An E-box Motif Residing in the Exon/Intron 1 Junction Regulates
Both Transcriptional Activation and Splicing of the Human
Norepinephrine Transporter Gene*
Chun-Hyung
Kim
§,
Paul
Ardayfio
, and
Kwang-Soo
Kim
¶
From the
Molecular Neurobiology Laboratory, McLean
Hospital, Harvard Medical School, Belmont, Massachusetts 02478 and the § Department of Anatomy and Neurobiology, University
of Tennessee, College of Medicine, Memphis, Tennessee 38163
Received for publication, February 9, 2000, and in revised form, March 20, 2001
 |
ABSTRACT |
The norepinephrine transporter (NET) is
responsible for the rapid NaCl-dependent uptake of
norepinephrine into presynaptic noradrenergic nerve endings. Recently,
we have characterized the structural organization of the 5' upstream
promoter region of the human NET (hNET) gene. A new intron of 476 base
pairs was found in the middle of the 5'-untranslated leader sequence
and was shown to robustly enhance the promoter activity. Here, we show
that the first hNET intron enhances both the homologous hNET and the
heterologous thymidine kinase promoter activities in an orientation-
and position-dependent manner. The first hNET intron exhibited a similar promoter-enhancing effect in both SK-N-BE(2)C (NET-positive) and HeLa (NET-negative) cell lines, showing that its
function is not cell-specific. Transient transfection assays of a
series of deletional constructs show that the first hNET intron
contains subdomains with either positive or negative regulatory functions. Furthermore, DNase I footprinting analysis demonstrated that
the 5' side of the intron, encompassing the splice donor site, is
prominently protected by nuclear proteins isolated from both
SK-N-BE(2)C and HeLa cells. The protected nucleotide sequence contains
a consensus E-box motif, known to regulate diverse eukaryotic genes,
which overlaps with the splice donor site of the first intron. We
demonstrate that two basic helix-loop-helix proteins, upstream
stimulatory factors 1 and 2, are major proteins interacting at this
site and that the E-box is at least in part responsible for the
promoter-enhancing activity of the first intron. Furthermore, site-directed mutagenesis of the splice donor site of the first intron
affects both correct splicing and transcriptional activity. Taken together, our results indicate that a
cis-element residing at the first exon/intron junction,
encompassing an E-box motif, has a unique dual role in basal
transcriptional activation and splicing of hNET mRNA.
 |
INTRODUCTION |
Norepinephrine (NE)1 is
directly involved in mood stabilization, sleep regulation, expression
of aggression, and the general degree of alertness and arousal, as well
as in exerting central control over the endocrine and autonomic nervous
system. NE neurotransmission is terminated by the norepinephrine
transporter (NET), located on NE nerve terminals, which is responsible
for the rapid reuptake of NE into the presynaptic neurons (1). The
clinical importance of noradrenergic transmission is suggested by the
therapeutic usefulness of tricyclic antidepressant drugs, which enhance
the synaptic availability of NE through the inhibition of NE transport into presynaptic terminals. These findings provided support for the
catecholamine hypothesis that depression is associated with a decrease
in the levels of catecholamines, particularly norepinephrine (2).
Consistent with this catecholamine hypothesis, a recent analysis showed
that the levels of norepinephrine and its metabolites dihydroxyphenylglycolaldehyde and
monohydroxyphenylglycolaldehyde are significantly reduced in
the internal jugular venous plasma of depression patients (3).
Furthermore, a missense mutation (G to C) in exon 9 of NET gene has
recently been identified and found to cause a 98% loss of function
(4). It is of great interest that this mutation is associated with
hyperadrenergic states, leading to orthostatic intolerance (4).
Collectively, regulation of NET expression may significantly affect NE
neurotransmission, and its abnormal expression may play a role in the
pathophysiology of major depression and cardiovascular disorder (4,
5).
NET gene expression is controlled by various physiological and
pharmacological signals in noradrenergic neurons and neurosecretory cells. Levels of NET mRNA in the locus coeruleus and the adrenal medulla were decreased by the treatment with reserpine or
glucocorticoid but were increased by nerve growth factor (6, 7).
Several studies suggest that both short and long term function of NET may be modulated by metabolic hormones, such as insulin and thyroid hormone (8-10). In addition, angiotensin II has been shown to play an important role in the stimulation of NET gene transcription mediated by Ras-Raf-MAP kinase and PKC pathways in neuronal cultures (11, 12). In the superior cervical ganglia, leukemia inhibitory factor
and ciliary neurotrophic factor suppressed the level of NET mRNA,
whereas retinoic acid increased NET mRNA expression (13). The NET
mRNA is transiently elevated in locus coeruleus neurons following
either kainic acid-induced status epilepticus (14) or
penytylenetetrazol-induced seizures (15). At present, the molecular
mechanisms of NET gene regulation by these physiological and
pharmacological stimuli are poorly understood.
The human NET (hNET) gene is a highly dispersed locus with ~2 kb of
coding exons spread across 45 kb of genomic DNA and located at
chromosome 16q12.2 (16). Recently, we have characterized the structural
organization of the 5' upstream promoter region of the hNET gene (17).
A new intron of 476 bp was shown to reside in the middle of the
5'-untranslated leader sequence, and two major transcription initiation
sites were determined. Whereas the 5' upstream 9.0-kb sequence contains
important regulatory information for the cell-specific expression of
the hNET gene, the first intron increases the transcriptional activity
by ~10-40-fold, depending on the cell lines tested. These
observations suggest that the first hNET intron may play an important
role as a typical enhancer in NET gene regulation.
To further elucidate its regulatory mechanisms, we initiated a
systematic functional analysis of the first hNET intron. In addition to
showing that the first hNET intron enhances the promoter activity in an
orientation- and position-dependent manner, this study
identifies a cis-regulatory element, an E-box motif residing in the junction of the first exon and intron, that is critical for
basal transcriptional activation of NET mRNA via interaction with
transcription factors USF1 and USF2. Furthermore, we demonstrate that
the same cis-element directly regulates correct splicing of
hNET mRNA because it coincides with the splice donor site of the
first intron.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transient Transfection Assays--
Human
neuroblastoma SK-N-BE(2)C and HeLa cell lines were used as the
NET-positive and NET-negative cell lines respectively. Cell lines were
grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum (Hyclone), 100 µg/ml streptomycin, and 100 units/ml penicillin in a CO2 incubator. Transfection was
performed by the calcium phosphate coprecipitation as previously
described (18, 19). Plasmids for transfection were prepared using
Qiagen columns (Qiagen Co., Santa Clarita, CA). For the SK-N-BE(2)C
cell line, each 60-mm dish was transfected with an equimolar amount (0.5 pmol) of each reporter construct, 1 µg of
pRSV-
-galactosidase, and pUC19 plasmid to a total of 5 µg of DNA.
For the HeLa cell line, twice as much DNA was used in transfection.
Cells were harvested 72 h after transfection, lysed by freeze-thaw
cycles, and assayed for CAT activities. To correct for differences in
transfection efficiencies among different DNA precipitates, CAT
activity was normalized to that of
-galactosidase. CAT and
-galactosidase activities were assayed as previously described (18,
19).
Plasmid DNA Constructions--
The constructions of pNET1400CAT
and pNET1400(i)CAT were described previously (17). In order to
investigate whether the first intron functions in conjunction with a
heterologous promoter, the 540-bp BamHI fragment containing
the first intron was also cloned into the BglII site of
pBLCAT2 (20) containing the herpes simplex virus thymidine kinase (TK)
promoter to form TK(i)CAT. The same BamHI fragment
was made blunt with Klenow fragment and subcloned into either upstream
NET promoter or downstream CAT encoding sequence to generate
pNET(i)1400CAT and pNET1400CAT(i), respectively. Similarly, the
blunt-ended fragment was cloned into either upstream TK promoter or
downstream CAT encoding sequences to generate (i)TKCAT and TKCAT(i),
respectively. The plasmid pNET1400(i-rev)CAT was constructed by
subcloning the SmaI-SmaI fragment covering positions +230 ~ +586 within the first intron in the reverse
orientation. The orientation of the first intron was confirmed by
restriction enzyme mapping and sequence analysis.
For the internal deletion constructs, intronic sequence was
amplified by PCR using sense primer
(5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3') containing
restriction enzyme SpeI and antisense primer (catoligo2; 5'-CATTTTAGCTTCCTTAGC-3'), digested with PmlI and
XhoI, and subcloned into pNET1400(i)CAT that had been
digested with PmlI and XhoI to yield
pNET1400(i-spe)CAT. The 120-bp fragment including the intact
splicing acceptor site was also amplified by PCR using sense primer
(5'-GGATAACTAGTTTATCCAAGCAGAGCCTCGGCGTG-3') and catoligo2 primer, cut
with SpeI and XhoI, and subcloned into
pNET1400- (i-spe)CAT, which was digested with SpeI and
XhoI to generate pNET1400(i
190-645)CAT. In order to
subclone the PCR fragments into the pNET1400(i
190-645)CAT, the 5'
primers were designed to contain a SpeI site, and the 3' primers were designed to contain a XbaI site. PCR products
that were digested by SpeI and XbaI were then
subcloned into the SpeI site of the
pNET1400(i
190-645)CAT. Nucleotide sequences of primers used in PCR
were 5'-TGCCCACTAGTTCGGTGAGTTCAATCCCAGC-3' and
5'-TGCTTTCTAGAAGGGAAAAGAGGTGGTTACC-3' for pNET1400(i
381-645)CAT,
5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3' and
5'-TGCCCTCTAGAGCTGGGATTGAACTCACCGA-3' for pNET1400(i
190-362)CAT, 5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3' and
5'-TTGAGACTAGTCGTGCCCCAACCTCTGTTTC-3' for pNET1400(i
190-482)CAT,
and 5'-CCGGACACGTGAGTGCGCACTAGTCCTGAGCGCGGGACAGGGCTAGGT-3' and
5'-GGAAATCTAGAGAAACAGAGGTTGGGGCACG-3' for
pNET1400(i
190-362/ 501-645)CAT. The PCR-derived fragments
were sequenced to ensure that no errors had been introduced. Base
substitutions in E-box and splicing donor site were generated in the
context of the 1400 bp upstream plus intron sequence using the
TransformerTM site-directed mutagenesis kit
(CLONTECH, Palo Alto, CA) according to the
manufacturer's procedure. The following oligonucleotides were used in
the mutagenesis procedure:
5'-GATCCCCTCGCCGCCGGAaAgGTGAGTGCGCCCTGAGCG-3' for E-box mutation
unaffecting the splice donor site and
5'-GATCCCCTCGCCGCCGGACACGacAGTGCGCCCTGAGCG-3' for E-box mutation
affecting the splice donor site (lowercase letters indicate the
substitutions in nucleotides). The mutation was confirmed by sequence analysis.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared from SK-N-BE(2)C and HeLa cells according to the procedure
described by Dignam et al. (21). The pellet was
resuspended in Buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol)
and dialyzed against the same buffer. The extracts were quick-frozen in
liquid nitrogen, stored in aliquots at
70 °C, and used within 3 months of extraction. Protein concentrations of the nuclear extracts
were determined by the Bio-Rad protein assay method using bovine serum
albumin as a standard (22).
DNase I Footprinting Analysis--
The first intron fragment was
prepared by PCR and used as a probe in the DNase I footprinting
experiment. For the coding strand probe, a primer
(5'-AGCTCTTCCCCGGCCCCGCCCGAACGCCACACGGCGGA-3') representing the coding
nucleotide sequence from +47 to +84 bp of the hNET gene was labeled by
polynucleotide kinase using [
-32P]ATP. This was used
in PCR, together with an unlabeled nucleotide, 5'-CCCTACTTGCAACTCCCAAGACCACCCGGGAGCGCCTTAG-3', representing the noncoding nucleotide sequence from +574 to +613 of the hNET gene. PCR
was performed with the denaturation, annealing, and DNA synthesis at
95 °C (40 s), 55 °C (30 s), and 72 °C (1 min), respectively, for a total of 30 cycles using the plasmid pNET1400(i)CAT as a template,. The end-labeled probe was isolated from a 7% polyacrylamide gel, as described previously (23). After incubating 30,000 cpm of
labeled probe with 10-20 µl of nuclear extracts in 40 µl of binding buffer for 25 min at room temperature, DNase I digestion was
carried out using freshly diluted DNase I in 1× binding buffer, which
contained 20 mM HEPES (pH 7.9), 2 mM
MgCl2, 50 mM NaCl, 1 mM
dithiothreitol, 0.1 mM EDTA, and 10% glycerol. 2 µg of
poly(dI-dC) was included in the reaction as a nonspecific competitor.
The amount of DNase I was empirically adjusted for each extract to produce an even pattern of partially cleaved products. The DNase I
reaction was stopped by adding 100 µl of stop buffer (50 mM Tris (pH 8.0), 1% SDS, 10 mM EDTA (pH 8.0),
0.4 mg/ml proteinase K, and 100 mM NaCl). Samples were then
extracted twice with phenol-chloroform, and the DNA was precipitated
with 3 volumes of ethanol. The DNA pellet was dried and resuspended in
sequencing stop buffer (0.05% xylene cyanol, 0.05% bromphenol blue,
10 mM Na2 EDTA, and 90% deionized formamide)
and incubated at 95 °C for 3 min. An aliquot of sample was then
loaded onto a 6% polyacrylamide-8 M urea sequencing gel.
The same probe was subjected to parallel digestion without prior
incubation with nuclear extracts, typically using 5-10% of the DNase
I used in the presence of nuclear extracts. Location of cleaved
products was determined by comparison with sequencing ladders run in
adjacent lanes on the gel.
Electrophoretic Mobility Shift Assay--
Sense and antisense
oligonucleotides corresponding to the sequences protected by DNase I
were synthesized (Gene Link, Inc., Thornwood, NY) with the following
nucleotide sequences: 5'-GCCGCCGGACACGTGAGTGCGCCC-3' and
5'-CGGGCGCACTCACGTGTCCGGCGG-3' for NI,
5'-GCACCCGGTCACGTGGCCTACACC-3' and 5'-GGGTGTAGGCCACGTGACCGGGTG-3'
for the consensus binding site for USF1 (UI).
Nucleotide sequences for mutant oligonucleotides were
5'-GCCGCCGGAAAGGTGAGTGCGCCC-3' and 5'-CGGGCGCACTCACCTTTCCGGCGG-3' for NIm. The consensus AP2 and Sp1 oligonucleotides were
previously described (24, 25). The sense and antisense
oligonucleotides were annealed, gel-purified, and
32P-labeled by T4 DNA kinase and used as probes in
electrophoretic mobility shift assays. Electrophoretic mobility shift
assay and antibody coincubation experiments were performed using
30,000-50,000 cpm of labeled probe (~0.05-0.1 ng) and nuclear
extracts (10-30 µg) in a final volume of 20 µl of 12.5% glycerol,
and 12.5 mM HEPES, pH 7.9, 4 mM Tris-HCl, pH
7.9, 60 mM KCl, 1 EDTA, and 1 mM dithiothreitol
with 1 µg of poly(dI-dC) as described (26). Competition binding
assays were performed by adding nonradioactive competitor
oligonucleotides in a molar excess before adding
32P-labeled oligonucleotides. For the supershift assay,
antibodies were coincubated with the nuclear extract mix for 30 min at
room temperature before adding the radiolabeled probe. Antibodies
against USF1, USF2, c-Myc, Sp1, and AP2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Reverse Transcription-PCR (RT-PCR)--
SK-N-BE(2)C cells were
transiently transfected with different CAT reporter gene constructs.
Poly(A+) RNA was prepared by oligo(dT)-cellulose affinity
column chromatography as previously described (23). The
poly(A+)RNA was reverse-transcribed with SUPERSCRIPT II
RNase H
reverse transcriptase (Life Technologies, Inc.)
by priming with the catoligo2 primer. The products were
subjected to the PCR using sense primer
5'-AGCTCTTCCCCGGCCCCGCCCGAACGCCACACGGCGGA-3' and antisense primer
5'-TCGCGGATCCGAATTCTGGCGAGAGGAACTTTACCGG-3'. The PCR product was
analyzed on 7% polyacrylamide gel. RT-PCR was also performed using
primers for
-actin gene messages as a control. Primers for the
-actin gene were 5'-GGTCAGAAGGACTCCTATGTG-3' (sense) and
5'-TGTAGCCACGCTCGGTCAGG-3' (antisense).
 |
RESULTS |
The First Intron of the Human NET Gene Increases Activities of Both
the Homologous NET and the Heterologous TK Promoters in a Position- and
Orientation-dependent Manner--
In the native position
(pNET1400(i)CAT construct), the first intron increased the reporter
expression driven by the 1.4 kb NET upstream promoter by 12-15-fold in
SK-N-BE(2)C and HeLa cell lines (Fig.
1A). However, the first hNET
intron was unable to activate promoter activity when placed either in
the 5' upstream position (pNET(i)1400CAT) or in a position 3' to the
reporter gene (pNET1400CAT(i)). Thus, the promoter-enhancing function
of the first hNET intron appeared to be dependent on its original position in the context of the homologous promoter. Notably, the first
hNET intron by itself did not have any promoter activity, as the
reporter gene expression driven by the first intron alone was no higher
than that obtained with promoterless construct (data not shown). When
placed downstream of the heterologous TK promoter (TK(i)CAT in Fig.
1B), which is analogous to the native position, the first
hNET intron increased the transcriptional activity by 5- and 8-fold in
SK-N-BE(2)C and HeLa cells, respectively. However, when placed either
in the upstream position ((i)TKCAT) or in a position 3' to the reporter
gene (TKCAT(i)), the first hNET intron again failed to show enhancing
activity (Fig. 1B).

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Fig. 1.
Position effect of the first hNET
intron. A, the intron was placed in different positions
in the context of the homologous hNET promoter. The bent
arrow represents the major transcription start site of hNET, which
had been determined previously (17). The boldface line
denotes the 5'-untranslated leader sequence of the hNET gene. The first
intron is shown in a dotted box. The structure of each
promoter construct is shown at the left side of the figure.
Constructs were transiently expressed in SK-N-BE(2)C (black
bar) and HeLa cells (open bar). CAT activities were
standardized relative to -galactosidase activity driven by Rous
sarcoma virus-LacZ plasmid as an internal control for variation
in transfection efficiencies. The normalized CAT activities are
expressed relative to pNET1400CAT containing 1400 bp of hNET promoter,
assigned a value of 1.0. This experiment was repeated in triplicate
using independently prepared plasmid DNAs, resulting in similar
patterns. In the native position (pNET1400(i)CAT construct), the first
intron increased the transcription of the NET promoter in both
SK-N-BE(2)C and HeLa cell lines, but not when placed in the 5' upstream
site of the hNET promoter or in the 3' position of the CAT gene.
B, the first intron was fused to different positions in
conjunction with the heterologous thymidine kinase promoter. The CAT
constructs were co-transfected with Rous sarcoma virus LacZ
plasmid into SK-N-BE(2)C (black bar) and HeLa cells
(open bar). The CAT activities normalized by
-galactosidase activity are expressed relative to pBLCAT2, assigned
a value of 1.0. The first intron exerted a prominent enhancer function
to the TK promoter when placed in the untranslated region. However, it
again failed to show any enhancer activity either in the 5' or in the
3' location. Comparable patterns were observed in SK-N-BE(2)C and HeLa
cell lines.
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In order to investigate whether orientation of the first hNET intron
affects its function, we generated a reporter construct containing an
internally inverted intron. To avoid the possibility that reporter gene
expression is affected by an improper splicing of mRNA, the
original donor and acceptor sites for splicing were kept intact. The
inverted sequence somewhat stimulated promoter activity, but less
effectively than that in the correct orientation (4.5-fold
versus 11-fold; Fig.
2A). To confirm that the
difference in reporter gene expression could be attributed to
transcriptional regulation but not to a defective splicing mechanism,
RT-PCR was performed. When the mRNAs isolated from SK-N-BE(2)C
cells transfected with the forward or reverse intron-containing
constructs were examined by RT-PCR, we detected only a mRNA species
with a size expected from a proper splicing event (Fig. 2B).
Taken together, our results suggested that the first hNET intron
robustly enhances the promoter activity, and its full activity requires
the original position and orientation. Therefore, it appears that the
regulatory elements within the first hNET intron fail to meet the
criteria for a "classical enhancer" but require the native
orientation and position for promoter-enhancing activity.

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Fig. 2.
Orientation effect of the first hNET
intron. A, the intron was placed in different
orientations relative to the hNET promoter. The bent arrow
represents the major transcription start site of hNET. The
boldface line denotes the 5'-untranslated leader sequence of
the hNET gene. The first intron is shown in a dotted box.
The arrow indicates the orientation of the first intron.
Levels of CAT reporter gene activity were determined by transient
transfection assays into SK-N-BE(2)C cells and expressed relative to
that of pNET1400CAT, assigned a value of 1.0. When residing in the
5'-untranslated area, the first intron exerted a prominent enhancer
function only in the correct orientation. B, SK-N-BE(2)C
cells were transfected with pNET1400(i)CAT and pNET1400(i-rev)CAT
plasmids, and the resultant mRNAs were subjected to RT-PCR,
as described under "Experimental Procedures." The size of amplified
products was ascertained by electrophoresis on a 7% polyacrylamide
gel. Lane 1, cells transfected with pNET1400(i)CAT plasmid;
lane 2, cells transfected with pNET1400(i-rev)CAT plasmid.
The -actin gene was used as a control. RT-PCR detected an mRNA
with a size of 176 bp, as expected from a proper splicing event.
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Potential Regulatory Domains Residing in the First hNET
Intron--
To locate the regulatory domains within the first hNET
intron, we next performed deletional analyses. To distinguish
transcriptional regulation from improper splicing mechanisms,
deletional constructs were generated such that the original splicing
donor and acceptor sites were kept intact (Fig.
3). Reporter gene activity of each construct was examined by transient transfection assays using SK-N-BE(2)C and HeLa cell lines. Deletion of a 5'-side domain between
+190 and +362 bp (pNET1400(i
190-362)CAT) decreased the CAT
expression by ~40%, suggesting that this subdomain contains positive
regulatory sequences. Interestingly, CAT expression driven by
pNET1400(i
190-482)CAT, in which the first hNET intron was further
deleted to +482 bp, was even higher than that by the wild type plasmid
pNET1400(i)CAT, suggesting the possibility that an internal subdomain
between +362 and +482 bp encompasses negative regulatory sequences.
Deletion of a 3'-side domain between +381 and +645 bp
(pNET1400(i
381-645)CAT) diminished CAT expression by about
half, suggesting that a 3' side subdomain between +483 and +645
bp contains positive regulatory sequences. The internal deletion of
most nucleotides of the first intron (pNET1400(i
190-645)CAT) shows
a reduction of approximately 50% of CAT activity. Finally, consistent
with the concept that sequences between +362 and +501 contain negative
regulatory sequences, insertion of this subdomain to
pNET1400(i
190-645)CAT further decreased the CAT expression (construct pNET1400(i
190-362/501-645)CAT). The first hNET intron therefore appears to contain several regulatory subdomains with either
positive or negative regulatory function, which collectively enhance
the promoter activity. It is noteworthy that pNET1400(i
190-645)CAT, in which only 10 bases of each splicing junction area are retained, still can enhance the promoter activity 4-5-fold, which is
approximately half the activity of the full first intron sequence. This
indicates that a cis-element important for the enhancing
activity may reside in or overlap with the splicing donor or acceptor
site (see below).

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Fig. 3.
The activity of plasmids bearing deletions in
the area of the hNET first intron. Several 5' and 3' first intron
deletion mutants in the context of the upstream 1400-bp sequence were
transiently transfected into SK-N-BE(2)C (black bar) and
HeLa cells (open bar). The normalized CAT activity driven by
pNET1400CAT in each cell line was set to a value of 1.0 to compare the
effect of each mutation. Transfections were performed in triplicate in
at least three independent experiments. The positive regulatory region
consists of at least two distinct subdomains, which are present at +181
to +190 bp and +482 to +656 bp. In addition, the sequences
between +362 and +481 bp appear to contain a subdomain mediating
negative control on transcription.
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Upstream Stimulatory Factors 1 and 2 Specifically Interact at the
Splicing Junction Area of the First hNET Intron--
To identify
specific transcription factor(s) responsible for the promoter enhancing
activity of the first hNET intron, we investigated DNA-protein
interactions by DNase I footprinting analysis using nuclear extracts
isolated from SK-N-BE(2)C and HeLa cells. A prominent DNA-protein
interaction was mapped at +173 to +190, using both the coding and
noncoding strands (Fig. 4 and data not
shown). This precisely overlaps with the splicing donor site,
suggesting that this site may be important for basal transcriptional
activation as well as correct splicing of NET mRNA. Nuclear
proteins from both SK-N-BE(2)C and HeLa cells footprinted the same
area. In contrast, no clear DNA-protein interaction was detected along
the whole internal sequence of the intron (data not shown), suggesting
that transcription factors involved in the transcriptional regulatory
function of the first hNET intron may have weak DNA binding affinity
and/or exist in a very low concentration.

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Fig. 4.
DNase I footprinting analysis of the first
intron region of the hNET gene. Nuclear extracts were prepared
from SK-N-BE(2)C and HeLa cells and used for DNase I footprinting of
the first intron area of the hNET gene. The coding strand probe was
labeled as described under "Experimental Procedures." The
sequencing reaction mixtures (lanes 1-4) were run on the
left side of the autoradiogram. Each labeled probe was
digested with DNase I in the absence (lane 5) or presence of
nuclear extracts prepared from SK-N-BE(2)C (lane 6) and HeLa
cells (lane7), as described under "Experimental
Procedures." The nucleotide sequence and number on the
right side represent the protected region. Uppercase
letters indicate the nucleotide sequences in exon, and
lowercase letters represent the nucleotide sequences in
intron. Nuclear proteins from both SK-N-BE(2)C and HeLa cells
footprinted the same area, which contains a consensus E-box (CACGTG)
and splicing donor site.
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The protected area at +173 to +190 contains a consensus E-box (CACGTG),
which is known to be a binding site for basic helix-loop-helix class
transcription factors, such as c-Myc and the upstream stimulatory factors USF1 and USF2 (27, 28). When an oligonucleotide NI encompassing
the protected nucleotides was used as the probe, a specific DNA-protein
complex (C1) was prominently formed using nuclear extracts from
SK-N-BE(2)C cell line (Fig.
5B). A similar DNA-protein
complex was formed with an identical mobility when an oligonucleotide
UI containing the consensus USF1 binding site (Santa Cruz
Biotechnology) was used as a probe (Fig. 5B). The formation
of the C1 complex was completely abolished when the oligonucleotide
NIm with two mutated bases within the core E-box motif was
used as the probe. When the nuclear extracts from HeLa cell line were used, a similar pattern was observed except that another band with a
faster mobility was also formed. Because this band was robustly formed
even when the mutated NIm oligonucleotide was used as the
probe, it may represent a nonspecific complex. In a competition assay,
a 100-fold molar excess of unlabeled NI or UI oligonucleotide almost
completely abolished formation of C1 (Fig. 5C). However, its
mutant form (NIm), the Sp1 oligonucleotide, or the AP1
oligonucleotide failed to compete for complex formation. In addition,
formation of the second complex with HeLa nuclear extracts was not
efficiently competed by the specific NI and UI oligonucleotides,
supporting the notion that it is a nonspecific complex. These
observations suggest that (i) C1 represents sequence-specific
DNA-protein complex, (ii) the E-box motif is essential for formation of
C1 complex, and (iii) the C1 complex may involve the USF1 or related
factors.

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Fig. 5.
Sequence-specific binding of protein to the
region protected by DNase I footprinting analysis. A,
oligonucleotides used in gel shift assays to identify factors binding
to the first intron. The oligonucleotide NI contained the sequence from
+169 to +192 of the hNET gene, including the consensus E-box
(underscored). E-box consensus oligonucleotide (UI) was used
as unlabeled competitors. The oligonucleotide NIm contained
the same sequence as NI except for 2 base pairs mutated as indicated by
lowercase. B, electrophoretic mobility shift
assays were conducted using nuclear extracts from SK-N-BE(2)C cells
(left) or HeLa cells (right) with labeled probes
for the NI (lanes 1 and 4), UI (lanes
2 and 5), and NIm oligonucleotides
(lanes 3 and 6). A sequence-specific complex
(C1) was formed by both nuclear extracts as indicated by the
arrow at left. Mutant oligonucleotide NIm failed
to form the sequence-specific complex with the nuclear extracts from
both SK-N-BE(2)C cells or HeLa cells. A nonspecific complex was
observed with the nuclear extracts from HeLa cells, as shown by the
arrow at right. Unbound free probe (F) is
indicated by an arrowhead. C, electrophoretic mobility shift
assays were performed using nuclear extracts from SK-N-BE(2)C cells
(left) or HeLa cells (right) with
32P-labeled NI oligonucleotide. DNA-protein complex formed
with nuclear extracts and the NI oligonucleotide was competed by molar
excesses of NI or UI unlabeled nucleotides but not by the mutant form
of NI (NIm), SP1, or AP2 consensus sequence.
|
|
USF1 and USF2 Interact with a Consensus E-box Site Located in the
First hNET Intron--
To identify protein factor(s) involved in
formation of the C1 complex with the E-box motif, we performed antibody
coincubation experiments using the oligonucleotide NI as the probe.
When nuclear extracts from SK-N-BE(2)C or HeLa cells were coincubated
with an antibody against c-Myc, formation of C1 was not affected, and no supershifted band was observed. In contrast, coincubation of nuclear
extracts with an antibody against USF1 diminished formation of C1 and
produced supershifted complexes (Fig. 6,
lanes 2 and 8). When nuclear extracts were
incubated with anti-USF2 antibody (Fig. 6, lanes 3 and
9), complex C1 was modestly diminished, and supershifted
complexes were formed to a lesser degree than the USF1 antibody.
Coincubation with antibodies against Sp1 or AP2 neither diminished the
complex C1 nor generated a supershifted band (Fig. 6, lanes
4-6 and 10-12). Based on these results, we conclude
that USF1 and USF2, but not c-Myc, participate in formation of the C1
complex via interacting with the E-box motif residing at the junction
area of the first hNET intron.

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Fig. 6.
Both USF1 and USF2 bind to the E-box in the
exon/intron 1 junction of the hNET gene. A mobility shift assay
was performed using NI as a radiolabeled probe and incubated with 10 µg of SK-N-BE(2)C nuclear extract (lanes 1-6) and 14 µg
of HeLa nuclear extract (lanes 7-12). 1 µg of anti-USF1
antibody was added to lanes 2 and 8, and 1 µg of anti-USF2 antibody was added to lanes 3 and
9. Anti-c-Myc, anti-Sp1, and anti-AP2 antibodies were added
to lanes 4 and 10, lanes 5 and
11, and lanes 6 and 12, respectively.
The main band (C1) resulting from the interaction between
the oligonucleotide NI and nuclear extract was partially supershifted
(asterisks) by anti-USF1 and anti-USF2 antibodies but not by
anti-c-Myc, anti-Sp1, or anti-AP2 antibodies.
|
|
A Single cis-Regulatory Element Encompassing the E-box Motif and
the Splicing Donor Site of the First hNET Intron Regulates Both
Splicing and Transcriptional Enhancement of hNET mRNA--
Given
that the E-box motif precisely overlaps with the splice donor site of
the first intron, the cis-element encompassing these sites
may regulate both splicing and basal transcriptional activation of the
hNET gene. To address this possibility, we generated two different
mutant reporter gene constructs and examined them by transient
transfection assay and RT-PCR analysis of mRNAs isolated from
transfected cells. In one of these mutant constructs
(pNET1400(iCaCg)CAT), a double point mutation was introduced into the
E-box core motif (CACGTG to aAgGTG). Whereas this mutation completely
disrupted the interaction with USF1 and USF2 (Fig. 5A, lanes
3 and 6), the splicing donor motif was still intact. In
contrast, the other construct (pNET1400(iTaGc)CAT) harbors a double
mutation right at the consensus splicing donor site, which disrupted
its interaction with USF1 and USF2 (data not shown) and, presumably,
also affected the proper splicing of the first intron. By analyzing and
comparing mRNAs from cell lines transfected with the wild type and
mutant reporter constructs, we sought to address the importance of
different nucleotides of the E-box motif on the enhancer activity
and/or on splicing mechanisms (Fig. 7).
As shown in Fig. 7B, reporter gene expression driven by
either mutant construct was ~40% of that driven by the wild type
construct. This observation clearly demonstrates that interaction of
the E-box motif with USF1 and USF2 is important for the promoter
enhancing activity of the first hNET intron. When mRNAs isolated
from transfected cells were analyzed by RT-PCR, it was shown that only
a properly spliced transcript (170 bp) was detected from mRNAs
isolated from both wild type and pNET1400(iCaCg)CAT constructs (Fig.
7C). In contrast, pNET1400(iTaGc)CAT generated an aberrant
transcript of 360 bp that resulted from retention of 184 bp of intronic
sequence. Sequence analysis of this transcript showed that the mutation
in the donor splice site at E-box led to the use of a cryptic donor
splice site (gt) starting at nucleotide position +365 (Fig.
7C).

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|
Fig. 7.
Effect of mutating the conserved E box
binding in exon/intron 1 junction on enhancer activity.
A, schematic diagram showing the cis-element
residing at the junction area of the first intron and exon,
encompassing the E-box motif and splice donor site, that was identified
by DNase I footprinting analysis. Nucleotides of the first exon are
indicated by uppercase letters, and those of the intron are
indicated by lowercase letters. The consensus splice donor
site is denoted by an open box, and the E-box motif is
underlined. B, SK-N-BE(2)C cells were transiently
transfected with the wild type (pNET1400(i)CAT) and mutated constructs
as indicated. CAT activities were normalized to the -galactosidase
activity driven by the co-transfected Rous sarcoma virus-LacZ
plasmid. The normalized CAT activity driven by the intron-less
construct (pNET1400CAT) was set to 1 to compare the enhancing activity
of the wild type and mutated intron. Reporter gene expression driven by
either mutant construct was ~40% of that by the wild type construct,
indicating that interaction of the E-box motif with USF1 and USF2 is
important for the promoter enhancing activity of the first hNET intron.
B, SK-N-BE(2)C cells were transfected with wild type and
mutated plasmids, and the resultant mRNAs were subjected to
RT-PCR as described under "Experimental Procedures."
Amplified products were subjected to 7% polyacrylamide gel
electrophoresis. Lane 1, cells transfected with wild type
plasmid; lane 2, cells transfected with the
pNET1400(iCaCg)CAT plasmid; lane 3, cells transfected with
the pNET1400(iTaGc)CAT plasmid. An mRNA species was detected with a
size exactly matching that expected from a proper splicing event from
both wild type and E-box motif mutant constructs (pNET1400(iCaCg)CAT).
In contrast, the other construct (pNET1400(iTaGc)CAT), which harbors a
double mutation that also targeted the consensus splicing donor site,
prevented proper splicing of the first intron and led to the use of a
cryptic donor splice site, which is indicated in
boldface.
|
|
 |
DISCUSSION |
We have previously reported that ~9 kb of the 5' flanking
sequences of the hNET gene can confer cell specificity but require the
presence of an intronic enhancer for high level reporter gene expression (17). In the present study, we investigated the functional and structural properties of the first hNET intron to further delineate
control mechanisms affecting hNET gene regulation. Our results reveal
several salient features of the first intron in NET gene regulation, as follows.
Our analysis of the first hNET enhancer in the context of either the
homologous NET promoter or the heterologous TK promoter demonstrated
that it enhances the transcriptional activity of both promoters in an
orientation- and position-dependent manner (Figs. 1 and 2).
This sequence therefore does not belong to the category of classical
enhancers, the action of which are independent of position,
orientation, or distance in relation to the transcription start site
(29). It is increasingly recognized that transcription of some
mammalian genes can be influenced by intronic sequences (30-35), and
many of them contain important regulatory sequences in the first
intron. For example, the first intron (1.8 kb) of the aldolase B
gene was shown to be crucial for tissue-specific expression,
independent of position and orientation (31). On the other hand, in
many other genes the first intron was shown to enhance or silence the
transcriptional activity in an orientation- and
position-dependent manner (36-41). It may be presumed that correct and efficient transcriptional regulation of these genes may
require specific spatial and directional arrangement between protein
factors binding to the intronic sequences and other regulatory elements
residing in the 5' region or near the transcription start site. In this
scenario, misplacement of the intronic sequences may disrupt the normal
spatial geometry of protein-protein interactions required for correct
transcriptional regulation. In addition, if an important
cis-element resides exactly in the exon/intron junction, the
structural integrity of the first intron may be crucial for intact
enhancer (or silencer) activity. The latter possibility appears to
apply to the first hNET intron.
Deletional analysis identified two subdomains with positive regulatory
function at +181 to +190 bp and at +482 to +656 bp (Fig. 3).
Surprisingly, a middle subdomain at +362 to +501 bp was found to have a
negative regulatory function. Therefore, the robust enhancing activity
of the first hNET intron seems to be a net effect of several subdomains
with either positive or negative regulatory function. At present, the
exact physiological role of the negative subdomain remains largely a
matter of speculation. In several genes that are known to have both
positive and negative elements in the first intron, the combined action
of these elements has been shown to be important for cell type-specific
gene expression (42-44).
Although several subdomains appear to participate in the regulatory
function of the first intron, only one domain was prominently protected
in our DNase I footprinting assay using nuclear proteins from
SK-N-BE(2)C and HeLa cells (Fig. 4). The protected area encompassed a
consensus E-box motif (Fig. 4), and detailed analyses of DNA-protein interaction showed that the basic helix-loop-helix transcription factors USF1 and USF2 interact with this site (Figs. 5 and 6). Site-directed mutational analyses demonstrated that interaction of this
E-box motif with USF1/USF2, at least in part, underlies the enhancer
activity of the first hNET intron. USF, originally purified as a
complex from HeLa cells (45-47), is composed of two polypeptides of 43 and 44 kDa. Protein factors belonging to the USF family homodimerize
and heterodimerize with each other and thus are able to generate
diverse sets of complexes with potentially different transcriptional
capacity (48, 49). However, it is to be noted that an in
vivo footprinting analysis is required to absolutely prove that
the E-box motif is bound in the endogenous NET gene, which awaits
further investigation. In addition, interaction of USF1/USF2 with the
E-box has been shown to alter the transcriptional activity of the
eukaryotic genes in response to various physiological stimuli. For
example, the interaction of USF and E-box is required for insulin
regulation of the fatty acid synthase gene transcription (50). In USF
knockout mice, USF factors have been shown to be involved in
transcriptional regulation of liver-specific genes in response to
glucose (51, 52). Thus, it is likely that USF1 and USF2 may
co-operatively regulate NET gene expression in the presence and/or
absence of physiological stimuli.
The E-box motif interacting with USF1 and USF2 resides at the first
exon/intron junction and overlaps with the consensus splice donor site.
This observation suggested the intriguing possibility that this region
may control not only transcriptional activation but also splicing of
hNET mRNA. To address this possibility, we generated mutant
reporter constructs in which different nucleotides of the E-box motif
were mutagenized. When the mutation blocked binding of USF factors
without affecting the consensus splice donor site, the transcription
enhancing activity was diminished, but splicing was intact. There was
no evidence for an altered splicing pattern with the USF binding site
mutations. This result indicates that reduced CAT activity does not
result from the defective splicing event. However, when the mutation
changed the splicing donor site, not only was the transcriptional
enhancing activity similarly diminished, but an aberrant transcript of
360 bp was generated instead of the correct 174-bp transcript.
Sequencing analysis of this transcript showed that the mutation in the
splice donor site, which resides within the E-box motif, led to the use of a cryptic donor splice site (gt) starting at nucleotide position +365. Taken together, these results demonstrate that a
cis-element residing at the junction of the first exon and
intron, encompassing the splice donor site and an E-box motif, has a
unique dual role in splicing and transcriptional activation of NET
mRNA and that the latter involves the interaction with
transcription factors USF1 and USF2. To our knowledge, this is the
first report that a single cis-element regulates both
transcription and splicing of mRNA. However, based on the potential
of the consensus splicing donor site (5'-GT-3') to
coincide with critical cis-elements, such as E-box
(5'-CACGTG-3') and consensus binding site for TEF-2 (5'-GGGTGTGG-3') (53), it is possible that more genes may
contain a similar sequence motif(s) with regulatory functions in both transcription and splicing.
 |
ACKNOWLEDGEMENT |
We thank Dr. Thaddeus Nowak at the College of
Medicine, University of Tennessee, for critically reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant MH48866 and a National Alliance for Research on Schizophrenia and
Depression (NARSAD) Independent Award (to K. S. K.), a Ford Foundation Predoctoral fellowship (to P. A.), and an LG Chem. Inc. fellowship (to C. H. K.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Molecular
Neurobiology Laboratory, McLean Hospital, Harvard Medical School, 115 Mill St., Belmont, MA 02478. Tel.: 617-855-2024; Fax: 617-855-2023; E-mail: kskim@mclean.harvard.edu.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M101279200
 |
ABBREVIATIONS |
The abbreviations used are:
NE, norepinephrine;
NET, norepinephrine transporter;
hNET, human NET;
CAT, chloramphenicol
acetyltransferase;
TK, thymidine kinase;
PCR, polymerase chain
reaction;
USF, upstream stimulatory factor;
RT, reverse transcription;
bp, base pair(s);
kb, kilobase(s).
 |
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