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The Journal of Neuroscience, October 15, 1998, 18(20):8247-8260
Noradrenergic-Specific Transcription of the Dopamine
 -Hydroxylase Gene Requires Synergy of Multiple
Cis-Acting Elements Including at Least Two Phox2a-Binding
Sites
Hee-Sun
Kim1,
Hyemyung
Seo1,
Chunying
Yang1,
Jean-Francois
Brunet2, and
Kwang-Soo
Kim1
1 Department of Neurology and Department of Anatomy and
Neurobiology, University of Tennessee, College of Medicine, Memphis,
Tennessee 38163, and 2 Laboratoire de Genetique et
Physiologie du Developpement, Institut de Biologie du Developpement de
Marseille, Centre National de la Recherche Scientifique, Institut
National de la Santé et de la Recherche Médicale,
Universite de la Mediterranee, Marseille, France
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ABSTRACT |
Dopamine  -hydroxylase (DBH) catalyzes the conversion of dopamine
to noradrenaline and is selectively expressed in noradrenergic and
adrenergic neurons and neuroendocrine cells. Recent data from this
laboratory showed that a paired-like homeodomain (HD) protein, Phox2a,
interacts with the HD-binding site residing within a composite promoter
of the human DBH gene, designated domain IV, in a cell-specific manner
and directly controls noradrenergic-specific DBH promoter activity. In
this report, we demonstrate that three additional protein-binding sites
(i.e., domains I, II, and III) between domain IV and the TATA box are
critical for intact DBH promoter activity in noradrenergic cells and
that they activate DBH transcription in a highly concerted manner.
Transient transfection assays of mutant DBH reporter constructs
indicated that domain I was active in every cell line tested, whereas
domain III was preferentially active in DBH-positive cells. Remarkably,
mutation of domain II was associated with inactivation of DBH promoter
activity exclusively in DBH-positive cell lines, defining it as another
noradrenergic-specific promoter element. The cell-specific profile of
the promoter function of these sequence motifs was further supported by
in vitro DNA-binding studies and Southwestern analysis.
Furthermore, competition and antibody supershift assays show that
transcription factors Sp1 and AP2 are the cognate nuclear factors
interacting with domains I and III, respectively. Parallel evidence
indicates that domain II is another Phox2a-binding site, demonstrating
at least two binding sites for this factor in the upstream DBH
promoter. Strikingly, four tandem copies of domain II increased the
promoter activity of a minimal DBH promoter by 100- to 200-fold in
DBH-positive cell lines without compromising cell specificity.
Cotransfection of Phox2a-expression vector dramatically increased the
activity of the multiple domain II promoter only in DBH-negative cell
lines, confirming that domain II is responsive to Phox2a. Collectively, this study emphasizes a critical role of Phox2a as well as its functional synergism with other transcription factors (e.g., CREB, AP2,
and Sp1) in transcriptional activation of the DBH gene.
Key words:
dopamine -hydroxylase; homeodomain protein; Phox2a; Phox2b; cell-type specific transcription; noradrenergic neuron; cis-acting element; AP2; Sp1; synergistic activation
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INTRODUCTION |
Dopamine -hydroxylase (DBH) is a
hallmark protein of noradrenergic neurons because noradrenaline is
synthesized by this enzyme (Kirshner and Goodall, 1957 ; Friedman and
Kaufman, 1965). The highly restricted pattern of DBH expression in the
nervous system predicts that this gene is subject to neuron-specific as
well as to cell type-specific control mechanisms. Transgenic mice
experiments have shown that 5.8 or 4 kb of the 5' flanking sequences of
the human DBH gene can drive expression of the reporter gene in neurons of the locus coeruleus as well as other noradrenergic neurons and
adrenal chromaffin cells, with some ectopic expression (Mercer et al.,
1991; Morita et al., 1993; Kobayashi et al., 1994). More recently,
comparison of reporter gene expression in transgenic animals generated
by using DBH 5' flanking regions of different lengths indicated that
the upstream region between  1.1 and 0.6 kb is necessary for
expression in adult and fetal noradrenergic neurons (Hoyle et al.,
1994). In addition, this and other laboratories, using cell culture
systems, demonstrated that the 5' upstream region of the DBH gene can
drive reporter gene expression in a cell-specific manner (Shaskus et
al., 1992, 1995; Ishiguro et al., 1993, 1995; Lamouroux et al., 1993;
Kim et al., 1994; Afar et al., 1996; Seo et al., 1996; Yang et al.,
1998a,b).
Deletional and site-directed mutational analyses indicated that as
little as 486 bp of the upstream sequence of the human DBH gene can
direct expression of a reporter gene in a cell-specific manner
(Ishiguro et al., 1993; Seo et al., 1996). In the 486 bp region of the
human DBH gene, the distal part spanning 486 to 263 bp appears to
have a cell-specific silencer function that contributed to suppression
of the promoter activity in non-neuronal cells (Ishiguro et al., 1993,
1995). A recent analysis has indicated that its cell-specific silencer
function may depend on several sequence motifs including two novel
cis-elements (Kim et al., 1998). Transient transfection
assays identified the proximal part spanning 262 to +1 bp as
sufficient and essential for the high-level DBH promoter activity in
DBH-positive cells (Ishiguro et al., 1993; Seo et al., 1996). In this
262 bp proximal area, four protein-binding regions (domains I-IV) have
been identified by DNase I footprinting analysis (Seo et al., 1996). A
cAMP response element (CRE), 5'-TGACGTCC-3', with a single base
deviation from the consensus octamer motif, 5'-TGACGTCA-3' (Roesler et
al., 1988; Goodman, 1990), was shown to be critical for both the basal
and cAMP-inducible transcription in DBH-expressing cell lines (Ishiguro
et al., 1993; Lamouroux et al., 1993; Kim et al., 1994; Seo et al.,
1996). This CRE is included in a composite promoter structure located
at 185 to 150 bp, designated domain IV, which contains several
additional cis-elements such as AP1, YY1, and two core
motifs of homeodomain (HD)-binding sites. Site-directed mutagenesis of
each sequence motif has revealed that the CRE is essential for basal
promoter activity in every cell line, YY1 is multifunctional, and the
AP1-like motif may be transcriptionally inactive (Seo et al., 1996;
Yang et al., 1998a). Most interestingly, mutation of either core ATTA motif within domain IV significantly reduced the promoter activity only
in DBH-positive cell lines, defining the HD-binding site within domain
IV as the first noradrenergic-specific promoter element (Yang et al.,
1998a).
The murine paired-like HD protein, Phox2a, is selectively expressed in
noradrenergic cells and is critical for development of several
noradrenergic neuron populations, including the locus coeruleus (Morin
et al., 1997). Phox2a, whose forced expression robustly activates the
DBH promoter activity, binds to the HD site of domain IV, strongly
suggesting a mechanism for noradrenergic-specific promoter function
(Yang et al., 1998a). In addition, Phox2b, containing an HD identical
to that of Phox2a, has been identified and shown to be widely
coexpressed with Phox2a in both the central and peripheral nervous
system (Pattyn et al., 1997). Cotransfection assays showed that Phox2a
and Phox2b transactivate the DBH promoter activity with a comparable
efficiency (Yang et al., 1998a). There is no direct biochemical
evidence that Phox2b binds to the HD-binding site of domain IV.
However, the fact that Phox2a and Phox2b have identical HDs and the
above cotransfection data strongly suggest that both Phox2a and Phox2b
directly interact with the HD-binding site of domain IV; accordingly,
we designate this site as PRS1 for
Phox2a/Phox2b-response site 1 in this
article.
Although domain IV contains several cis-elements critical
for DBH transcription, when placed 5' to the TATA and transcription start site, it was able to recapitulate neither intact basal level nor
noradrenergic cell-specific transcription of the reporter gene (Yang et
al., 1998a). In addition, domain IV by itself mediated Phox2a/Phox2b-induced transcription only modestly (approximately threefold) compared with the intact DBH promoter (10- to 15-fold). In
the present study, we sought to characterize the contributions of
domains I, II, and III to DBH gene expression and identify their
cognate nuclear factors. We now show that all three domains are
critical for maximal DBH promoter activity in noradrenergic cell lines.
We identify Sp1 and AP2 as the transcription factors binding to domains
I and III, respectively. Domain II is found to be exclusively active in
DBH-expressing cells, defining it as another noradrenergic-specific
promoter element, and we provide several lines of evidence supporting
domain II as an additional Phox2a-binding site. Interestingly, four
tandem copies of domain II increased the DBH minimal promoter activity
by 150-fold in DBH-positive cells, which is at least twofold of that by
the intact DBH promoter. The promoter activity of this multiple domain
II was similar to that of the DBH minimal promoter in DBH-negative cells and increased by 200-fold after cotransfecting the
Phox2a-expressing plasmid. These results demonstrate that domain II is
indeed a noradrenergic-specific promoter and another
Phox2a/Phox2b-response site; accordingly, we designate domain II as
PRS2.
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MATERIALS AND METHODS |
Cell culture and transient transfection assays. Human
neuroblastoma SK-N-BE(2)C and SK-N-BE(2)M17 and mouse central
noradrenergic neuron-derived CATH.a cell lines were maintained as
described (Ishiguro et al., 1993; Suri et al., 1993; Kim et al., 1994)
and used as the DBH-positive system. The HeLa and rat C6 glioma cell lines were grown in DMEM supplemented with 10% fetal calf serum (Hyclone), streptomycin, and penicillin and used as the DBH-negative system in this study.
Transfection was performed by the calcium phosphate coprecipitation
method as previously described (Ishiguro et al., 1993; Seo et al.,
1996). For the SK-N-BE(2)C and SK-N-BE(2)M17 cell lines, each 60 mm
dish was transfected with 2 µg of the reporter construct, 1 µg of
pRSV -galactosidase, varying amounts of the effector plasmid,
and pUC19 plasmid to a total of 5 µg DNA. For the other cell lines,
twice as much DNA was used in transfection. Plasmids used for transient
transfection assays were prepared using Qiagen (Santa Clarita, CA)
columns. To correct for differences in transfection efficiencies among
different DNA precipitates, chloramphenicol acetyltransferase (CAT)
activity was normalized to that of -galactosidase. CAT and
-galactosidase activities were assayed as previously described
(Ishiguro et al., 1993; Seo et al., 1996).
DNA constructions. The DBH978CAT and DBH262CAT reporter
constructs contain the 978 and 262 bp upstream sequences of the human DBH gene, respectively, fused to the bacterial CAT gene (Ishiguro et
al., 1993). A series of human DBH promoter-CAT reporter constructs with
progressive deletions of proximal protein-binding sites were generated
using pBLCAT3-1 (Yang et al., 1998a), which drives significantly lower
background CAT activity compared with pBLCAT3 (Luckow and Schutz,
1987). 262'CAT construct was generated by ligating the 271 bp
SphI-XbaI fragment of 262CAT plasmid with the
4.3 kb SphI-XbaI backbone of pBLCAT3-1 plasmid.
114'CAT construct was made by ligating the 726 bp
HindIII-NcoI fragment with
HindIII-NcoI backbone of pBLCAT3-1 plasmid. To
generate 142'CAT and 62'CAT plasmids, PCR was performed using
oligonucleotides 5'-GACATGCATGCGCAGGCTGAGTGCTTGGC-3' and
5'-CATTTTAGCTTCCTTAGC-3', and 5'-GACATGCATGCGCTGCCTGGACCCACCCC-3' and
5'-CATTTTAGCTTCCTTAGC-3', respectively, using DBH978CAT as the
template. The 163 and 71 bp fragments were isolated after digesting the
PCR products with SphI and XbaI and were
subcloned to pBLCAT3-1 that had been digested with SphI and
XbaI, resulting in 142'CAT and 62'CAT plasmids,
respectively. 38'CAT plasmid, which is identical to the TATA-CAT
plasmid, has been described previously (Yang et al., 1998a). The
upstream sequences and junction regions of these deletional constructs
were confirmed by sequencing analysis.
Base substitutions in domains I, II, or III were generated in the
context of the 978 bp upstream sequence using the QuickChange PCR-based
site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to
the manufacturer's procedure. The following oligonucleotides were used
in the mutagenesis procedure using DBH978CAT plasmid as the template:
5'-CCTGGACCCACTATGTTCAGGACCAG-3' and
5'-CCTGGTCCTGAACATAGTGGGTCCAG-3' for domain I mutant,
5'-CCGCTAGACAAGCAGACGTACCCGTGCTG-3' and
5'-GCAGCACGGGTACGTCTGCTTGTCTAGCG-3' for domain II
mutant, and 5'-TGAGTGCTTGGCCTGGTTAGCAAGCTTGTGGGAGG-3' and 5'-CCCTCCCACAAGCTTGCTAACCAGGCCAAGCACTC-3' for
domain III mutant. The first set of primers represents coding strand
sequences of the promoter containing the desired mutations (underlined
bases), and the second set of primers represents the corresponding
noncoding strand sequences. Constructs with correct mutations were
screened by restriction enzyme digestion and sequencing analysis.
A single copy of the domain II oligonucleotide (see below) was
subcloned to the SphI site of 38'CAT plasmid. After
restriction and sequencing analyses, the 1xII-CAT construct containing
a single copy of domain II in correct orientation was selected. In
addition, the same domain II oligonucleotide was ligated after Klenow
reaction. A DNA fragment of 92 bp was isolated and subcloned to the
same SphI site of 38'CAT plasmid. 4xII-CAT plasmid, which
contains four copies of domain II (three copies in the right
orientation and one in the opposite orientation; see Fig. 8), was
isolated and confirmed by sequence analysis. pRC/Phox2a and pRC/Phox2b, which express full lengths of Phox2a and Phox2b protein factor, respectively, under control of the CMV promoter, have been described previously (Yang et al., 1998a) and were used as effector plasmids.
Preparation of nuclear extracts, EMSA, and DNase I
footprinting. Nuclear extracts were prepared from different cell
lines according to the procedure described by Dignam et al. (1983). Sense and antisense oligonucleotides corresponding to the sequences of
domains I, II, and III were synthesized (Gene Link, Inc., Thornwood, NY) with the following nucleotide sequences: 5'-GCCTGGACCCACCCCATTCA-3' and 5'-CTGAATGGGGTGGGTCCAGG-3' for domain I (DI),
5'-CCGCTAGACAAATGTGATTACC-3' and 5'-GGGTAATCACATTTGTCTAGCG-3' for
domain II (DII), and 5'-TGAGTGCTTGGCCTGGGGCGCAAGCTTGTGGGAGG-3' and
5'-CCCTCCCACAAGCTTGCGCCCCAGGCCAAGCACTC-3' for domain III
(DIII). Nucleotide sequences for mutant oligonucleotides were
5'-GCCTGGACCCACTATGTTCA3' and
5'-CTGAACATAGTGGGTCCAGG-3' for DIm,
5'-CCGCTAGACAAGCAGACGTACC-3' and
5'-GGGTACGTCTGCTTGTCTAGCG-3' for
DIIm, and
5'-TGAGTGCTTGGCCTGGTTAGCAAGCTTGTGGGAGG-3' and
5'-CCCTCCCACAAGCTTGCTAACCAGGCCAAGCACTC-3' for
DIIIm. Additional mutant oligonucleotides for domain
II were used and described in Figure 7A. In addition,
oligonucleotides 5'-GATCGAACTGACCGCCCGCGGCCCG-3' and
5'-ACGGGCCGCGGGCGGTCAGTTCGAT-3' were synthesized as the consensus AP2
(Williams et al., 1988), and Sp1 oligonucleotides were previously described (Seo et al., 1996). HD oligonucleotide, which contains the
HD-binding site within domain IV, was also described previously (Yang
et al., 1998a). 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 (EMSA). EMSA 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 (in
mM) 12.5 HEPES, pH 7.9, 4 Tris-HCl, pH 7.9, 60 KCl, 1 EDTA,
and 1 DTT with 1 µg of poly(dI-dC) as described (Yang et al., 1998a).
Competition-binding assays were performed by adding nonradioactive
competitor oligonucleotides in a molar excess before adding
32P-labeled oligonucleotides. For supershift assay,
antibody was coincubated with the nuclear extract mix for 30 min at
room temperature before adding the radiolabeled probe. Antibodies
against Sp1, Sp3, Sp4, and AP2 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). A Phox2a-specific antibody (number 59)
raised against a polypeptide (Y75-R88) residing immediately upstream of the HD was used in the supershift assay. Recombinant AP2 proteins were purchased from Promega (Madison, WI) and used for EMSA with 32P-labeled DIII oligonucleotide and consensus AP2 sequence
as probes. DNase I footprinting assay was performed using the wild-type
and mutant human DBH 5' proximal promoter fragments that had been prepared by PCR as the probe as described (Seo et al., 1996). After
incubating ~30,000 cpm of labeled probe with 150-200 µg of nuclear
extracts from different cell lines, freshly diluted DNase I (1.5-2.5
units) was added to a final volume of 40 µl and incubated for 90 sec
at the room temperature. The precise amount of DNase I was empirically
determined for each extract to ensure an even pattern of digested
bands. For the sample without nuclear extracts, much lower amounts of
DNase I (approximately one-tenth) were used. The probe DNA treated with
DNase I was purified, and an aliquot (~10-20%) of each sample was
analyzed on a 6% polyacrylamide/8 M urea-sequencing gel
followed by autoradiography with an intensifying screen. Location of
the protected area was determined by Maxam-Gilbert sequencing of
labeled probes.
Southwestern blot analysis. Southwestern blotting was
performed as described (Michael et al., 1988). Two sets of nuclear
proteins prepared from different cell lines (100 µg each per lane)
were mixed with 10 µl of 2 × sample loading buffer (4% SDS,
14% glycerol, 0.16 M Tris, pH 6.8, 0.1% brome phenol
blue, 5 mM DTT) and buffer D (Dignam et al., 1983)
to a final volume of 20 µl, heated to 95°C for 5 min and then
separated on a denaturing SDS-10% polyacrylamide gel. The protein
bands were transferred to a nitrocellulose membrane, and the
nonspecific protein bands on the membrane were blocked by three washes
of 45 min in 10 mM Tris, pH 7.5, 5% nonfat dry skim milk,
10% glycerol, 2.5% Nonidet P-40, 0.1 mM DTT, and 150 mM NaCl at 25°C. The membrane was then rinsed briefly in
binding buffer (in mM: 10 Tris, pH 7.5, 40 NaCl, 1 EDTA,
and 1 DTT, 8% glycerol, and 0.125% nonfat dry skim milk) and was
incubated in 10 ml of binding buffer containing 500,000 cpm/ml
end-labeled domain II oligonucleotide probe and 10 µg/ml poly(dI-dC).
After incubation overnight at room temperature, the membranes were
removed from the bag, washed with 10 ml of 10 mM Tris, pH
7.5, and 50 mM NaCl three times. The specific
protein-domain II interactions were visualized by autoradiography. The
specificity of these interactions was determined by adding a 100-fold
molar excess of unlabeled domain II oligonucleotide to the second set
of separate hybridization bag.
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RESULTS |
Transcription of the human DBH gene requires synergy of four
proximal protein-binding sites
In transient transfection assays using DBH-expressing SK-N-BE(2)C
and SK-N-BE(2)M17 cell lines (Fig.
1B), deletion of domain IV resulted in a dramatic decrease (20- to 30-fold) of the
transcription activity in both cell lines. Further deletion of domains
III, II, and I resulted in a progressive decrease of the promoter
activity, indicating that all three domains may act as positive
regulators of the DBH promoter function in DBH-positive cells, but the
proportional decrease in promoter activity was minor (30-50%) for
successive deletions. Base substitutions within each motif were then
introduced in the context of the intact 978 bp DBH promoter and
examined for effects on promoter activity in both DBH-positive and
DBH-negative cells (Fig. 1C). The CAT activity driven by the
intact 978 bp DBH promoter in DBH-positive cell lines was much higher
than that in DBH-negative cell lines (typically >10-fold; Ishiguro et
al., 1993; Yang et al., 1998a), but was given the relative value of 100 in each cell line to compare the relative effect of individual mutation
on the promoter activity. Base substitutions within domain I, II, or
III diminished most (>80%) promoter activity in DBH-positive cells,
suggesting that the proximal cis-elements activate DBH transcription in an interdependent manner. Mutation of domain I equally
diminished most of DBH promoter activity in DBH-negative cell lines,
whereas domain III mutation was severalfold less effective in the
negative cell lines. Base substitutions in domain II diminished DBH
promoter activity only in DBH-expressing cells, indicating that domain
II is a critical noradrenergic cell-specific cis-acting element.
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Figure 1.
Interdependent activation of DBH transcription by
proximal protein-binding sites in DBH-expressing cells.
A, Nucleotide sequences and the locations of domains I,
II, and III of the human DBH gene, as identified by DNase I footprint
analysis. The Sp1-binding motif and AP2-binding motif residing in
domain I and III, respectively, are indicated by brackets. In contrast,
domain II did not show any significant sequence homology to known
binding motifs except an ATTA motif at its 3' side. Base substitutions
within each domain that are analyzed by EMSA and transient transfection
assays in this study are also indicated. B, Promoter
activities of deletional DBH-CAT reporter constructs were determined by
transient transfection assays in DBH-expressing SK-N-BE(2)C and
SK-N-BE(2)M17 cell lines and expressed relative to that of the minimal
38'CAT construct. C, Effect of site-directed mutation of
each cis-regulatory element on DBH promoter activity in
the context of the upstream 978 bp sequences in DBH-expressing
[SK-N-BE(2)C and CATH.a] and nonexpressing (HeLa and C6) cell lines.
The normalized CAT activity driven by 978CAT in each cell line was set
to 100 to compare the effect of each mutation on cell-specific promoter
function of the DBH upstream sequence. The relative values are
presented as mean ± SEM values from six to eight independent
samples. Base substitutions in domain II diminished DBH promoter
activity exclusively in DBH-expressing cells, virtually rendering the
upstream DBH sequence a nonspecific promoter.
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Using different nuclear extracts, we next performed DNase I footprint
analyses of the wild-type and mutant promoters (Fig. 2) to address (1) whether there is a
positive correlation between promoter function and DNA-protein
interaction and (2) whether cognate nuclear factors synergistically
bind to these proximal protein-binding sites. Consistent with our
previous report (Seo et al., 1996), patterns of DNA-protein
interaction appeared to be significantly different between DBH-positive
and DBH-negative cell lines in this study using multiple cell lines; a
hypersensitive site at 161 bp appeared only with DBH-positive
extracts, and footprinting at domains II and III was much more evident
with DBH-positive extracts (Fig. 2, compare lanes 5 and 9 with 13 and 17). Mutation
of each motif specifically blocked footprinting at that site,
demonstrating a direct correlation between promoter function and
DNA-protein interaction at each motif. Mutation of one site did not
impair DNA-protein interactions at other sites, including domain IV,
suggesting that the transcription factors bind to the corresponding
sites independently of each other. This conclusion was further
supported by additional footprinting experiments using suboptimal
amounts of nuclear extracts, which protected these domains only
incompletely (data not shown).
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Figure 2.
Protein factors bind independently to domains I,
II, III, and IV. Nuclear extracts from SK-N-BE(2)C, CATH.a, HeLa, and
C6 cells were used for DNase I footprinting analyses of the wild-type
and mutant upstream promoters of the human DBH gene. The coding strand
probes were prepared using wild-type (lane 1), domain
Im (lane 2), domain
IIm (lane 3), and domain
IIIm (lane 4) mutant
constructs. Each 32P-labeled probe was digested with DNase
I in the presence of each nuclear extract. The TATA box and four
footprinted domains are denoted by brackets at the right
side of the panel. The patterns of DNA-protein interaction at these
domains appear to be conserved among DBH-expressing cell lines
[SK-N-BE(2)C and CATH.a] and significantly differed from those of
nonexpressing (HeLa and C6) cell lines.
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Domain I interacts with the transcription factor Sp1 without cell
type specificity
In EMSA, nuclear extracts prepared from SK-N-BE(2)C and HeLa cells
formed a similar complex (CI) with an oligonucleotide representing domain I, indicating that domain I interacts with nuclear proteins in
both DBH-positive and DBH-negative cells (Fig.
3A). This interaction of
domain I with protein factor(s) in DBH-positive and DBH-negative nuclear extracts is consistent with its general promoter function as
identified in Figure 1C. In a competition assay, a 100-fold excess of cold domain I oligonucleotide almost completely abolished formation of CI, but its mutant form (Im) or the CRE
oligonucleotide did not affect it at all (Fig. 3B), demonstrating that CI is a sequence-specific complex. Molar excesses of
the consensus Sp1 motif competed formation of CI with a slightly higher
efficiency than that of domain I oligonucleotide (Fig. 3B,
compare lanes 2 with 6 and 3 with 7). In addition, coincubation of SK-N-BE(2)C and
HeLa nuclear extracts with specific antibody against Sp1, but not with
that against AP2, diminished formation of CI and produced a new
supershifted complex in a dose-dependent manner (Fig. 3C;
data not shown). In addition, coincubation with different
concentrations of antibodies against Sp3 and Sp4 neither diminished the
complex C1 nor generated a supershifted band (data not shown).
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Figure 3.
Domain I is an Sp1-binding site and interacts with
the transcription factor Sp1 without cell type-specificity.
A, 20 bp oligonucleotide containing domain I sequence
was radiolabeled and incubated with 10 µg (lanes 1,
3) or 20 µg (lanes 2,
4) of SK-N-BE(2)C or HeLa nuclear extracts. A
sequence-specific complex (CI) was formed by both
nuclear extracts. Unbound free probe (F) is
indicated by an arrowhead. B, Complex CI
was competed by cold domain I (I) or Sp1
oligonucleotide but not by the mutant form of domain I
(Im) or the CRE oligonucleotide, indicating that
CI is a sequence-specific complex. For competition, 10-fold
(lanes 2, 4, 6,
8) or 100-fold (lanes 3,
5, 7, 9) molar excesses of
cold oligonucleotides were added to the reaction mixture containing 20 µg of SK-N-BE(2)c nuclear extracts before the addition of the
radiolabeled probe. C, Twenty micrograms of SK-BE(2)C
nuclear extracts were coincubated with 0.03 (lane 2),
0.1 (lane 3), or 0.3 (lane 4) µg
of Sp1-specific antibody or with 0.1 µg (lane 5) of
AP2-specific antibody. Coincubation with Sp1 antibody but not with AP2
antibody diminished formation of CI and resulted in formation of a
supershifted band (arrowhead) in a dose-responsive
manner.
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Domain II interacts with cognate protein factor(s) in a
cell-specific manner
In contrast to domain I, DNase I footprint analysis suggests that
domain II selectively interacts with nuclear proteins from noradrenergic cells (Fig. 2). As shown in Figure
4A, nuclear extracts from DBH-positive cells robustly formed the complex CII. In competition assays (Fig. 4B), molar excesses of cold domain II
oligonucleotide, but not its mutant form or unrelated Sp1
oligonucleotide, abolished formation of CII, strongly suggesting that
it represents a sequence-specific complex. To further test whether the
cognate protein factor(s) of domain II exist in a
noradrenergic-specific manner, Southwestern analysis was performed
using nuclear proteins prepared from DBH-positive and DBH-negative
cells (Fig. 5). This analysis
demonstrated that a few nuclear protein factors interact with domain
II. Two protein bands of 39 and 40 kDa were detected in SK-N-BE(2)C
cells, and those of 38 and 35 kDa were evident in CATH.a cells (Fig.
5A). In contrast, nuclear proteins from DBH-negative cells
did not show prominent signals. A 100-fold molar excess of cold domain II oligonucleotide abolished most of the signals (Fig. 5B),
suggesting that the bands represent sequence-specific associations
between domain II and cognate protein factors.
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Figure 4.
Domain II, a potentially novel
cis-regulatory element, interacts with nuclear
protein(s) in a cell-specific manner. A, 22 bp
oligonucleotide containing the domain II sequence formed a complex
(CII) with nuclear extracts (20 µg) from BE(2)C
or CATH.a cells but not from HeLa or C6 cells, indicating that domain
II interacts with nuclear proteins in a cell-specific manner.
B, CII is a sequence-specific complex. Twenty micrograms
of SK-BE(2)C nuclear extracts were incubated with the radiolabeled
domain II oligonucleotide. We used 40-fold (lanes 2,
4, 6) or 400-fold (lanes
3, 5, 7) molar excesses of
indicated cold oligonucleotides for competition. CII was competed by
domain II cold oligonucleotide but not by its mutant form
(IIm) or Sp1 sequence.
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Figure 5.
Identification of noradrenergic-specific protein
factors that interact with domain II by Southwestern blot analysis.
A, One hundred micrograms of nuclear proteins extracted
from the indicated cell line were separated on a denaturing
polyacrylamide gel, transferred to two sets of nitrocellulose
membranes, and probed with the P32-labeled domain II
oligonucleotide probe (~500,000 cpm/ml) in the absence
(A) or presence (B) of a
100-fold molar excess of unlabeled domain II oligonucleotide. Two major
proteins of ~35-40 kDa were identified to specifically interact with
domain II only in DBH-positive cells.
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Domain III is a high-affinity AP2-binding site and preferentially
interacts with nuclear proteins from noradrenergic cells
Domain III was more evidently footprinted by DBH-expressing cells
(Fig. 2) and contains a sequence patch at its middle, which perfectly
matches the consensus AP2 motif, 5'-GCCNNNGGC-3' (Williams and
Tjian, 1991). Figure 6A
demonstrates that nuclear extracts from SK-N-BE(2)C and CATH.a cells
generated prominent complexes with an oligonucleotide that contains
domain III sequence. At present, it is not clear whether these bands
represent single or multiple proteins interacting with domain III, and
we collectively designated these complexes as CIII. Affinity-purified
human AP2 protein also generated multiple bands that migrated with
mobilities similar to those formed by SK-N-BE(2)C nuclear proteins
(Fig. 6A, compare lanes 1 and
5). Using SK-N-BE(2)C nuclear proteins in EMSA, formation of
CIII was efficiently competed by molar excesses of cold domain III or
consensus AP2 oligonucleotide but not by those of mutated domain III or
unrelated Sp1 sequence (Fig. 6B). These findings
suggest that CIII represents sequence-specific complex(es) and that
domain III is an AP2-binding site. Consistent with this, coincubation
of SK-N-BE(2)C and CATH.a nuclear extracts with specific antibody
against AP2, but not with that against Sp1, significantly diminished
the DNA-protein complex(es) and generated a supershifted band,
demonstrating that AP2 is involved in formation of CIII (Fig.
6C; data not shown). Nuclear extracts from DBH-negative
cells formed either much weaker bands (HeLa) or diffuse bands with
faster mobilities (C6) (Fig. 6A). However, these
complex(es) were shown to contain AP2 by antibody coincubation experiments (data not shown). Our competition assay indicates that cold
domain III oligonucleotide competes formation of CIII more efficiently
than the consensus AP2 sequence (Fig. 6B, compare lanes 2 and 6). To test whether
domain III has higher affinity for AP2 protein, we incubated the
affinity-purified recombinant AP2 protein with radiolabeled domain III
or consensus AP2 sequence. Figure 6D indeed shows
that domain III has a significantly higher affinity for AP2 than the
consensus AP2-binding oligonucleotide. In addition, coincubation with
AP2-specific antibody, but not with Sp1-specific antibody, completely
abolished both complexes formed by recombinant AP2 protein and
generated supershifted bands (Fig. 6E), demonstrating
that the AP2 protein is involved in forming the complex(es) with both
the domain III and the consensus AP2 sequence.
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Figure 6.
Domain III preferentially interacts with nuclear
extracts from DBH-expressing cells and binds to the transcription
factor AP2. A, 32P-labeled domain III
oligonucleotide was incubated with 20 µg of nuclear extracts from
each cell line as indicated at the top of the
lanes. Three nanograms of the recombinant human AP2
protein (rhAP2) were also used in a control experiment (lane
5). B, CIII was competed by 40-fold
(lanes 2, 4, 6,
8) or 400-fold (lanes 3,
5, 7, 9) molar excesses of
domain III cold oligonucleotide or AP2 consensus sequence but not by
the mutant form of domain III (IIIm) or Sp1
sequence. Twenty micrograms of SK-N-BE(2)C nuclear extracts were used
for each binding reaction. C, In supershift assays, 0.03 (lane 2), 0.1 (lane 3), or 0.3 (lane 4) µg of AP2-specific antibody were used
in each reaction along with 20 µg of SK-N-BE(2)C extracts. In a
control experiment (lane 5), 0.1 µg of Sp1-specific
antibody was used. D, Two nanograms (lanes
1, 3) or 10 ng (lanes 2,
4) of recombinant AP protein is incubated with a
32P-labeled domain III oligonucleotide (lanes
1, 2) or the consensus AP2 sequence
(lanes 3, 4). AP2 protein bound to
domain III sequence much more strongly than to the consensus sequence.
E, Incubation of the recombinant AP2 protein with 0.03 (lanes 2, 7) or 0.1 (lanes
3, 8) µg of AP2-specific antibody
completely diminished the specific complex and produced supershifted
bands (arrows). In control experiments, 0.03 (lanes 4, 9) or 0.1 (lanes
5, 10) µg of Sp1-specific antibody did not
affect the patterns of AP2-specific complexes formed with either domain
III (lanes 4, 5) or the consensus AP2
sequence (lanes 9, 10).
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Domain II is a Phox2a-binding site
Our findings that domain II binds to nuclear proteins in a
cell-specific manner and that its mutation is associated with a severe
loss of DBH promoter function only in noradrenergic cells prompted us
to characterize and identify the cognate nuclear factor(s). The
nucleotide sequence of domain II is A/T-rich, and our previous sequence
search did not reveal significant homology to any known cis-acting motif (Seo et al., 1996). To determine nucleotide
bases important for domain II-protein factor interaction, we performed EMSA using domain II or mutant oligonucleotides containing double or
triple base substitutions at different locations as probes (Fig.
7A). Mutant m1 and m2 probes
containing base substitutions at the 5' side of domain II were able to
form complexes with an efficiency comparable to that of the wild-type
domain II oligonucleotide with nuclear proteins prepared from
SK-N-BE(2)C (Fig. 7D, lanes 1-3)
or CATH.a cells (data not shown). In contrast, m3 and m4 probes no
longer generated signals as prominent as the wild-type sequence (Fig.
7D, lanes 4-5), indicating that
nucleotides residing at the 3' side are critical for domain II-protein
interactions. The m4 probe showing the most severe defect in forming
the DNA-protein complex has base substitutions within the ATTA motif
of the HD-binding site at the 3' side, raising the possibility that
domain II may represent another Phox2a/Phox2b-binding site in addition
to the PRS1 within domain IV (Yang et al., 1998a). To test this, we
further analyzed DNA-protein interaction at domain II using
competition and supershift assays. Using the DII oligonucleotide as the
probe, the cold PRS1 oligonucleotide was able to compete
formation of DNA-protein complex(es) even more efficiently than the
cold DII oligonucleotide (Fig. 7B; lanes
1-10). These competition assays thus suggest that
common nuclear factor(s) interact with PRS1 and, with a lower affinity,
with domain II, presumably caused by the fact that PRS1 contains two
ATTA core motifs, whereas domain II has only one such motif.
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Figure 7.
Domain II is another Phox2a-binding site.
A, Nucleotide sequences of the PRS1 and wild-type and
mutant of domain II oligonucleotides. The ATTA motifs are indicated by
asterisks. Mutated bases are shown in the mutant
oligonucleotides. Dots represent unchanged sequences.
B, DNA-protein complexes formed with SK-N-BE(2)C
nuclear proteins and the DII oligonucleotide were competed by molar
excesses of different cold oligonucleotides as indicated above each
panel. Thirty micrograms of nuclear proteins were used in each binding
reaction. C, Antibody supershift assays indicate that
Phox2a binds to both domain II and PRS1 sequences. Coincubation of
nuclear proteins with increasing amounts of 1 µl of
10 2 (lanes 2,
6), 101 (lanes
3, 7) and 1:3 dilution (lanes
4, 8) of Phox2a-specific antibody resulted in
the generation of a supershifted band (arrowhead) in a
dose-responsive manner with both PRS1
(C-1) and DII oligonucleotide
(C-2). In addition, formation of domain
II-specific complex, CII, was significantly diminished. In contrast,
coincubation with either SP1 or AP2-specific antibody (0.1 µg each)
neither generated the supershifted band nor diminished formation of CII
(lanes 9, 10). Phox2a-specific antibody
by itself was unable to form any complex with the radiolabeled probes
(data not shown). D, Determination of nucleotide bases
important for domain II-protein interaction in the absence
(D-1) or presence
(D-2) of Phox2a-specific antibody. m1 and
m2 probes formed DNA-protein complexes as efficiently as the wild-type
DII probe. m3 probe showed a limited ability to form complexes, and m4
probe shows the most severe defect for forming specific complexes
(lanes 4, 5). In the presence of 1 µl
of 1:3 dilution of Phox2a-specific antibody, even m3 was able to
produce a robust band of supershifted complex as indicated by an
arrowhead. m4 was able to generate little, if any,
supershifted band, suggesting that the ATTA motif is critical for its
interaction with Phox2a. Two nonspecific bands are indicated by
arrows.
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To address whether domain II-protein complex(es) contain Phox2a, a
supershift assay was performed using Phox2a-specific antibody. In a
control experiment using radiolabeled PRS1 oligonucleotide as the
probe, coincubation with Phox2a-specific antibody generated a
supershifted band in a dose-response manner as described previously (Fig. 7C, lanes 1-4; Yang et
al., 1998a). When the radiolabeled domain II oligonucleotide was used
as the probe, coincubation of SK-N-BE(2)C nuclear extracts with
Phox2a-specific antibody diminished formation of CII and generated a
supershifted band in a dose-dependent manner (Fig. 7C,
lanes 6-9). In contrast, coincubation with
specific antibodies against Sp1 (Fig. 7C, lane 9) or AP2 (Fig. 7C, lane 10)
neither diminished CII nor generated a supershifted band. Coincubation
of Phox2a-specific antibody with nuclear proteins from CATH.a or PC12
cells similarly resulted in generation of a robust supershifted band
(Yang et al., 1998a; data not shown). In contrast, coincubation
with preimmune serum or nuclear extracts from C6 or HeLa cells did not
produce any detectable signal of supershifted band using either
radiolabeled PRS1 (Yang et al., 1998a) or domain II
oligonucleotide (data not shown). Taken together, these data
demonstrate that Phox2a is directly involved in formation of CII.
The signals of the supershifted band were significantly stronger than
those of original DNA-protein complex(es) using either PRS1 or domain
II probe. In the supershift assay using wild-type and mutant domain II
oligonucleotides, all probes except m4 formed a robust supershifted
band, strongly suggesting that the ATTA motif is the only subregion
that is essential for interaction of domain II with Phox2a (Fig.
7D). Similar results were obtained using lower amounts of
antibodies (data not shown). Although the m3 mutant did not itself form
intact amounts of DNA-protein complex(es), it was able to generate a
supershifted band with a comparable signal (Fig. 7D, compare
lanes 4 and 9 with 1 and
6). One interpretation for this finding is that m3
has comparable affinity to Phox2a but does not bind to other binding
proteins as efficiently as the wild-type domain II. Alternatively,
association of Phox2a with the specific antibody may have overcome its
low affinity to m3 sequence.
Domain II upregulates the DBH minimal promoter activity in a
noradrenergic-specific manner and mediates
Phox2a/Phox2b-induced transcriptional activation
A single copy of domain II was subcloned in the correct
orientation in front of the minimal promoter region of the DBH gene containing the TATA box and transcription start site (Fig.
8A, 1xII-CAT), and its transcriptional
activity was examined by transient transfection assays in DBH-positive
and DBH-negative cell lines. As shown in Figure 8B,
1xII-CAT drives expression of the reporter gene threefold higher than
that driven by 38'CAT in DBH-expressing SK-N-BE(2)C and CATH.a cells,
but not in DBH-negative HeLa and C6 cells. Furthermore, cotransfection
assay shows that Phox2a or Phox2b activates the reporter gene
expression driven by 1xII-CAT plasmid threefold to fourfold in
DBH-negative HeLa (Fig. 8C) and C6 (data not shown) cells.
In DBH-expressing cell lines, in contrast, cotransfection of Phox2a
or Phox2b activated the promoter activity of 1xII-CAT construct only
marginally, if at all (Fig. 8C; data not shown). These data
confirm that domain II is a noradrenergic-specific promoter element and
mediates Phox2a-responsive transcriptional activation. However, the
promoter activity of a single copy of domain II by itself represented
~5% of the intact DBH promoter activity in the DBH-positive cell
lines (Fig. 8B). To address whether multiple copies
of domain II may synergistically activate the DBH minimal promoter
activity in a cell-specific manner, we next subcloned four tandem
copies of domain II using the same 38'CAT plasmid (Fig.
8A). The resulting plasmid, 4xII-CAT plasmid, increased the DBH minimal promoter activity by 100- to 200-fold in
DBH-positive cell lines. Thus, four tandem copies of domain II
exhibited at least twofold of the promoter activity of the intact DBH
promoter in our transient transfection assay. Strikingly, the CAT
activity driven by 4xII-CAT plasmid in DBH-negative cell lines was
comparable to that of 38'CAT, demonstrating a tight cell specificity.
Furthermore, cotransfection with Phox2a/Phox2b-expression plasmid
increased its promoter activity by 200- to 300-fold only in
DBH-negative cell lines. We conclude that domain II is another Phox2a-binding site and designate it as PRS2. These results suggest that the multiple domain II promoter may be used as a valuable tool for
targeted transgene expression to noradrenergic neurons.
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Figure 8.
Domain II mediates transactivation of the promoter
activity by Phox2a and Phox2b. A, Diagram of reporter
plasmids. 38'CAT, identical to TATA-CAT
(Yang et al., 1998a), is a minimal DBH-CAT reporter plasmid that
contains the TATA box and the transcription start site of the human DBH
gene. A single copy of domain II oligonucleotide is cloned at the
SphI site upstream of the TATA box, resulting in
1xII-CAT. Likewise, four tandem copies of
domain II oligonucleotide are cloned at the SphI site.
Sequence analysis of 4xII-CAT showed
that, among the four copies of domain II, the third copy was in
opposite orientation as indicated by the direction of
arrows. B, Domain II sequence motif(s)
can activate the promoter activity in a noradrenergic-specific manner.
Transcription activities driven by reporter constructs were compared by
transient transfection assay using two DBH-positive and two
DBH-negative cell lines. 4xII-CAT plasmid drives the CAT
gene expression 100- to 200-fold higher than the 38'CAT plasmid, which
represent at least twice activity of that by the intact DBH
promoter-CAT construct (978CAT), without
compromising cell specificity. The CAT activity driven by each
construct is presented relative to that of 38'CAT, with mean ± SEM for six to eight determinations plotted on a logarithmic scale.
This experiment was repeated once more in triplicate, using plasmid
DNAs independently prepared, and resulted in similar patterns.
C, Domain II can mediate transactivation by Phox2a and
Phox2b. HeLa and SK-N-BE(2)C cells were transiently cotransfected with
reporter plasmids and pRC/Phox2a or pRC/Phox2b with a molar ratio of
0.5. The CAT activity driven by each reporter construct itself was set
to 1.0 to compare transactivation by Phox2a or Phox2b. Fold induction
by Phox2a/Phox2b cotransfection is presented as mean ± SEM values
from six to eight samples on a logarithmic scale.
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DISCUSSION |
Our present study characterizes the potential promoter
function of multiple protein-binding sites residing within the 5'
proximal area of the human DBH promoter and demonstrates that their
combinatorial interplay is crucial for noradrenergic-specific
transcriptional activity. These protein-binding sites have been
identified as protected regions in DNase I footprinting analysis using
nuclear extracts prepared from DBH-expressing SK-N-BE(2)C and
DBH-negative HeLa cells (Seo et al., 1996). One of these footprinted
regions (domain IV), located at 185 to 150 bp upstream of the
transcription start site, is a composite promoter that contains
overlapping cis-acting elements, including a CRE,
YY1-binding site, and two core ATTA motifs of the HD-binding site. Our
previous work showed that the HD-binding site within domain IV is a
noradrenergic-specific promoter element and that paired-like HD protein
factors, Phox2a and Phox2b, directly transactivate DBH transcription
through this sequence (Yang et al., 1998a). The studies described here
further characterize the promoter function of the proximal
protein-binding sites residing between domain IV and the TATA box of
the human DBH gene and identify the cognate nuclear factors that
interact with these sequences.
Protected region I (domain I) is located at 58 to 40 bp in the
human DBH proximal promoter region and is only 10 bases from the TATA
box (Figs. 1A, 2; Seo et al., 1996). The nucleotide
sequences of domain I are G/C-rich and include a 9 bp fragment at the
center, 5'-ACCCACCCC-3', the opposite sequence of which shows an 8/9
match to the consensus Sp1-binding site,
5'-(G/T)(G/A)GGC(G/T)(G/A)(G/A)(G/T)3' (Faisst and Meyer, 1992).
Consistent with the imperfect sequence match, domain I appeared to have
a slightly lower affinity for the transcription factor Sp1 than for the
consensus Sp1 motif. Several lines of evidence establish that domain I
is an authentic Sp1-binding site: (1) formation of DNA-protein complex
(CI) was specifically competed by the consensus Sp1 motif-containing
oligonucleotide and domain I oligonucleotide (Fig. 3), (2) CI is shown
to contain Sp1 in antibody supershift assay (Fig. 3), and (3) the
recombinant Sp1 protein binds to domain I, and the complex shows an
identical mobility to that of CI (data not shown). Our mutational
analyses demonstrate that the Sp1-binding site in domain I is critical for the intact DBH promoter activity. Sp1 is known to be a ubiquitous transcription factor (Kadonaga et al., 1987) and controls, by interacting with its binding site(s), numerous eukaryotic genes including housekeeping, signal pathway-induced, as well as
tissue-specific genes. Based on its frequent occurrence in CpG islands,
one possible mechanism underlying transcriptional control of a variety
of eukaryotic genes by Sp1 may be its role in maintaining
methylation-free CpG islands of active genes (Macleod et al., 1994;
Graff et al., 1997). Another potential structure/function link of the
Sp1-binding site is that it is mostly located in proximity to the core
promoter region and, thus, may facilitate the formation of the active
transcriptional machinery. In support of this, Sp1 is found to directly
interact with TAFII110 (Hoey et al., 1993; Gill et al.,
1994).
Intriguingly, the rat phenylethanolamine N-methyltransferase
gene, encoding the last step of catecholamine biosynthesis, has been
shown to be regulated by Sp1 through two Sp1-binding sites at 45 and
165 bp (Ebert and Wong, 1995). In addition, our recent analysis of
the tyrosine hydroxylase promoter identified a proximal Sp1-binding
site that is critically required for intact promoter activity (Yang et
al., 1998b). Taken together, Sp1 may represent the first known
transcription factor that coregulates transcription of all three
catecholamine-specific synthesizing genes.
Domain III, at 136 to 102 bp, resides immediately downstream of the
composite promoter, domain IV. This region includes a nucleotide patch,
5'-GGCCTGGGGCGC-3', which perfectly matches the consensus AP2 site
(Williams and Tjian, 1991; Faisst and Meyer, 1992). In contrast to Sp1,
which is ubiquitously expressed in most cells, AP2 is primarily
expressed in neural crest cells and their major derivatives in a cell
type-specific manner (Mitchell et al., 1991). In addition, our EMSA and
DNase I footprint analyses demonstrate that domain III is more
efficiently bound by nuclear proteins prepared from DBH-expressing
SK-N-BE(2)C and CATH.a than by those from DBH-negative HeLa and C6
cells (Fig. 2). However, AP2 was originally purified and cloned from
HeLa cells (Williams et al., 1988) and, thus, we previously proposed
that a protein factor other than AP2 may bind to domain III and
regulate DBH transcription (Seo et al., 1996). Nevertheless, our
present studies demonstrate that AP2 interacts with the domain III
sequence, because the specific DNA-protein complex was eliminated by
the unlabeled consensus AP2-binding site and supershifted by
AP2-specific antibody (Fig. 6). Antibody coincubation experiments
showed that the DNA-protein complex(es) formed by HeLa and C6 nuclear
extracts also contain, albeit to a lesser extent, AP2 (data not shown).
Base substitutions within domain III strikingly reduced the DBH
promoter activity in DBH-positive cell lines and reduced it modestly in
DBH-negative cells (Fig. 1C). Collectively, these data
indicate that HeLa and C6 cells express low levels of AP2 compared with
DBH-positive cells. Consistent with these results, Greco et al. (1995)
have shown that the recombinant AP2 protein binds to the corresponding motif of the rat DBH promoter and an AP2 expression vector
preferentially transactivates the DBH promoter activity in DBH-negative
HepG2 cells. Our EMSA and competition assays (Fig. 6) indicate that domain III has a significantly higher affinity (>5 times) for AP2 than
a well characterized AP2 consensus motif residing in the human
metallothioneine-IIA gene (Williams et al., 1988; Williams and Tjian,
1991). We conclude that the transcription factor AP2 is abundantly
expressed in noradrenergic cells and activates transcription of the
human DBH gene via interaction with this high-affinity AP2-binding site
residing within domain III.
Domain II is identified as a positive noradrenergic-specific
cis-element in that its mutation diminishes the DBH promoter activity exclusively in DBH-expressing cell lines. DNA binding studies
using EMSA, Southwestern, and DNase I footprint analyses show that
domain II interacts with nuclear factor(s) in a noradrenergic-specific manner (Figs. 4, 5, 7). We originally proposed that domain II may
represent a novel cis-regulatory element because it did not show any significant homology to known protein-binding motifs (Seo et
al., 1996). Domain II contains an ATTA motif at the 3' side but beyond
these four bases does not bear any sequence homology with the PRS1
residing within domain IV. However, several lines of evidence support
the identification of domain II as another Phox2a/Phox2b-binding site:
(1) DNA-protein complex (CII) formed by domain II oligonucleotide is
specifically competed by the PRS1 sequence, (2) coincubation of nuclear
extracts from DBH-positive cells with Phox2a-specific antibody produces
supershifted complexes using domain II oligonucleotide as the probe,
(3) domain II oligonucleotides containing mutation within the ATTA
motif was unable to form complex CII, (4) the same mutant
oligonucleotide does not produce the supershifted complex in the
presence of the Phox2a-specific antibody, and (5) cotransfection assay
shows that domain II confers Phox2a- or Phox2b-mediated transactivation
in the DBH-negative cells. In a previous study, forced expression of
Phox2a or Phox2b in DBH-negative cell lines activated the reporter gene
expression driven by a single copy of the PRS1 approximately fourfold
while it activated that driven by the intact DBH promoter 10- to
15-fold (Yang et al., 1998a). Similarly, coexpression of Phox2a or
Phox2b stimulated reporter expression driven by 1xII-CAT plasmid
~3.5-fold in DBH-negative HeLa or C6 cells (Fig. 7; data not shown).
Taken together, we conclude that Phox2a/Phox2b activates DBH
transcription through at least two contacts on the DBH upstream
promoter.
The finding that PRS1 and domain II, both critical for
noradrenergic-specific DBH promoter activity, are binding sites for Phox2a/Phox2b (Figs. 7, 8; Yang et al., 1998a) was unexpected, because
these two sequence elements do not show any sequence identity beyond
the single ATTA motif at the 3' side (Fig. 7A). Because this
motif may occur quite often (statistically once in every 128 bases in
either orientation), it is unlikely that this motif by itself can
determine noradrenergic-specific transcriptional activity of the DBH
gene. Several different, but mutually nonexclusive, mechanisms may
explain this rather surprising finding. First, the DBH promoter
contains multiple binding sites for Phox2a/Phox2b, and their
simultaneous occupation may be critical for intact transcriptional activity. In line with this, PRS1 contains two ATTA motifs, both of
which are important for the intact promoter activity (Yang et al.,
1998a). Thus, PRS1 may in fact represent two Phox2a/Phox2b-binding sites that may interact with Phox2a/Phox2b alternately or
simultaneously. If so, the proximal DBH promoter may include three or
even more PRSs, and combinatorial action of these sequences may be
critical for DBH transcription. Second, as-yet-unidentified cofactor(s) may modulate DNA-binding properties, e.g., affinity and/or specificity, of Phox2a/Phox2b in vivo. This possibility is supported by
our finding that, with both PRS1 and domain II, Phox2a forms
DNA-protein complex(es) much more robustly when it is bound by a
Phox2a-specific antibody. Many HD proteins are known to have relaxed
DNA-binding specificities in vitro despite their functional
specificity in vivo (Hayashi et al., 1990; McGinnis and
Krumlauf, 1992). Several such cofactors, including extradenticle and
 Ftz-F1, have recently been identified to modify DNA-binding
specificity or selectivity of several HD protein factors (Chan et al.,
1994; van Dijk and Murre, 1994; Chan and Mann, 1996; Guichet et al.,
1997; Yu et al., 1997).
A schematic diagram of DBH promoter activation via multiple
protein-binding sites is depicted in Figure
9. Based on DNase I footprinting analyses
using the upstream promoters containing mutations at each site, it is
unlikely that synergistic binding of the cognate transcription factors
to their binding sites underlies their concerted activation of DBH
transcription (Fig. 2). Rather, we infer that protein-protein
interactions between the nuclear factors, their putative cofactors,
and/or the components of the basal RNA polymerase II machinery may be
pivotal for intact transcription of the DBH gene. In this model, lack
of any one component diminishes transcriptional activation as when
mutation of PRS2 abolishes most of the promoter activity in
noradrenergic cells (Fig. 9B). In DBH-negative cells, lack
of a cell-specific nuclear factor(s), e.g., Phox2a and AP2, causes RNA
polymerase II machinery to be largely inactive for the DBH gene (Fig.
9C,D). In support of synergistic activation of DBH transcription by multiple cis-regulatory
elements, four tandem copies of domain II increased the minimal
promoter activity by 100- to 200-fold in DBH-positive cell lines, which far exceeds the additive activity of individual four copies of domain
II (Fig. 8). Remarkably, the cell specificity of this multiple domain
II promoter was not compromised at all, suggesting that it may be of
great value for targeted expression of therapeutic genes to
noradrenergic neurons. We are currently testing the promoter activity
and cell-specificity with additional copies of domain II.
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Figure 9.
Proposed interaction among multiple transcription
factors in the 260 bp upstream region of the human DBH gene.
A, In DBH-positive cells, transcription factors
interacting with domains I-IV, including cell type specific factors
(e.g., Phox2a, Phox2b, and AP2) and general transcription factors
(e.g., CREB and Sp1), synergistically activate the DBH transcription
(+++). B, When any of these domains, domain II motif in
this example, is mutated, the synergism is disrupted and robust
transcription cannot occur (+/) although DNA-protein interactions at
the other sites are intact (Fig. 2). C,
D, In DBH-negative cells, cell-specific Phox2a/Phox2b
and AP2 are absent or present in lower amount compared with
DBH-positive cells, respectively. Accordingly, synergistic activation
of DBH gene transcription cannot occur whether or not domain II is
intact (+/).
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Most mutant mice lacking DBH die in utero, indicating that
proper expression of DBH is critical for fetal development and survival
(Thomas et al., 1995). Strikingly, clinical scientists have reported
that DBH is deficient in some patients and have come to recognize that
DBH deficiency is a genetic disorder of cardiovascular regulation
(Robertson et al., 1986; Man in't Veld et al., 1987; for review, see
Robertson, 1995). Neonates with DBH deficiency had episodic
hypothermia, hypoglycemia, and hypotension. Survivors cope relatively
well until late childhood, when overwhelming orthostatic hypotension
profoundly limits their activities. At present, mechanisms underlying
DBH deficiency are not known. Although this disease may be associated
with mutation of the coding sequence that leads to expression of
nonfunctional protein, it is also possible that mutation of critical
promoter element(s) or a cognate protein factor may cause this genetic
disease.
In summary, our results suggest that multiple nuclear factors,
including general transcription factors (Sp1 and CREB) as well as
cell-specific factors (AP2, Phox2a, and Phox2b) bind to proximal cis-acting elements and synergistically activate
transcription of the DBH gene in noradrenergic cells. We have
previously shown that the upstream region spanning from 486 to 263 bp
of the human DBH gene preferentially exhibits a silencer function in
DBH-negative cells, and this region may contain several cell-specific
silencer motifs (Ishiguro et al., 1993, 1995; Kim et al., 1998).
Furthermore, transgenic mice experiments showed that the upstream
region between 1.1 and 0.6 kb of the human DBH gene is required for
expression of the reporter gene in vivo (Hoyle et al.,
1994). Thus, DBH gene regulation appears to require multiple layers of
regulatory mechanisms to ensure its accurate expression pattern in
quite limited locations in the nervous system, and promises to be an
excellent system to study neural-specific gene regulation in the normal
and diseased state of the brain, as well as in response to
environmental stimuli.
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FOOTNOTES |
Received June 12, 1998; revised July 31, 1998; accepted Aug. 6, 1998.
This work was supported by National Institutes of Health Grant MH48866
and the Research Contingency Fund from the University of Tennessee. We
thank Drs. T. S. Nowak Jr and E. A. Park for critical reading
of this manuscript.
Correspondence should be addressed to Dr. Kwang-Soo Kim, Department of
Neurology, University of Tennessee, College of Medicine, 855 Monroe
Avenue, Memphis, TN 38163.
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Top
Abstract
Introduction
