 or
MEF cells (Fig. 7B). DNase I footprinting experiments
indicate that this region binds both constitutive and
Me2SO-inducible factors (Fig. 8B), and sequence
comparisons indicate that this region is similar to a neural specific
regulatory element identified in the Drosophila
dopadecarboxylase (Ddc) gene (64). The Ntr-1 gene
is expressed at high levels in midbrain dopamine neurons (31), and the
homology to the Drosophila neural element raises the
intriguing possibility that this region may be required for expression
in catecholamine-producing neurons (N1E-115 cells produce catecholamines).
Although there were significant differences in the promoter elements
required for basal expression, certain regions of the mPAL are
important in all the lines examined. The most critical sequence is
defined by mPal mut-6 which had a severe impact on basal expression in
all lines but had only a modest effect on Me2SO induction
in N1E-115 cells (Figs. 6A and 7, A and
B). The sequence affected by this mutation is similar to the
consensus NGFI-A/Egr-1/Krox 24-binding site (65), and DNase I footprint analysis indicates that it is bound by a factor that is regulated by
Me2SO but also expressed in uninduced N1E-115 cells (Fig.
8A). Neurotrophic factors and other stimuli induce
ngfi-A gene expression (65); however, further work will be
required to determine whether NGFI-A plays a role in Ntr-1
gene expression.
Transcription Initiates within the mPAL--
A combination of
RNase protection experiments and RT-PCR were used to map the
transcription initiation site of the Ntr-1 gene to a
position that is either at or near a consensus initiator sequence at
position
, and
TOP
ABSTRACT
INTRODUCTION
