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INTRODUCTION |
The synaptic action of norepinephrine
(NE)1 is terminated primarily
by rapid, NaCl-dependent re-uptake into presynaptic nerve terminals. NE reuptake is mediated by the norepinephrine transporter (NET) located in the plasma membrane of noradrenergic neurons (1). The
NET is a major target for tricyclic antidepressant drugs and for
illicit drugs such as cocaine and amphetamine. These agents block NE
transport by the NET, resulting in enhancement of the synaptic activity
of NE. Blockade of NET is an important mechanism of therapeutic action
of tricyclic antidepressants, and this observation provided support for
the catecholamine hypothesis of depression (2). Recently, Klimek
et al. (3) reported that the level of NET is significantly
reduced in the locus coeruleus from major depressive subjects. These
observations suggest that regulation of NET expression could play an
important role in human mental illnesses such as major depression and
schizophrenia (3, 4). In rodent studies, the mouse NET (mNET) locus,
Slc6a5, overlapped with a quantitative trait locus (Lore4) for
differential ethanol sensitivity in long-sleep and short-sleep mice
(5-7). Together with previous studies indicating ethanol-induced
changes of noradrenergic neurotransmission (8, 9), these findings suggest the additional possibility that regulation of NET expression may contribute to ethanol tolerance and/or addiction.
A cDNA encoding human NET (hNET) was isolated by an expression
cloning approach (10), and subsequently, cDNAs for the bovine and
the rat NET genes were cloned on the basis of their sequence homologies
(11, 12). NET belongs to a superfamily of Na+- and
Cl
-dependent transporters that includes
transporters for dopamine, serotonin,
-aminobutyric acid, betaine,
glycine, proline, and taurine (13-16). Recently, genomic clones of the
hNET and mouse placental NET (mNET) have been isolated and
characterized. The hNET gene, spanning approximately 45 kb, consists of
14 exons separated by 13 introns and is located on chromosome
16q12.2 (17). The structure of the mNET gene was found to be similar to
that of the hNET gene. The mNET gene is composed of 14 exons spanning approximately 36 kb and is located on chromosome 8 which is homologous with 16q12.2 of the human genome (5).
In the brain, NET is specifically expressed in noradrenergic neurons
but is not expressed either in dopaminergic neurons (e.g. substantia nigra) or in adrenergic neurons (e.g. C1 and C2
cell groups), demonstrating that NET is a hallmark protein of
noradrenergic cells (18). Another protein selectively expressed in
noradrenergic (and adrenergic) neurons is dopamine
-hydroxylase
(DBH) which catalyzes the conversion of dopamine to noradrenaline (19,
20). Collectively, NET and DBH are tightly co-expressed in
noradrenergic neurons but differentially in adrenergic neurons, such as
those in the C1 and C2 cell groups (18). These findings suggest that NET gene expression is subject to stringent cell type-specific control
mechanisms that overlap with, but are not identical to, those
regulating DBH gene expression.
Levels of NET mRNA in noradrenergic neurons and neurosecretory
cells are acutely or chronically regulated in response to various physiological and pharmacological signals including reserpine (21),
glucocorticoids (22), leukemia inhibitory factor, and ciliary
neurotrophic factor (23), angiotensin II (24), retinoic acid (23),
nerve growth factor (22), and protein kinase C (25). The foregoing
observations suggest that changes in NET gene expression induced by
physiological or pharmacological stimuli may alter noradrenergic
transmission depending on physiological demand.
Despite the clinical and physiological significance of NET gene
regulation, little is known about the transcriptional control mechanisms governing its expression. To investigate transcriptional regulation of the human NET gene, we isolated and analyzed genomic clones that encompass the 5'-flanking promoter region of the human NET
gene. Here we report that the human NET gene contains an
as-yet-unidentified intron of 476 bp in the middle of the
5'-untranslated region. Multiple transcription start sites of the hNET
gene were detected by both primer extension analysis and the 5'-RACE
technique. On the basis of this information, we constructed several
hNET-chloramphenicol acetyltransferase reporter constructs. Transient
transfection assays of these reporter constructs in both NET-positive
and NET-negative cell lines show that combination of the 5' upstream
sequences and the first intron is critical for high level noradrenergic cell type-specific promoter activity of the hNET gene. However, the
paired-like homeodomain transcription factor Phox2a, which is critical
for noradrenergic cell type-specific DBH promoter activity (26, 27),
appears not to regulate the promoter activity of the hNET gene.
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EXPERIMENTAL PROCEDURES |
Isolation and Characterization of hNET Genomic Clones--
A
0.5-kb rat NET cDNA fragment corresponding to the base pairs
990-1512 of the coding sequence (21) was radiolabeled by the random
primer method and used as the probe to screen a
EMBL3 human genomic
library derived from placenta chromosomal DNA
(CLONTECH, Mountain View, CA). Positive clones were
isolated and characterized by restriction mapping, using the same rat
NET cDNA probe. Then these clones were retested by genomic Southern
blot analyses. An oligonucleotide HN3-1 with the nucleotide sequence,
5'-GCATGGATGCGGCTGGCGAGAGGAA-3', which encompasses the start codon of
hNET cDNA (see Ref. 10; Fig. 6), was radiolabeled by T4
polynucleotide kinase using [
-32P]dATP and was used
as the probe. This probe discriminates the genomic clones containing
the 3' side of the gene, thereby facilitating identification of
candidate clones that contain the transcription start site and the 5'
upstream region of the hNET gene. The final positive clones (clones 3 and 7), which hybridized with both cDNA and oligonucleotide probes,
were isolated and further analyzed by restriction enzyme mapping and
sequence determination. Nucleotide sequences were analyzed using the
MacDNASIS 3.0 program (Hitachi Software).
Cell Culture and mRNA Isolation--
Human neuroblastoma
SK-N-BE(2)C and SK-N-BE(2)M17, rat pheochromocytoma PC12, mouse central
noradrenergic neuron-derived CATH.a, and peripheral nervous
system-derived PATH.2 cell lines were maintained as described
previously (28-30). Human cholinergic neuroblastoma SK-N-MC (31),
HeLa, thyroid carcinoma, rat C6 glioma, and mouse mastocytoma 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. Poly(A)+ RNA was prepared from each cell line by
oligo(dT)-cellulose affinity column chromatography as described (32)
and used for determination of the transcription start site and Northern
blot analysis.
Primer Extension--
Primer extension analysis was
performed based on the procedure previously described (32) with some
modification. Briefly, an oligonucleotide primer 8A,
5'-GTGTCCGGCGGCGAGGGGATCCCTGGTGCCTTGCGCCCT-3', complementary to hNET
nucleotides +142 to +180 (Fig. 6), was 5'-end-labeled with
[
-32P]dATP and T4 polynucleotide kinase. Approximately
1 × 105 cpm of 32P-labeled
oligonucleotide was mixed with 5 µg of poly(A)+ RNA
isolated from SK-N-BE(2)M17, SK-N-BE(2)C, and HeLa cells in a 30-µl
reaction containing 10 mM Tris-HCl, pH 8.3, 150 mM KCl, and 1 mM EDTA. Five µg of yeast tRNA
served as a negative control. The oligonucleotide was annealed to RNA
by heating the mixture at 100 °C for 5 min followed by incubation at
42 °C for 16 h. The annealed primer was extended by incubating
with SUPERSCRIPT II RNase H
Reverse Transcriptase (Life
Technologies, Inc.) in a 20-µl reaction containing 50 mM
Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 40 units of RNasin, and 1 mM
dNTPs (1 mM 7-deaza-dGTP, 1 mM dCTP, 1 mM dATP, and 1 mM dTTP) at 42 °C for 1 h. The reaction products were analyzed on a 6% denaturing
polyacrylamide gel. The sizes of the extension products were determined
by counting the number of nucleotides in a concurrently run sequencing
ladder using a control template (the bacteriophage M13mp18) and a
control primer with the nucleotide sequence, 5'-GTTTTCCCAGTCACGAC-3'. In an additional experiment, another oligonucleotide primer 7A, 5'-TCCGCCGTGTGG CGTTCGGGCGGGGCCGGGGAAGAGCT-3', complementary to nucleotides from +47 to +84 bp of the hNET gene (Fig. 6) was used as
the primer.
5'-Rapid Amplification of cDNA Ends (5'-RACE)--
To
identify the 5'-end of the hNET mRNA, 5'-RACE was carried out as
described (33) with the following modifications (Fig. 4A).
Poly(A)+ RNA from SK-N-BE(2)M17 cells was
reverse-transcribed with SUPERSCRIPT II RNase H
Reverse
Transcriptase by priming with the oligonucleotide HN3-1. Because the
5'-flanking region of hNET gene is highly GC-rich, 7-deaza-dGTP was
used along with the other 3 dNTPs for synthesizing cDNA. A 3'
poly(A) tail was added to the cDNA by terminal
deoxynucleotidyltransferase (Life Technologies, Inc.). The product was
subjected to the first PCR using an adaptor-(dT)17 primer
HN5A, 5'-GGAGACTCGAGTCGACATCGA(T)17 -3', and HN3-1. 40 cycles of PCR were performed with the temperature profile of 95 °C
for 40 s, 55 °C for 40 s, and 72 °C for 1 min, which
was followed by additional incubation at 72 °C for 9 min. An aliquot
of the product was subsequently used for the PCR using an adaptor
primer HN5, 5'-GGAGACTCGAGTCGACATCG-3', and an hNET gene-specific inner
primer HN3-2, 5'-TGGCGAGAGGAACTTTACCGG-3' (Fig. 6). The second PCR was
run for 25 cycles of the same temperature profile as that of the first
PCR. To improve the specificity of DNA amplification,
MasterampTM PCR Optimization Kit (Epicentre Technologies)
was used according to the manufacturer's protocol for both the first
and second rounds of PCR. The final PCR products were directly cloned
into pCRII (Invitrogen). Insert DNAs were isolated from individual
colonies and sequenced (34).
Northern Blot Analysis--
Two micrograms of
poly(A)+ RNA prepared from each cell line were fractionated
on a formaldehyde agarose gel, transferred to a nylon membrane, and
hybridized with radiolabeled probes. To detect DBH messages, cDNA
probes for the rat (35) and human (29) DBH genes were labeled and used
as a mixture. To detect NET messages, the rat NET cDNA fragment
(21) was radiolabeled and used as a probe. In addition, a cDNA
fragment of 700 bp, encompassing the mouse NET cDNA from middle of
exon 1 to exon 6 (a kind gift from Dr. Randy Blakely, Vanderbilt
University), was also used as a probe. A cDNA fragment for the
mouse glyceraldehyde-3-phosphate dehydrogenase was used as a positive
control. These probes were labeled with [
-32P]dATP
using Klenow fragment and random hexamer priming. Blots were
autoradiographed on an intensifying screen for 6 to 24 h at
70 °C according to the relative strength of signals. For
rehybridization, previous signals were removed by thoroughly washing
the membrane at 80 °C in 1.5 mM NaCl, 0.15 mM sodium citrate, pH 7, 0.01% SDS until no signal was
detected after overnight exposure.
Reporter Gene Constructs and Transient Transfection
Assay--
pBLCAT3-1 is a derivative of pBLCAT3 (36) which was
constructed by deleting the CRE-like sequence and TATA-like sequence upstream of the multiple cloning site (26). Several NET-CAT reporter
constructs were prepared using pBLCAT3-1 as follows. A 14-kb
SalI fragment of hNET clone 3 (Fig. 1) was cloned into pBLCAT3-1 that was cut with SalI. The resulting plasmid was
digested with BamHI, ligated, and named as pNET9000CAT. For
the construct containing promoter plus the first intron, intronic
sequence was amplified by PCR using sense primer
(5'-ACCAGGGATCCCCTCGCCGCCGGACAC-3') and antisense primer
(5'-TCGCGGATCCGAATTCTGGCGAGAGGAACTTTACCGG-3'), digested with
BamHI, and subcloned into pNET9000CAT that had been digested
with BamHI to yield pNET9000(i)CAT. Based on previous observations that less than 1.5-kb upstream sequence of the hDBH gene
drives cell-specific expression (29, 37, 38), we also made pNET-CAT
constructs that contain an hNET upstream sequence of comparable length.
For this purpose, approximately 1.4-kb upstream region of the hNET gene
was amplified by PCR using pNET9000CAT as the template with a sense
primer of 5'-GTGCCAAGCTTGATTCGGATAGCGATGTAA-3' and an antisense primer
of 5'-CGAGGGGATCCCTGGTGCC-3'. The PCR product was digested with
HindIII and BamHI and subcloned into the
pBLCAT3-1 that had been cut with the same enzymes, resulting in
pNET1400CAT. The first intron was similarly subcloned into pNET1400CAT,
generating pNET1400(i)CAT. The orientation of the first intron in
pNET9000(i)CAT and pNET1400(i)CAT was confirmed by sequence analysis.
Transfection was performed by the calcium phosphate coprecipitation
procedure as described previously (28, 29). Plasmids for transfection
were prepared using Qiagen columns (Qiagen Co., Santa Clarita, CA). For
the SK-N-BE(2)C and SK-N-BE(2)M17 cell lines, each 60-mm dish was
transfected with an equimolar amount (0.5 pmol) of each reporter
construct, 1 µg of pRSV-
gal, varying amounts of the effector
plasmid, and pUC19 plasmid to a total of 5 µg of DNA. For the other
cell lines, twice as much DNA was used in transfection. To compare the
NET promoter activity in NET-positive and NET-negative cell lines, the
CAT activity driven by reporter constructs are compared with that
driven by pRSV-CAT plasmid which contains the Rous sarcoma virus (RSV)
enhancer/promoter. Due to its strong promoter activity, a one-tenth
molar amount of the reporter construct (0.05 or 0.1 pmol) was used for
pRSV-CAT plasmid in transient transfection assay. In cotransfection
analysis, a half molar amount of the reporter construct was used for
the Phox2a-expressing plasmid, pRc/Phox2a, which was described
previously (26, 27). 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 described
previously (28, 29).
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RESULTS |
Isolation and Sequencing of hNET Genomic Clones That Contain the
5'-Flanking Sequences--
Eight positive clones, containing insert
DNAs of 6-14 kb length, were isolated by screening approximately
1.5 × 106 plaque-forming units of a
EMBL3 human
genomic library with a 0.5-kb cDNA probe corresponding to the
N-terminal 167 amino acids of the rat NET gene. On subsequent Southern
blot analysis, clones 3 and 7 hybridized to an oligonucleotide probe
encompassing the translational start codon of hNET. Clones 3 and 7 contained insert DNAs of 14 and 12 kb, respectively, of overlapping
sequence (Fig. 1A). Further
restriction mapping and Southern blot analyses indicated that the
BamHI-SacI fragment residing at the 3' side of
both clones contained the start codon of hNET (Fig. 1, A and
B). Clones 3 and 7 contained approximately 9.5- and 7.5-kb
sequence upstream of the start codon of hNET, respectively.

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Fig. 1.
Southern blot analysis and organization of
the hNET genomic clones. A, the structure of the hNET
genomic clones #3 and #7. Black boxes
denote the first three exons as described by Porzgen et al.
(17). The relative position of the translation start codon is indicated
by the arrow. Clones 3 and 7 contained substantially
overlapping insert DNAs of 14 and 12 kb. Locations of restriction
enzyme sites are shown as follows: S, SalI;
E, EcoRI; B, BamHI;
C, SacI. B, Southern blot analysis of
restriction fragment. The genomic DNA was cut with SalI
(lane 1), SalI and EcoRI (lane
2), SalI and BamHI (lane 3), or
SacI (lane 4) and was probed with a radiolabeled
oligonucleotide HN3-1 that encompasses the start codon. Positions of
size markers are indicated on the left. C,
comparison of nucleotide sequences of the cDNA and genomic clones
of hNET gene immediately 5' to the start codon. The nucleotide sequence
of the corresponding region of the mouse NET genomic clone (5) is also
shown. The translation start codon (ATG) is boxed. The
nucleotide sequences of the cDNA and genomic clones of hNET diverge
after a perfect match of 51 bases. In contrast, the sequence homology
continues beyond this junction between the genomic sequences of human
and mouse NET genomic clones. At the junction of divergence, a
consensus splicing acceptor sequence, 5'-AG-3', is found and denoted by
a circle.
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Sequence analyses of the BamHI-SacI fragment of
both clones 3 and 7 demonstrated that it includes the start codon,
untranslated leader sequence, and an exon described as the first exon
in previous reports (10, 17), and a part of an intron (Fig.
1A) previously described as the first intron (17).
Surprisingly, however, the nucleotide sequences of the genomic clone
diverged from the published untranslated leader sequence starting at
the base 52 bp upstream of the start codon, following perfect matches
of 51 bases (Fig. 1C). Both clones 3 and 7 contained the
same nucleotide sequences in this area, indicating that these are not
cloning artifacts. In addition, the corresponding nucleotide sequence
of the mouse genomic NET clone shows significant homology with that of
hNET clone (Fig. 1C; see Ref. 5). The presence of a
consensus splice acceptor site (5'-AG-3') in the genomic sequence at
the junction of divergence suggested the possibility that the human NET
gene contains an as-yet-unidentified intron(s) in the middle of the leader sequence. To investigate these findings in detail, the insert
DNA of clone 3 was subjected to structural and functional analyses as follows.
An Intron of 476 bp Resides in the 5'-Untranslated Leader Sequence
of hNET--
To assess whether the hNET gene contained an additional
intron(s) in the leader sequence, we determined additional nucleotide sequences upstream of the start codon and compared this sequence to the
remaining 9 bases of the previously described leader sequence of hNET
(see Ref. 10; Fig. 1C). As shown in Fig.
2 (also see Fig. 6), the sequence of
clone 3, corresponding to 528-536 bp upstream of the start codon,
perfectly matched the remaining leader sequence, 5'-GCCGGACAC-3' (Fig.
1C). This observation strongly suggests that the intervening
476-bp region represents an as-yet-unidentified intron of the hNET
gene. Also consistent with this hypothesis, the putative intervening
sequence contains a consensus splicing donor site (5'-GT-3') at its 5'
end in addition to an acceptor site (5'-AG-3') at the 3' end (Fig. 2).
Furthermore, eight out of nine nucleotides at the potential exon/intron
junction area match the consensus sequence (5'-(A/C)AG/GT(A/G)AGT-3';
see Ref. 39) and as would be expected, a pyrimidine-rich sequence
precedes the acceptor site (Fig. 6). Based on these observations, we
conclude that the first intron of 476 bp length resides in the middle
of the untranslated leader sequence of hNET. Primer extension and 5'-RACE analyses further substantiated this conclusion and identified the full untranslated leader sequences of hNET, as follows.

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Fig. 2.
Comparison of the sequences of the hNET
genomic clone and hNET cDNA. The remaining 9 bases of the 5'
side of the leader sequence of hNET is observed in the upstream region
of the genomic sequence after the intervening region of 476 bases,
suggesting that this region represents an as-yet-unidentified intron.
In support of this, the conserved GT/AG splice donor/acceptor motifs
were found at these junctions and are denoted by
boxes.
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Mapping of the Transcription Start Sites of hNET mRNA--
To
identify the transcription start site(s) of hNET mRNA, primer
extension analysis was performed using poly(A)+ RNAs
prepared from SK-N-BE(2)M17, SK-N-BE(2)C, and HeLa cell lines. When the
mRNAs from SK-N-BE(2)M17 and SK-N-BE(2)C were primed with the
primer 8A, extended cDNA products appeared at multiple locations,
clustered in two areas. The first of these clusters occurred between
176 and 184 bp and the second between 208 and 212 bp, upstream of the
3' utmost base of the primer 8A, respectively (Fig.
3). In a separate experiment using the
distinct primer 7A, two clusters of multiple bands were similarly
detected at corresponding locations (data not shown). In contrast, no
such clusters of extension products were detected when mRNA from
HeLa cells was used as the template, suggesting that these bands
represent catecholamine cell-specific cDNA products of NET
mRNA. Several bands detected in both NET-positive and -negative
samples (denoted by asterisks in Fig. 3) appear to be
nonspecific products, probably generated due to the GC-rich structure
of the proximal hNET promoter (Fig. 6). None of these bands, specific
or nonspecific, were detected when yeast tRNA was used as the template.
Together, the data suggest that the two catecholamine cell-specific
clusters of bands, located approximately 180 and 210 bp upstream of the
primer 8A, represent multiple transcription start sites for hNET
mRNAs.

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Fig. 3.
Determination of the hNET gene transcription
start sites by primer extension analysis. Primer extension was
performed with the 39-nucleotide antisense primer 8A using
poly(A)+ RNA from the human neuroblastoma SK-N-BE(2)C
(lane 3) and SK-N-BE(2)M17 (lane 4) as
NET-positive system, and from human HeLa (lane 2) as
NET-negative system. In addition, the same amount of yeast tRNA was
used in parallel as the negative control (lane 1). The
sequencing reaction on the left side of the autoradiogram was carried
out using M13 primer ( 40) on an M13mp18 single-stranded DNA template
and used to size the extension products. Two brackets at the
right side of the panel, together with indicated
sizes, indicate two clusters of several start sites. Nonspecific
products generated with both NET-positive and -negative mRNAs are
indicated by asterisks.
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To establish whether the multiple bands identified in the above primer
extension analyses represent genuine transcription start sites of hNET,
we next performed nested 5'-RACE experiments using poly(A)+
RNAs prepared from SK-N-BE(2)M17 cells by priming with the antisense oligonucleotide HN3-1, the sequence of which corresponds to the translation start codon and its immediate upstream 21 nucleotides (Figs. 4A and 6). Following
reverse transcription and poly(A) tailing, PCR was performed using the
adaptor primer HN5A and the antisense primer HN3-1. This first PCR
generated two major products of approximately 310- and 280-bp lengths
(data not shown), which were used as the template in the second PCR
with another adaptor primer HN5 and an inner hNET primer HN3-2 which
partially overlaps with HN3-1 (Figs. 4A and 6). Two major
products of approximately 300- and 270-bp lengths were prominently
detected by gel analysis (Fig. 4B). Assuming that the
potential intron of 476-bp length is spliced out, the sizes of the PCR
products match well with the putative two clustered transcription start
sites identified in the above primer extension analyses.

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Fig. 4.
Determination of the hNET gene transcription
start sites by 5'-RACE. A, a diagram showing the
procedure of 5'-RACE analysis of hNET mRNA. 1, single-stranded cDNA was synthesized from SK-N-BE(2)M17 mRNA by
priming with the oligonucleotide HN3-1 and subsequently digested with
RNase H. 2, the cDNA was then tailed with poly(A) at its
3' side and used as template in two consecutive nested PCR using
adaptor primers and hNET-specific inner primers as indicated (steps
3 and 4). B, ethidium bromide staining
of 5'-RACE products. Two RACE products (300 and 270 bp in size) were
generated as denoted by two arrows. A 1-kb DNA ladder (Life
Technologies, Inc.) was run in parallel as a size marker. C,
sequence analysis of 22 independent subclones of the smaller product
(~270 bp) identified 5 different transcription start sites and all
contained additional G residue, indicating that they represent mature
hNET RNA molecules. Nucleotide sequences around the transcription start
site of the 5'-RACE products are shown at the right side of each
sequence panel. Next to these sequences are also shown the
nucleotide sequences of the corresponding genomic sequences of the hNET
gene. The additional G residue is indicated by an asterisk.
D, in contrast, all 10 subclones of the larger product
(~300 bp) included the same sequence and did not contain an
additional G residue.
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We isolated and subcloned the second PCR products of 5'-RACE into pCRII
vector. Sequence analysis of 32 individual clones showed that all these
clones contained the previously identified leader sequence of hNET,
indicating that they were derived from the intact hNET cDNAs (see
below). The putative first intron which represents the genomic
sequences at 527 to 52 bp upstream of the start codon were missing in
all RACE products, demonstrating that this sequence indeed represent
the first intron of the hNET gene. Along with the above gel analysis of
PCR products (Fig. 4B), our sequencing analysis of 5'-RACE
products indicates that there is no alternate splicing of this newly
identified first intron in hNET mRNAs of the SK-N-BE(2)M17 cell
line. Among 22 individual isolates that were cloned from the smaller
molecular weight (~270 bp) products, 5 transcription start sites were
found to be clustered at 233 to 227 bp upstream of the start codon in
hNET mRNAs (Fig. 6; it is to be noted that transcription start
sites are shown to be at 709 to 703 bp upstream of the start codon
since it includes the intronic sequence); 1 clone contained an apparent
initiation site at the A located 233 bp upstream of the ATG codon, 10 clones at the G 231 bp upstream, 6 clones at the G 230 bp upstream, 2 clones at the C 228 bp upstream, and 3 clones at the A 227 bp upstream.
Remarkably, all these 22 clones were found to contain an additional G
residue expected for intact mRNA capped at the 5' end (Fig.
4C and data not shown; see Ref. 40). These results strongly
suggest that the foregoing nucleotides represent authentic start sites
of mature hNET transcripts. We next isolated 10 individual clones from
the larger product (~300 bp) and compared their nucleotide sequences.
All 10 clones included the same nucleotide sequences with the 5'
termini residing at the C residue 259 bp upstream of the start codon,
suggesting that this site is another major transcription start site of
the hNET gene (Fig. 4D). However, in contrast to the smaller
RACE products, these clones did not contain the additional G residue.
Therefore, the authenticity of this C residue as the transcription
start site remains open to question. Given that all 10 clones isolated
from the larger product exhibited the same sequence without the
additional G residue and that the size matches well with the primer
extension result, we favor the possibility that the larger form(s) of
hNET mRNA are generated without capping at the 5' site. Taken
together, we conclude that hNET transcription starts at multiple sites
clustered at two areas approximately 30 bases apart from each other.
The extra G residues at the 5' positions of most RACE products further supports the authenticity of these transcription start sites. Among
these, we arbitrarily designated the G residue 231 bp upstream of the
translation start site as +1 because both primer extension analysis and
RACE indicated this site is the most common apparent initiation site
that contains a capping G residue.
NET Is Co-expressed with DBH in Several Catecholaminergic Cell
Lines--
As a first step toward investigating transcriptional
regulation of the hNET gene, we tested several cell lines for
expression of NET mRNA. Northern blot analysis of
poly(A)+ RNA, from five catecholamine-synthesizing (human
neuroblastoma SK-N-BE(2)C and SK-N-BE(2)M17, rat pheochromocytoma PC12,
and mouse CATH.a and PATH.2) and five noncatecholamine-synthesizing (human HeLa, human cholinergic neuroblastoma SK-N-MC11, rat glioma C6,
human thyroid carcinoma, and mouse mastocytoma) cell lines, showed that
SK-N-BE(2)C and SK-N-BE(2)M17 cells express two NET mRNAs (3.6 and
5.8 kb in length), whereas PC12 cells preferentially express the larger
5.8-kb transcript (Fig. 5A).
All three of those cell lines also robustly express tyrosine
hydroxylase (TH) and DBH messages (Fig. 5B; 27). NET
mRNA was not detectable in mouse CATH.a and PATH.2 whether hNET
(Fig. 5A) or mouse NET cDNA (data not shown) was used as
the probe, despite clear expression of TH and DBH transcripts (Fig.
5B; 27). Thus, most, but not all, catecholamine-synthesizing
cell lines express NET at levels detectable by Northern blot analysis.
None of the five non-catecholaminergic cell lines expressed NET or DBH
mRNA, despite abundant expression of glyceraldehyde-3-phosphate
dehydrogenase mRNA, used as a control for mRNA recovery (Fig.
5C).

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Fig. 5.
Cell-specific expression of NET mRNAs in
catecholaminergic cell lines. Poly(A)+ RNAs were
isolated from five catecholaminergic cell lines including human
neuroblastoma SK-N-BE(2)C, SK-N-BE(2)M17, rat pheochromocytoma PC12,
mouse CATH.a, and mouse PATH.2 cell lines and five noncatecholaminergic
cell lines including human HeLa, SK-N-MC, thyroid carcinoma, rat C6,
and mouse mastocytoma cell lines and were analyzed by Northern blot as
described under "Experimental Procedures." The membrane was
consecutively hybridized for mRNAs encoding NET (A) and
DBH (B) and then hybridized with glyceraldehyde-3-phosphate
dehydrogenase probe (C). The location of each mRNA was
determined by comparison with RNA size standards (Life Technologies,
Inc.).
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Sequence Analysis of the 5'-Flanking Promoter Region of the hNET
Gene--
We determined the nucleotide sequence up to 4.8-kb upstream
of the start codon (GenBankTM accession number AF061198).
Nucleotide sequences including the first intron and the 5' proximal
554-bp region are presented in Fig. 6. We
numbered the position of each nucleotide based on the major
transcription start site which is arbitrarily designated as +1 site
(denoted by the bent arrow in Fig. 6). Two areas
encompassing two clustered transcription start sites centered at
28
and +1, respectively, and individual nucleotides identified as
transcription start sites in primer extension and 5'-RACE analyses are
indicated by asterisks above each base. A TATA-like sequence
(TTAAT) is found at
64/
60, which may control transcription
initiation at around
28 bp. Intriguingly, a second TATA-like sequence
(TACATTA) is found at
30/
24bp, that presumably controls
transcription initiation at around +1. This suggests the interesting
possibility that the second TATA sequence area may work both as the
TATA box and as a transcription initiation site. These analyses show
that mature hNET mRNAs, once the first 476-bp intron is spliced
out, contain untranslated leader sequences of 227-259 bp (Fig. 6). Like other neurotransmitter transporter genes (41-44), the proximal promoter region of hNET is highly GC-rich. The leader sequence contains
77% GC residues and the first 200-bp upstream region contains 72% GC
residues.

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Fig. 6.
Nucleotide sequences of the 5'-flanking
region of the hNET gene. The first 18 amino acids of hNET are
shown under the nucleotide sequences in a
single-letter code. The nucleotide sequences of the newly
identified first intron are indicated by lowercase letters.
Numbers at the right and left side are
the nucleotide positions relative to one of the major transcription
initiation sites designated as +1 and is indicated by a bent
arrow. All nucleotide bases identified as transcriptional start
sites are denoted by asterisks. Two TATA-like sequences,
residing approximately 30 bp upstream of the transcription start sites,
are indicated by boxes and designated as TATA1 and TATA2.
Potential transcription factor binding sites such as Sp1-binding site
(GC box), E-box, C/EBP-binding site, CRE-like sequence, and CCAAT box
are indicated by boxes. The nucleotide sequences
of oligonucleotides used as primers in the primer extension and 5'-RACE
analyses are underlined. The GenBankTM accession
number for this proximal and additional upstream sequences is
AF061198.
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The location and nucleotide sequence of potential cis-regulatory
elements are summarized in Table I, and
those residing in the proximal promoter area are shown in Fig. 6. The
potential cis-regulatory elements residing in the upstream sequence
include four AP1-binding sites, seven CCAAT boxes, two C/EBP motifs, a cAMP response element (CRE)-like motif, a Oct1-binding site, seven Sp1-binding sites, and a sterol response element. Notably, the first
intron area also contains multiple cis-regulatory elements such as
sterol response element, C/EBP, E-box, CRE, and Sp1-binding sites. Our
sequence search, however, did not identify sequence motif(s) with
significant homology to restrictive element/neuron-restrictive silencer
element that appears to regulate a large number of neuronal genes
(45-47).
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Table I
Potential transcription factor recognition sites in the hNET gene
The location of each potential cis-element is indicated relative to the
transcription start site. (c) at the right side of the location shows
that the cis-element is found in the complementary sequence. We
included two consensus Sp1 sites here because both motifs are shown to
be authentic Sp1-binding sites.
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Cell-specific Promoter Activity of the hNET Gene Requires Both the
5' Upstream Sequences and the First Intron--
Based on the
structural organization of the hNET genomic clone, we wished to
identify the promoter/enhancer region(s) containing critical regulatory
function for cell-specific transcriptional activity. For this purpose,
we assembled four reporter gene constructs, containing hNET 5'
sequences, with or without the putative first intron. NET9000CAT and
NET1400CAT plasmids contained 9.0- and 1.4-kb nucleotide sequences
upstream of the transcription start site, respectively, fused to the
reporter gene, chloramphenicol acetyltransferase (CAT). NET9000(i)CAT
and NET1400(i)CAT plasmids, in addition to those upstream sequences,
included the newly identified first intron as well (Fig.
7A). The transcriptional
activities of these constructs were examined by transient transfection
assay in four different cell lines. As shown in Fig. 7B,
NET9000CAT-driven reporter gene expression was approximately 4-7-fold
higher in NET-expressing cell lines compared with that in NET-negative
cell lines. In NET/DBH-positive cell lines, the relative CAT activity driven by this construct was approximately 50% that driven by the
DBH-CAT construct (DBH978CAT) containing the 978-bp upstream sequence
of the human DBH gene (29, 38). In negative cell lines, the CAT
activities driven by NET9000CAT and DBH978CAT are comparable. These
results strongly suggest that the upstream 9.0-kb sequence contains
important regulatory information for the cell-specific expression of
the hNET gene. CAT activity driven by NET1400CAT was approximately 20%
that by NET9000CAT in NET-positive cell lines, indicating that the
upstream region between
9.0 and
1.4 kb may contain positive
regulatory sequence element(s) required for the full promoter activity
of the hNET gene. In contrast the CAT activity driven by NET1400CAT is
not significantly higher (less than 2-fold) in NET-positive cell lines
compared with that in NET-negative cell lines, suggesting that the
proximal 1.4-kb flanking region may have some, but not sufficient,
information for driving cell-specific NET gene expression (Fig. 7,
B and C). This is in sharp contrast to the case
of hDBH gene regulation, in which a relatively short upstream sequence
(<1.1 kb) contains sufficient information for the cell-specific
expression both in transgenic mice and transient transfection
experiments (26, 29, 37, 38). Taken together, it appears that the
upstream region at
9.0 to
1.4 kb imparts substantial cell-type
specificity to hNET expression, and the proximal 1.4-kb sequence may
have general promoter function (Fig. 7C).

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Fig. 7.
Promoter activities of the 5'-flanking and
upstream regions of the hNET gene. A, diagrams showing
the structures of hNET-CAT reporter constructs. The bent
arrow represents the major transcription start site of hNET,
designated as +1 in this study. The bold line denotes the
5'-untranslated leader sequence, and the thin line denotes
the 5' upstream sequence of the hNET gene. The shaded box
represents the first intron of hNET, identified in this study, which
resides in the leader sequence. B, promoter activities of
hNET-CAT constructs in NET-positive SK-N-BE(2)C (BE) and SK-N-BE(2)M17
(M17) and NET-negative C6 and HeLa cell lines. Levels of CAT reporter
gene activity were determined by transient transfection assays and
expressed relative to that of pRSV-CAT plasmid which shows universally
high promoter activity in all cell lines used. Due to its strong
promoter activity, only one-tenth the relative amount of the pRSV-CAT
construct was used in the transient transfection assays (see
"Experimental Procedures"). The normalized CAT activity driven by
pRSV-CAT in each cell line was set to 100 to compare the relative
strength of each reporter construct. The numbers shown in
the table are the average of triplicate samples, with variation less
than 20%. This experiment has been repeated twice more in triplicate
using independently prepared plasmid DNAs, resulting in similar
patterns. Each sample was assayed in different dilutions so that the
final cpm were in the linear range. C, schematic diagrams
showing the location of potential regulatory elements in the 5'
upstream region and the first intron of the human NET gene. The
relative transcription-promoting/enhancing activities in NET-positive
(NET+) and NET-negative (NET ) cell lines are
indicated as very strong (+++), strong (++), moderate (+), and weak
(+/ ). As indicated in this diagram, the 1.4-kb proximal sequence was
not sufficient for driving cell-specific NET gene expression. Instead,
the upstream region at 9.0 to 1.4 kb of the hNET gene appears to
have cell type-specific promoter activity, whereas the first intron
exhibits a strong transcription-enhancing activity.
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The First Intron of the hNET Gene Exerts Important Transcription
Enhancing Activity--
Inclusion of the first intron in NET9000CAT
reporter constructs increased transcriptional activity 30-40-fold in
NET-positive cell lines (Fig. 7B). It also increased the CAT
activity by approximately 3-15-fold in NET-negative cell lines. When
the first intron was included in the shorter NET1400CAT construct, it
increased the transcriptional activity by approximately 15- and
10-fold, respectively, in NET-positive and NET-negative cell lines. Our
results suggest that the first intron of the hNET gene exerts a
moderate general enhancer effect on transcription and a stronger
cell-specific enhancing effect in noradrenergic cell lines in the
context of the long upstream sequence. Collectively, our transient
transfection assay indicates that the fullest cell-specific
transcriptional activity of the hNET gene so far examined requires both
the 5' upstream region up to 9.0 kb and the first intron residing in the untranslated leader sequence.
The combination of the 9.0-kb upstream sequence and the first intron
drove a remarkably high level of noradrenergic cell-specific expression
of the reporter gene, comparable to 30-40% that driven by the intact
RSV promoter/enhancer (Fig. 7B). At present, it is not clear
why NET9000(i)CAT drove much higher CAT expression than that by
DBH978CAT in SK-N-BE(2)C and SK-N-BE(2)M17 cell lines, given that the
steady state mRNA levels of DBH and NET are comparable in these
cells (Fig. 5).
Phox2a Does Not Influence NET Transcriptional Activity, a Marked
Contrast to DBH--
Based on the critical role of the paired-like
homeodomain transcription factor Phox2a for noradrenergic-specific
promoter activity of the DBH gene (26, 27, 48), and the co-expression pattern of DBH and NET in most noradrenergic neurons (18), we hypothesized that Phox2a may co-regulate DBH and NET transcription. To
test this hypothesis, we examined the effect of forced expression of
Phox2a on NET promoter function. In agreement with previous studies
(26, 27), cotransfection of Phox2a significantly increased (approximately 10-fold) the transcriptional activity of DBH978CAT in
DBH/NET-negative HeLa and C6 cell lines (Fig.
8; data not shown). In contrast, forced
expression of Phox2a in these cell lines did not affect hNET promoter
activity, in either NET-expressing or non-expressing cells, regardless
of the presence or absence of the first intron in the reporter
construct (Fig. 8; data not shown). These data suggest that in contrast
to the DBH gene, the Phox2a does not exert a strong influence on
cell-specific expression of the NET gene.

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Fig. 8.
Differential transactivation of the hDBH and
hNET promoters by the paired-like homeodomain transcription factor
Phox2a. The DBH-CAT or NET-CAT reporter construct was
cotransfected with Phox2a-expressing plasmid (pRc/Phox2a), in a molar
ratio of 0.5, in each cell line indicated. pRc/CMV plasmid is used as
an empty control. The CAT activity driven by each reporter construct by
itself was set to 1.0 to compare transactivation by Phox2a. Fold
induction by Phox2a cotransfection is presented as mean ± S.E.
values from six samples.
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DISCUSSION |
To understand molecular mechanisms regulating transcription of the
hNET gene, we isolated a genomic clone containing approximately 9.5 kb
of the 5' upstream region of the hNET gene. The experiments presented
here have defined critical aspects of the promoter structure and
function of hNET gene, including the following: (i) the location of
transcription start site(s); (ii) the sequence of the full 5'-untranslated leader sequence; (iii) the sequence of approximately 5 kb of the 5'-flanking promoter region, including a search for potential
cis-regulatory motifs; (iv) most unexpected, an as-yet-unidentified region of the hNET gene that appears by multiple criteria to be the
first intron and which resides in the middle of the 5'-untranslated leader sequence; and (v) that the combination of the upstream 9-kb
sequence and the first intron is critical for the high level, noradrenergic-specific transcriptional activity of the hNET gene. Finally, we tested, and rejected, the hypothesis that Phox-2a transactivation regulates hNET expression, as it does DBH expression.
Promoter Structure of the hNET Gene--
Data from previous
studies have shown that the coding region of the human NET gene
consists of 14 exons disrupted by 13 introns (10, 17), with the entire
5'-untranslated leader sequence present in the first exon, and the
intron immediately 3' to that first exon designated intron 1 (17). In
contrast to these earlier findings, the studies described here
demonstrate that the hNET gene contains an additional 476-bp-long
intron in the middle of the 5'-untranslated leader sequence. Several
pieces of evidence support this conclusion as follows: (i) the
nucleotide sequences of two independent genomic clones (clones 3 and 7)
diverge from that of the 5' leader sequence previously identified (10),
following a perfect match of 51 bases immediately upstream of the start codon; (ii) the remaining 9 bases of the leader sequence appear at the
5' end of the putative intron of 476 bp; (iii) the intervening sequence
contains the consensus splicing donor and acceptor sites at both
junctions; and (iv) this 476-bp sequence is missing in all RACE
products isolated and cloned in the present studies. We therefore
conclude that the hNET gene contains an additional exon at the 5'
utmost area and that the first intron of 476 bp resides in the middle
of the 5'-untranslated leader sequence. In addition, Porzgen et
al. (49) recently reported that the hNET mRNA is alternatively
spliced at its 3' region, in agreement with the Northern blot
experiment showing more than one transcript (Fig. 5), and thus contains
an additional exon at its 3' side of the gene.
The region both upstream and downstream from the transcriptional start
sites is rich in GC sequence, having an average GC content of 77 and
72% in the leader sequence and the first 200-bp upstream region,
respectively. Previous studies likewise reported that the proximal
upstream regions of other transporter gene family contain high GC
content (41-44). It seems likely that secondary structure in these
GC-rich regions might explain why previous analyses did not identify
the putative first intron described here (17). In support of this
possibility, successful determination of the transcription start sites
by 5'-RACE and primer extension analyses described in this study was
possible only after extensive optimization of these procedures,
including substitution of 7-deaza-dGTP in the synthesis of cDNA:RNA
template from poly(A)+ RNA in the primer extension and
5'-RACE experiments. In addition, specific PCR-enhancing agents
included in the MasterampTM PCR Optimization Kit (Epicentre
Technologies) were necessary to amplify specific products of the
correct size. Furthermore, we found that both the sequence and the
length of the primer were crucial for generating the specific products
in primer extension analysis (data not shown). Data from our primer
extension analysis and 5'-RACE experiments strongly suggest that
transcription of the hNET gene initiates at multiple sites clustered at
two locations, one at 703-711 bp and the other at 735-739 bp upstream
of the start codon in the genomic sequence. Given that the first intron is missing in all cDNA isolates from 5'-RACE, this indicates that the 5' leader sequences of hNET mRNAs are of approximately 230- and
260-bp lengths. In support of this, PCR analysis of the 5'-RACE generated two major products of corresponding lengths (Fig. 3). Two
TATA-like sequence motifs reside approximately 30 bp upstream of each
location, respectively, suggesting that the hNET gene contains two
alternate promoters. The locations of transcription start sites are
further confirmed by sequence analyses of individual clones of 5'-RACE
and PCR products, which all mapped to the above two locations. Most of
these clones (22 out of 32 isolates) contained an extra G residue at
their 5' ends, which was not present in the hNET genomic sequence (Fig.
3). Given that most mature eukaryotic mRNAs are capped at the 5'
end (40), these findings strongly support these locations as authentic
transcription start sites of hNET mRNAs. Among these multiple
sites, we designated the G residue which is 707 bp upstream of the
start codon as +1 (Fig. 6). It is worth noting that the 5' leader
sequences do not contain any ATG codons, indicating that the ATG codon
previously identified by Pacholczyk et al. (10) is the
genuine start codon of hNET.
Largely Overlapping but Also Distinct Expression Pattern of NET and
DBH--
DBH and NET are both hallmark proteins of noradrenergic cell
types because they are responsible for biosynthesis and reuptake of
noradrenaline, respectively, in the nervous system. In contrast to NET,
DBH is also expressed in cells that synthesize and release epinephrine.
An exhaustive in situ hybridization study showed that the
majority (>90%) of noradrenaline-containing cell bodies in the
brainstem express NET mRNA, whereas it is not detected at all in
dopaminergic or adrenergic cell bodies (18). Outside the nervous
system, some non-neuronal tissues, such as the placental syncytiotrophoblast (50), also express NET mRNA. Most, but not all,
norepinephrine-synthesizing cells in the adult nervous system co-express DBH and NET. The vast majority of cells that express NET,
with only rare exceptions, also express DBH. Understanding similarities
and differences in the molecular mechanisms governing cell
type-specific expression of DBH and NET promises to shed light on how
different classes of neurons express overlapping but distinct
biochemical phenotypes.
Various cell lines derived from catecholaminergic cell types are
available and have been used in different laboratories to study
catecholamine gene regulation and neuronal differentiation. Among
these, PC12, from the rat pheochromocytoma (51), is the prototype cell
line and has been extensively used in different laboratories. Several
human neuroblastoma cell lines, e.g. SK-N-BE(2)C and
SK-N-BE(2)M17, are of neural crest origin and express
catecholamine-synthesizing enzyme genes (29, 52). CATH.a and PATH.2
cells derived from brain and adrenal tumors, respectively, in
transgenic mice carrying the SV40 T antigen gene driven by a portion of
the TH promoter were also shown to express catecholamine-synthesizing
enzyme genes (30). To assess whether NET is co-expressed in these
catecholaminergic cell lines along with catecholamine synthetic enzyme
genes, Northern blot analysis was performed using NET and DBH cDNAs
as the probe. As expected, all five noncatecholaminergic cell lines
tested in this study showed no detectable levels of TH, DBH, or NET,
suggesting that expression of these genes is strongly suppressed in
these cell lines. Also as expected, all five catecholaminergic cell lines abundantly expressed DBH (Fig. 3) and TH message (27). In
contrast, and to our surprise, NET message, although easily detected in
PC12, SK-N-BE(2)C, and SK-N-BE(2)M17, was not detected in CATH.a and
PATH.2 cells. Thus, most, but not all, of the catecholaminergic cell
lines examined expressed NET. The CATH.a cell line, derived from a
central noradrenergic neuronal population, did not express any
detectable level of NET. It is possible that the CATH.a cell line has
lost its capability to express NET while it continues to express
synthesizing enzymes, TH and DBH, or that NET mRNA expression
occurs at a low level in this cell line, below the limits of detection
of Northern blot analysis. Alternatively, based on the in
vivo observation that small portions (approximately 10% or less)
of noradrenergic cells of the brainstem express DBH but not NET (18),
the CATH.a cell line may have originated from these
DBH+/NET
cell populations. Likewise, PATH.2
cells did not express detectable levels of NET mRNA.
Cell Type-specific Expression of NET, Potential Role of the 5'
Upstream Sequence and the First Intron--
To define promoter regions
important for driving cell-specific transcriptional activity of the
hNET gene, transient transfection assays using multiple cell line
systems were performed. The results are summarized schematically in
Fig. 7. 5' sequence up to 9.0 kb appears to contribute to cell-specific
promoter activity of the hNET gene. In addition, the newly described
first intron contains information that appears necessary for full high
level expression of the reporter gene in all cell lines examined.
Interestingly, the 1.4-kb upstream sequence of the hNET gene was not
sufficient at all for the high level, noradrenergic-specific expression
of the reporter gene. This is in contrast to that of the hDBH gene, in
which a relatively short upstream sequence (<1.1 kb) is necessary and
sufficient for cell-specific expression of the reporter gene both in
transgenic mouse experiments (37, 53-56) and in transient transfection
assays using cultured cell lines (26, 29, 38). Similarly, a relatively
short upstream sequence of the rat DBH gene was shown to drive the
cell-specific expression of the reporter gene (57). In addition, there
is no evidence available to suggest a role of the intronic sequence for
cell-specific transcription of the DBH gene. In this regard, it is
worth noting that reporter gene expression driven by NET9000(i)CAT was
much higher than that driven by DBH978CAT or other longer DBH-CAT
constructs (Fig. 7B; data not shown), despite the fact that
steady state mRNA levels of DBH and NET were comparable in the cell
lines used in this assay (Fig. 5). Thus, one interesting possibility to
explain this discrepancy is that full expression of the DBH gene may
also require as yet undiscovered sequence information residing in areas
other than the 5' upstream region, e.g. intron(s).
Alternatively, the enhancer activity of the first intron of the hNET
gene may be modest in the intact chromatin and may have been
exaggerated in our transient transfection assay. Finally, it is
possible that NET mRNA is less stable than DBH mRNA and
therefore requires higher levels of transcription for maintenance of
similar steady state levels. Further work is necessary to address these
issues. In other eukaryotic genes, ever increasing evidence suggests
that the intronic sequences play a crucial role in cell type-specific regulation of a variety of genes (58, 59). Given the high degree of
structural similarity among the NaCl-dependent transporter genes, and their generally extremely specific patterns of expression, it will be of great interest to see whether cell type-specific expression of transporter genes other than NET also requires intronic sequence information.
Cell Type-specific Expression of NET: How Similar to or Distinct
from DBH Is It?--
In the nervous system, noradrenergic neurons
constitute a tiny proportion (<0.0001%) of the total brain neurons.
Therefore, it is striking that the majority (>90%) of
noradrenaline-containing cell bodies co-express DBH and NET. Based on
these observations, we and others (18) have postulated that NET gene
expression may be closely linked to control mechanisms that specify
other hallmark phenotypes of noradrenergic neurons, such as DBH
expression. Recent studies showed the paired-like homeodomain
transcription factor Phox2a is restrictively expressed in noradrenergic
and adrenergic cell types (60, 61) and regulates development of major
noradrenergic populations including the locus coeruleus (48).
Furthermore, Phox2a apparently contributes to the phenotypic specification of noradrenergic cells by directly transactivating the
DBH promoter activity (26, 27). The above observations prompted us to
examine whether Phox2a co-regulates the promoter activities of the hNET
and hDBH genes (Fig. 8). Intriguingly, forced expression of Phox2a in
NET/DBH/Phox2a-negative cell lines did not change the promoter activity
of the hNET gene at all, although it significantly up-regulated that of
the hDBH gene. Based on these data, we hypothesize that Phox2a may not
regulate the cell-specific expression of the NET gene and that at least partially distinct control mechanisms underlie cell-specific expression of NET and DBH. In this context, it is worthwhile to note that these
genes are subject to different modes of regulation in response to some
pharmacological and physiological stimuli. For instance, reserpine
treatment in rat decreases the NET mRNA both in the adrenal gland
and the locus coeruleus (21) whereas it increases TH and DBH expression
(62-64). Although the mechanism of induction of TH by reserpine is
trans-synaptic, the down-regulation of NET expression is not (65).
Likewise, dexamethasone treatment appeared to regulate DBH and NET
mRNA expression in opposite directions (22, 66). Collectively,
these observations suggest the modes and mechanisms controlling NET and
DBH gene expression in response to cell-specific as well as various
physiological signals may differ in important ways. Further work to
define the similarities and differences in these mechanisms will
contribute to a better understanding of the regulation of
specification of catecholamine cell phenotypes.
In summary, our study defined the structural organization of the
5'-flanking promoter of the hNET gene. (i) We identified a new intron
of 476 bp that resides in the middle of the 5'-untranslated leader
sequence; (ii) we determined multiple transcription start sites,
clustered at two loci, by primer extension and 5'-RACE analyses; and
(iii) we determined the nucleotide sequence of the approximate 5-kb
upstream sequence. We have shown that the combination of the upstream
sequence and the first intron is required for driving the cell
type-specific transcriptional activity of the hNET gene. Furthermore,
despite their striking co-expression in most noradrenergic
neurons, our results suggest that differential control mechanisms may
underlie the cell type-specific expression of the DBH and NET genes.
The present study will serve as a basis for future studies of NET gene
regulation and for studies of coordinate regulation of
catecholamine-specific neuronal phenotypes.