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INTRODUCTION |
The neurotransmitter/neurohormone epinephrine is a
physiologically significant neuroregulator that assumes an important
role in the stress response and is a major component in cardiac
disease, immune dysfunction, and neuropsychiatric illness.
Consequently, phenylethanolamine N-methyltransferase
(PNMT)1 (EC 2.1.1.28), its
biosynthetic enzyme, and the mechanisms by which PNMT is genetically
regulated have been of interest in attempts to better understand the
etiologies of these disorders.
Examination of the PNMT gene from a variety of species (human (1, 2),
cow (3, 4), mouse (5), and rat (6)) suggests that there may be common
transcriptional regulatory proteins that control PNMT gene expression
based on the presence of canonical DNA binding sequences for several
known factors. We have previously demonstrated for the rat PNMT gene
that one stimulatory transcriptional regulator is the factor Sp1 for
which two binding elements exist within the proximal -863 bp of 5'
upstream PNMT promoter/regulatory sequence at -48 and -168 bp
relative to the site of transcription initiation, +1 (7, 8). Binding
elements for the immediate early gene transcription factor Egr-1
overlap 6 bp of the 3' end of each of these Sp1 sites. Although both
sites can function as Sp1 or Egr-1 activation sites, it appears that
in vivo, the distal overlapping Sp1/Egr-1 binding element
(-168 bp) may preferentially serve as a site for Egr-1 induction,
whereas the proximal binding element (-48 bp) likely functions as an
Sp1 site. This motif of overlapping or closely adjacent Sp1 and Egr-1
binding elements seems to be common among genes that possess these
regulatory sites (9-12), and Sp1 or Egr-1 either activates or inhibits
gene expression through interaction with their cognate DNA sequences.
The present studies further examine the role of Sp-1 in the regulation
of PNMT gene expression. We demonstrate that the high basal activity of
the PNMT promoter in the neuroblastoma-derived Neuro2A cell line is due
to Sp1 acting through its -48 and -168 bp binding elements. In the
case of the -48 bp Sp1 binding element, another transcription factor
can bind to a recognition site 3' to this Sp1 site, thereby preventing
Sp1 binding and activation of the PNMT promoter through this proximal
element. Consensus sequence match, Southwestern analysis, and gel
mobility shifts assays with antibodies indicate that this factor is the
transcription protein MAZ (13). Together, these findings suggest that
Sp1 may be an important regulator of tissue-specific PNMT gene
expression and that the exclusionary competition between Sp1 and MAZ
may provide additional tissue-specific control.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides and Plasmid Constructs--
The 21-mer WTA
(5'-CCTCCCCGCCCCCGCGCGTCC-3') (rat PNMT promoter sequences from -180
to -160 bp), WTB (5'-GTCTGGGCGGGGGGGAGGGGA-3') (PNMT promoter
sequences from -59 to -39 bp), and mut45, 5'-GTCTGGGCGGGGaaaAGGGGA-3' (point mutation in -45 bp Egr-1 consensus sequence) oligonucleotides have been previously described (7). Two additional 21-bp
oligonucleotides were synthesized to use as mutant probes for the
distal and proximal Sp1 sites at -168 and -48 bp, respectively: 1)
mut168 (5'-CCTCaaCGCCCCCGCGCGTCC-3'; mutated -168 bp Sp1 binding
element), and 2) mut48 (5'-GTCTaaGCGGGGGGGAGGGGA-3'; mutated -48 bp
Sp1 binding element). The duplex WTA, WTB, mut45, mut168, and mut48
oligonucleotides were subcloned into the SmaI site of the
vector pBluescript II SK(-) (Stratagene, La Jolla, CA), yielding
pBSIISK(-)RPWTA, pBSIISK(-)RPWTB, pBSIISK(-)RPmut45, pBSIISK(-)RPmut168, and pBSIISK(-)RP48 respectively, to
generate double-stranded DNA for gel mobility shift assays.
For all plasmid constructs, standard recombinant DNA technologies were
used. The wild-type PNMT promoter-luciferase reporter gene construct
(pGL3RP863) was obtained by subcloning the
XhoI-HindIII restriction fragment from pRP863LUC
(7), containing the rat PNMT promoter sequences from -863 to +18 bp,
into the corresponding restriction sites in the multiple cloning region
of the vector pGL3Basic (Promega, Madison, WI). Two deletion
constructs, pGL3RP444 and pGL3RP392, containing 444 and 392 bp of
proximal PNMT promoter sequence, respectively, were then generated by
digesting pGL3RP863 with KpnI or NheI, followed
by self-ligation. The unaltered plasmid, pGL3Basic, was used as a
promoterless control, whereas the plasmid pRSV-LacZ, containing the
-galactosidase gene, was used as a normalization control to correct
for variable transfection efficiency as described previously (7) and below.
The Sp-1 expression construct, pCGNSp-1, and the vector construct,
pCGN, were kindly provided by Dr. Thomas Shenk (Howard Hughes Medical
Institute, Princeton University).
Cell Culture and Transient Transfection Assays--
The rat
pheochromocytoma PC12 and RS1 cells have been previously described (7).
The neuroblastoma-derived Neuro2A, embryonic mouse fibroblast NIH/3T3,
and monkey kidney COS1 cells were obtained from the American Type
Culture Collection (Rockville, MD). All cells were maintained in
Dulbecco's modified Eagle's medium containing gentamycin sulfate (50 µg/ml, United States Biochemical Corp., Cleveland, OH) at 37 °C in
an atmosphere of 7% CO2-93% air (7). In the case of the
RS1 cells, 200 units/ml of hygromycin B (Calbiochem, La Jolla, CA) was
also included. In addition, 5% bovine calf serum and 5% equine serum
(Hyclone, Logan, UT) were added to the medium for the PC12, RS1, and
NIH/3T3 cells, whereas 10% fetal bovine serum (Gemini Bio-Products
Inc., Calabasas, CA) was used for the Neuro2A and COS1 cells.
For transient cotransfection assays, cells were plated in 24-well
culture dishes at a density of 2 × 105 cells/well and
maintained at 37 °C in an atmosphere of 7% CO2-93% air
overnight, and transfection was performed using SuperFect according to
the manufacturer's recommendations (Qiagen, Inc., Chatsworth, CA).
Each transfection assay included 1.0 µg of wild-type or mutant PNMT
promoter-luciferase reporter gene construct, 0-1.5 µg of expression
construct, and 0.3 µg of pRSV-LacZ. Following transfection, the cells
were exchanged to culture medium and maintained for 36 h at
37 °C and 7% CO2-93% air. Cells were processed and assayed for luciferase and
-galactosidase as described below
Luciferase and
-Galactosidase Assay--
Following two
phosphate-buffered saline (PBS) washes, cells were lysed in 120 µl of
lysis buffer (Promega), and luciferase activity was measured as
previously using the luciferase assay system (7). Purified luciferase
was used to define the linear range of the assay, and luciferase
activity was then determined in 20 µl of appropriately diluted cell
lysate. Protein was measured by the Bradford method (14), and
luciferase activity was expressed as pg of product/µg of protein.
-Galactosidase activity was determined as described previously (7),
and luciferase activity was then corrected for transfection efficiency
and expressed relative to control values as follows (7). Luciferase
activity expressed from the plasmid vector pGL3Basic was first
subtracted from the luciferase activity generated from each of the PNMT
promoter-luciferase reporter gene constructs. Then, luciferase activity
was expressed relative to
-galactosidase expressed from the
pRSV-LacZ control. To correct for variable transfection within the same
cell line,
-galactosidase activity from the pRSV-LacZ construct used
for cotransfection with the full-length pGL3RP863 was set as unity and
-galactosidase activity in the cotransfections with the other constructs expressed relative to it. The ratios so generated were then
used to correct each luciferase:
-galactosidase ratio so that
-galactosidase activity in each transfection was effectively identical. In a similar fashion, luciferase activity was normalized across cell lines. In this case,
-galactosidase activity from the
pRSV-LacZ construct for the cotransfection using the full-length pGL3RP863 in the RS1 cell line was used as unity. Luciferase activity was then expressed relative to luciferase activity for the pGL3RP863 construct in RS1 cells set to unity. At least six replicates were included for each sample group, and experiments were repeated at least twice.
Site-directed Mutagenesis--
Two single-stranded 21-mer
mutagenic primers corresponding to the mut168 and mut48 sequences
identified above were used for site-directed mutagenesis to inactivate
the distal and proximal Sp1 sites, respectively. First, mutagenic
megaprimers were generated for each mutant sequence by PCR using the
21-bp mutagenic primers paired with GLprimer2, a primer binding to the
extreme 22 bp of 5' coding region of the firefly luciferase gene
contained within the pGL3Basic vector (Promega), pGL3RP863 as template,
and VentR® DNA polymerase (New England BioLabs, Beverly,
MA) to amplify the DNA (30 cycles, 94 °C for 1 min, 58 °C
for 1 min, and 72 °C for 1 min). Then, a second PCR (10 cycles) was
performed to extend each megaprimer using pGL3RP392 as template,
followed by 20 cycles of PCR using RVprimer3 and the GLprimer2 once
again to produce 392-bp oligonucleotides with either the -48 or -168
bp Sp1 mutated. PCR products were separated by agarose gel
electrophoresis, and agarose was removed using a QIAXII Gel
Extraction Kit (Qiagen Inc.). The PCR products were then
subcloned into the pGL3Basic plasmid to generate pGL3RP392mut168 and
pGL3RP392mut48. The double mutant construct pGL3RP392
mut168/48 was generated identically by using the mut48 primer and
pGL3RP392mut168 as a template. Following selection and screening, the
mutant constructs were verified by DNA sequencing (16).
Nuclear Extracts--
Cells were propagated as described above
using 100-mm culture dishes and nuclear extracts prepared according to
the procedure of Andrews and Faller (15). Briefly, after the cells
reached a density of 5 × 105-1 × 107 per dish, they were collected into 1.5 ml of ice-cold
PBS, and washed once with cold PBS. The cells were then lysed in 400 µl of 10 mM HEPES-KOH, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride on ice
for 10 min. Following collection of the nuclei by centrifugation for
10 s at 17,000 × g and 4 °C in a
microcentrifuge, nuclear extract was prepared by hypotonic lysis. The
nuclei were resuspended in 20-100 µl of 20 mM HEPES-KOH,
pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride and
maintained on ice for 20 min, followed by centrifugation for 2 min at
17,000 × g and 4 °C. Extracts were separated from pelleted debris and stored at
70 °C until use for footprinting or
mobility shift assays.
DNase I Footprinting Analysis--
To generate the DNA
probes for footprinting analysis, two oligomers,
5'-AAAGGGCGCCCTCCACATCTC-3' (nucleotides -204 to -184) and
5'-TCCTGTTGAGGCCGCCTATCT-3' (nucleotides +18 to -3) were
uniquely labeled at their 5' terminus using [
-32P]ATP
(NEN Life Science Products) and T4 polynucleotide kinase (Life
Technologies, Inc), and PCR was performed as described for site-directed mutagenesis. The resulting PNMT promoter fragments (222 bp) were isolated by agarose gel electrophoresis and Qiagen gel
extraction as described above. Approximately 7.5 × 104 dpm of each DNA probe was incubated with 70 µg of
nuclear extract from Neuro2A or COS1 cells in 30 µl of binding buffer
consisting of 25 mM HEPES buffer, pH 7.9, 50 mM
KCl, 0.05 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol and
containing 2 µg of poly(dI-dC)-poly(dI-dC) for 30 min on ice. After
10 min at room temperature, DNase I footprinting was performed. The
optimal RQ1 DNase I (Promega) concentration (0.3 units/reaction) was
determined empirically in preliminary experiments. DNase I cleavage
patterns were identified by polyacrylamide gel electrophoresis (7%
sequencing gel), mapping protected sequences by comparison of the
autoradiographic banding patterns to sequencing ladders run in adjacent
lanes on the gel (16).
Gel Mobility Shift Assays--
Gel mobility shift assays were
performed using 1 ng of the probes described above, end-labeled with
[
-32P]ATP using T4 polynucleotide kinase
(3 nM, specific activity = 2.5 × 108
dpm/µg) and 3 µg of nuclear extract in 20 µl of binding buffer (see DNase I footprinting) containing 0.5 µg poly
(dI-dC)-poly(dI-dC). To identify the transcription factors constituting
the protein-DNA binding complexes, anti-Egr-1 (C-19, Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), anti-Sp1 (PEP2, Santa Cruz
Biotechnology) or anti-MAZ (Dr. Kenneth Marcu, State University of New
York, Stonybrook, NY) antibody was included in the binding reactions. Purified human Sp-1 protein was used as a control (Promega).
Protein-DNA complex formation and antibody supershifts were analyzed on
5% polyacrylamide gels by autoradiography.
Southwestern Analysis--
Twenty µg of Neuro2A cell nuclear
protein was separated by SDS-polyacrylamide gel electrophoresis (10%
gel) as described previously (17), along with human Sp1 as a control.
Proteins were transferred to nitrocellulose and renatured by incubation
in binding buffer consisting of 20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 10% glycerol,
0.2% Nonidet P-40 at room temperature for 45 min. After
prehybridization with binding buffer containing 0.5% nonfat dry milk,
1 µg/ml salmon sperm DNA, and 1 × 106 cpm/ml of
[32P]WTB probe at 4 °C overnight, the filter was
rinsed and washed twice for 15 min with binding buffer. Protein-DNA
complexes were then visualized by autoradiography, and the relative
molecular weight of the complexing proteins determined using the
prestained protein standards. Specifically, the ln
Mr for each protein standard was plotted as a
function of electrophoretic migration. Regression analysis was used to
generate a linear equation from which the Mr of
the unknown proteins could be determined by interpolation.
Statistic Analysis--
All data are presented as the mean ± S.E. with an n
6 for each experimental group. The
statistical significance of the difference between two groups was
determined using Student's t test. A p value
of
0.05 was considered statistically significant.
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RESULTS |
Cell-specific Basal PNMT Promoter Activity--
To examine
the dependence of PNMT promoter activity on potential
cell-specific transcription factors, three nested deletion PNMT
promoter-luciferase reporter gene constructs, pGL3RP863, pGL3RP444, and pGL3RP392, containing 863, 444, and 392 bp of PNMT 5' upstream promoter sequence, respectively, were cotransfected into
PC12, RS1, Neuro2A, 3T3 or COS1 cells along with the
-galactosidase control construct pRSV-LacZ, and relative luciferase activity was
determined (Fig. 1). Fig. 1A
provides a schematic representation for each PNMT promoter-luciferase
reporter gene construct, identifying the KpnI and
NheI restriction sites used to generate the deletion constructs. Because transfection efficiency may vary between
transfections and with different constructs, luciferase activity was
corrected for variable expression of both the control pRSV-LacZ
construct and PNMT promoter-luciferase reporter gene construct as
detailed under "Experimental Procedures." When transfection
efficiency for each construct was normalized within a given cell line
(Fig. 1B), the highest basal luciferase activity was
observed in the Neuro2A cells with 10-100-fold lower expression
(p
10
3) of luciferase from the nested
deletion constructs transfected into the COS1, RS1, 3T3, and PC12
cells. Transfection efficiency may also vary between different cell
lines. Two control constructs, pRSVLacZ and pRSVLUC (data not shown),
showed that the COS1 and Neuro2A cells had the highest transfection
efficiency, i.e. expressed higher
-galactosidase or
luciferase activity, with lower transfection efficiency in the 3T3
cells, and the lowest transfection efficiency in PC12 and RS1 cells.
When PNMT promoter driven luciferase reporter gene activity was
corrected to account for these inter-cell line differences (Fig.
1C), the Neuro2A cells still showed the highest levels of
luciferase expression, although luciferase levels expressed from the
constructs in the PC12 and RS1 cells approached that in the Neuro2A
cells. However, luciferase activity generated from the same PNMT
promoter-luciferase reporter gene constructs in the 3T3 and COS1 cells
remained markedly lower and barely detectable. Thus, PNMT promoter
activity appears highest in cell lines derived from tissues likely to
express PNMT.

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Fig. 1.
Cell-specific luciferase activity of PNMT
promoter-luciferase reporter gene constructs. The PNMT
promoter-luciferase gene constructs pGL3RP863, pGL3RP444, and
pGL3RP392, containing 863, 444, and 392 bp of rat 5' upstream PNMT
promoter/regulatory sequences were transfected into COS1, PC12, RS1,
NIH/3T3 and Neuro2A cells and luciferase activity determined as
described under "Experimental Procedures." A, schematic
of the nested deletion constructs pGL3RP863, pGL3RP444, and pGL3RP392.
B, luciferase activity corrected for variable transfection
efficiency within each cell line. Luciferase activity was first
corrected for variable transfection efficiency within each cell line
using the normalization control plasmid, pRSV-LacZ. Luciferase activity
was then expressed relative to luciferase expression for the
full-length pGL3RP863 in RS1 cells (7). C, luciferase
activity corrected for variable transfection efficiency across cell
lines. In this case, luciferase activity was first normalized across
all cell lines for variable transfection efficiency using the
normalization control plasmid, pRSV-LacZ and setting -galactosidase
expression from this construct in the RS1 cells as unity. Then,
luciferase activity was expressed relative to luciferase expression of
the pGL3RP863 construct in the RS1 cells as previously.
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In addition, in the Neuro2A cells, the construct pGL3RP444 showed the
highest basal luciferase expression. By comparison, luciferase activity
expressed from the pGL3RP863 and pGL3RP392 plasmid constructs in the
Neuro2A cells was approximately 20 and 50% lower, respectively.
Together these results suggest that basal PNMT promoter activity varies
depending on the host cell line as well as the extent of PNMT promoter
sequences included in the reporter gene constructs. This
specificity is likely due to the fact that the tissues from which these
cell lines were derived express different transcriptional proteins,
some of which may be important for PNMT promoter activity and, hence,
PNMT gene expression.
PNMT Promoter-specific Transcriptional Activators Expressed by
Neuro2A Cells--
As PNMT promoter activity was greatest in the
Neuro2A cells and low in the COS1 cells, DNaseI footprinting was
performed using uniquely labeled [32P]DNA probes spanning
nucleotides +18
-204 bp and nuclear extracts from these cell lines
to identify proximal DNA sequences to which transcription factors
specific to the Neuro2A cells bind to stimulate the PNMT promoter. As
shown in Fig. 2B, (lane
2), protected sequences using the Neuro2A cell nuclear extract
included the nucleotide sequences from -32 to -55 bp, -97 to -110
bp, and -156 to -200 bp. On the complementary DNA strand (lane
5), corresponding footprints were observed spanning nucleotides
-55 to -31 bp, -111 to -95 bp, and -195 to -161. In contrast,
only sequences spanning nucleotides -32 to -55 bp (-55 to -31 bp on
the complementary strand) showed a definite footprint with the COS1
cell extracts (lanes 3 and 6). The footprinted
regions coincided with previously identified binding elements for
several transcription factors shown to activate PNMT gene expression,
including Sp1 (-168 bp)/Egr-1 (-165 bp), AP-2 (-103 bp), and Sp1
(-48 bp)/Egr-1 (-45 bp).

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Fig. 2.
DNase I footprinting with Neuro2A and COS1
cell nuclear extracts. A 222-bp DNA fragment spanning nucleotides
+18 to -204 of the rat PNMT gene was generated by PCR using two 21-mer
primers uniquely labeled with 32P at their 5' terminus (5'
3', -204 to -184 bp and +18 to -3 bp). The fragments were
incubated with nuclear extracts from Neuro2A and COS1 cells and then
subjected to DNase I footprinting. A, schematic of the PNMT
promoter depicting binding elements for known transcriptional
regulators of the PNMT gene. B, autoradiogram from DNase I
footprinting. Lanes 1 and 4, no extract;
lanes 2 and 5, Neuro2A cell nuclear extract;
lanes 3 and 6, COS1 cell nuclear extract. DNase I
concentrations used for the footprinting were 0.001 unit/µl for
lanes 1 and 4 and 0.02 units/µl for lanes
2, 3, 5, and 6. The protected nucleotides were mapped
by comparison to sequencing products generated using the PCR primers
above and the position of the footprints denoted by the vertical bars
and nucleotide number to the left of each footprint.
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Sp1, a Transcriptional Activator of the PNMT Promoter in Neuro2A
Cells--
The putative AP-2 site at -103 bp shows low identity to
the AP-2 consensus sequence and low affinity for AP-2 protein. In addition, gel mobility shift assays using an oligonucleotide spanning this region and nuclear extracts from the Neuro2A and COS1 cells showed
no protein-DNA complex formation (data not shown). Therefore, attention
was focused on the proximal and distal overlapping Sp1 and Egr-1
consensus sites. To determine whether Sp1 or Egr-1 was expressed in the
Neuro2A cells and responsible for the high basal levels of PNMT
promoter activity in these cells, gel mobility shift assays were
performed in the presence of specific antibodies. 32P-labeled 21-bp oligonucleotide probes for both
the -168/-165 and -48/-45 bp Sp1/Egr-1 sites (WTA,
5'-CCTCCCCGCCCCCGCGCGTCC-3'; WTB, 5'-GTCTGGGCGGGGGGGAGGGGA-3',
respectively, Fig. 3A)
were combined with Neuro2A or COS1 cell nuclear extract in the presence or absence of an anti-Egr-1 antibody (C-19) or an anti-Sp1 antibody (PEP2). As shown in Fig. 3B, the WTA probe, spanning the
distal Sp1/Egr-1 site, produced several protein-DNA complexes with the nuclear extract from the Neuro2A cells (lanes 1 and
2). The major protein-DNA complex was supershifted by
anti-Sp1 antibody (lane 4), whereas no supershifted
complexes (Ref. 7, Santa Cruz Biotechnology) were observed with
anti-Egr-1 antibody (lane 3). Several protein-DNA complexes
were also observed using the duplex oligonucleotide probe WTB spanning
the proximal Sp1/Egr-1 binding element and Neuro2A cell nuclear extract
(Fig. 3B) or the WTA oligonucleotide probe and COS1 cell
nuclear extract (Fig. 3C). However, neither the anti-Egr-1
nor anti-Sp1 antibody supershifted any of these complexes (Fig. 3,
B and C, lanes 3 and 4). Finally, no
protein-DNA complexes were observed when the WTB oligonucleotide probe
was combined with nuclear extract from the COS1 cells (data not
shown).

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Fig. 3.
Neuro2A cell nuclear proteins binding to the
Sp1 consensus elements. Gel mobility shift assays were performed
as described under "Experimental Procedures" using the 21-bp WTA
(-168/-165 bp Sp1/Egr1 site) and WTB (-48/-45 bp Sp1/Egr1 site)
oligonucleotide probes end-labeled with 32P and nuclear
extract from Neuro2A and COS1 cells. Antibodies were used to identify
the proteins constituting the protein-DNA complexes. A,
schematic of the WTA and WTB oligonucleotide probes. The nucleotide
sequences for the WTA and WTB oligonucleotide probes are depicted. Sp1
and Egr-1 binding elements are identified. B and
C, autoradiograms from gel mobility shift assays for Neuro2A
(B) and COS1 (C) cell nuclear extracts using the
[32P]WTA and [32P]WTB probes. Lanes
1-4, nuclear extract without antibody (Ab), IgG,
anti-Egr-1 antibody (C-19, Santa Cruz Biotechnology), or anti-Sp1
antibody (PEP2, Santa Cruz Biotechnology), respectively.
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Thus, it would appear that the transcription factor Sp1 is one factor
expressed by the Neuro2A cells that may be responsible for the higher
basal activity of the PNMT promoter observed in these cells. However,
binding of Sp1 was only evident at -168 bp Sp1 site and not at the
-48 bp Sp1 site.
Inhibition of Sp1 Binding at Proximal Sp1 Site--
The proximal
-48 bp Sp1 binding element has been reported to be the higher affinity
Sp1 site, and evidence suggests that it may be the biologically
functional Sp1 site (8). It was therefore puzzling why the WTB probe
spanning this Sp1 site did not form an Sp1-DNA complex with the Neuro2A
cell nuclear extract. The possibility that another transcription factor
expressed in the Neuro2A cells might be binding to the WTB
oligonucleotide probe, thus preventing Sp1 binding, was examined. Gel
mobility shift assays were performed with both the oligonucleotides WTA
and WTB (Fig. 4A) and Neuro2A
cell nuclear extract or purified Sp1 protein. In addition, an
oligonucleotide, mut45 (Fig. 4A), with point mutations in
the DNA sequences 3' to the proximal -48 bp Sp1 consensus site (GCGGGGaaa, within the Egr-1 site), leaving the Sp1 element intact, was
also examined in the gel mobility shift assays. As shown in Fig.
4B, the WTA oligonucleotide probe again formed several
protein-DNA complexes with the Neuro2A cell nuclear extract (lane
3). The major complex showed the same electrophoretic mobility as
the protein-DNA complex formed with pure Sp1 protein (lane
2) and, as described above, was supershifted by anti-Sp1 antibody.
The WTB oligonucleotide again formed several protein-DNA complexes with
the Neuro2A cell nuclear extract as well (lane 6). The major and slowest migrating complex was not identical to the Sp1 complexes formed with the Neuro2A cell nuclear extract (Fig. 4B, lane
3) or Sp1 protein (lane 2) because it had a faster
electrophoretic mobility and was not supershifted by either anti-Sp1 or
anti-Egr-1 antibody as described above. However, Sp1 protein could form
a protein-DNA complex with the WTB probe that did migrate identically. Moreover, the mut45 probe also formed a protein-DNA complex with both
Sp1 protein (lane 8) and the Neuro2A cell nuclear extract (lane 9) with identical electrophoretic mobilities to the
Sp1-DNA complexes seen with the WTA oligonucleotide. A second, less
intense protein-DNA binding complex with slower electrophoretic
mobility was also observed with both pure Sp1 protein and Neuro2A cell nuclear extract. The Neuro2A cells therefore appear to contain a
transcriptional protein, other than Egr-1, that binds to DNA sequences
overlapping the -45 bp Egr-1 site and thereby precludes Sp1 from
binding to its consensus element at -48 bp.

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Fig. 4.
Displacement of Sp1 binding at 48 bp Sp1
consensus element. Gel mobility shift assays were performed with
purified human Sp1 protein (Santa Cruz Biotechnology) or nuclear
extract from Neuro2A cells and the double-stranded DNA probes, WTA,
WTB, and mut45, generated by digestion of pBSKII(-)WTA, pBSKII(-)WTB,
and pBSKII(-)mut45 with EcoRI and BamHI,
followed by end labeling with [ -32P]ATP and
T4 polynucleotide kinase. Mut45 has point mutations in the
-45 bp Egr-1 target sequences as described under "Experimental
Procedures." A, schematic of WTA, WTB and mut45
oligonucleotide probes. The nucleotides sequence for each of the probes
is depicted, and Sp1 and Egr-1 binding elements are identified as
described above. B, autoradiogram of gel mobility shift
assay. Lanes 1, 4, and 7, no Sp1 or nuclear
extract; lanes 2, 5, and 8, Sp1 protein;
lanes 3, 6, and 9, Neuro2A cell nuclear
extract.
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To confirm that the protein-DNA complex formed with Neuro2A cell
nuclear extract and mut45, with identical electrophoretic mobility to
the complex formed between Sp1 and mut45, does contain Sp1, gel
mobility shift assays were executed with the WTB and mut45
oligonucleotide probes and Neuro2A cell nuclear extract in the absence
or presence of anti-Sp1 antibody (Fig.
5). Multiple protein-DNA complexes were
again apparent with the WTB oligonucleotide (lanes 1 and
2). However, none of the complexes were supershifted by the
anti-Sp1 antibody (lanes 3 and 4). In contrast,
the mut45 probe with a mutated Egr-1 site and intact Sp1 site showed
the same two slower migrating complexes as described previously
(lanes 5 and 6), both of which were supershifted
by anti-Sp1 antibody (lanes 7 and 8). The fastest
migrating, predominant complex was nearly completely supershifted,
whereas the slower complex appeared only partially supershifted.

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Fig. 5.
Confirmation of Sp1 binding to the 48 bp
Sp1 consensus element. Gel mobility shift assays were performed
with the 32P-labeled WTB or mut45 oligonucleotide probes
and nuclear extract from Neuro2A cells in the absence or presence of
anti-Sp1 antibody. Autoradiogram of gel mobility shift assay.
Lanes 1 and 5, no nuclear extract; lanes
2 and 6, IgG; lanes 3 and 7, 0.1 µg of anti-Sp1 antibody; lanes 4 and 8, 0.2 µg of anti-Sp1 antibody.
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Thus, mutation of the 3' -45 bp Egr-1 sequences prevents a DNA-binding
protein in the Neuro2A cell nuclear extract from binding to the mut45
oligonucleotide, thereby permitting Sp1, present in the Neuro2A cell
nuclear extract, to bind to the intact proximal -48 bp Sp1 site in the
PNMT promoter.
Role of Sp1 in PNMT Promoter Activation--
To further define
the role of Sp1 in PNMT promoter activation, the effect of
mutation of the -168 bp and/or the -48 bp Sp1 consensus sites on
luciferase reporter gene expression was examined (Fig.
6). Changes in basal luciferase activity
as well as the effects of Sp1 over expression were determined by
cotransfecting the Neuro2A cells with the wild-type (pGL3RP392) or
mutant PNMT promoter-luciferase reporter gene constructs
(pGL3RP392mut168, pGL3RP392mut48, and pGL3RP392mut168/48,
Fig. 6A) and the vector pCGN or the Sp1 expression construct
pCGNSp1. In the case of basal luciferase activity, mutation of the
-168 bp Sp1 binding element reduced luciferase activity to 75% of
wild-type levels, whereas mutation of the -48 bp Sp1 binding element
reduced luciferase to 50% of wild-type levels (Fig. 6B). If
both Sp1 sites were altered, luciferase expression decreased to very
low levels (28% of control). When intracellular levels of Sp1 were
increased by cotransfection of the Sp1 expression plasmid pCGNSp1 along
with the PNMT promoter-reporter gene constructs, luciferase expression
could be further elevated, even in the case of the mutant constructs.
Luciferase rose 2.5-fold above basal levels for the wild-type construct
pGL3RP392 and each single mutant construct. However, each single mutant
construct showed reduced luciferase activity by comparison to the
wild-type construct (2.0- and 1.4-fold basal wild-type control for the
-168 and -48 Sp1 mutant constructs, respectively), although
luciferase activity expressed by the wild-type and the -168 bp mutant
construct was not significantly different. In contrast, the PNMT
promoter-luciferase reporter gene construct with the double Sp1
mutation showed no significant changes in luciferase in response to Sp1
over expression.

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Fig. 6.
Basal and exogenous
Sp1-stimulated luciferase activity from wild-type and mutant Sp1 PNMT
promoter-luciferase reporter gene constructs. The
wild-type plasmid construct pGL3RP392 or mutant plasmid constructs
pGL3RP392mut168, pGL3RP392mut48, or pGL3RP392mut 168/48 were
cotransfected with the control plasmid pCGN and/or the Sp1 expression
plasmid pCGNSp1 into Neuro2A cells, and luciferase activity was
determined as described under "Experimental Procedures."
A, schematic of pGL3RP392 (wild-type),
pGL3RP392mut168, pGL3RP392mut48, and pGL3Rpmut168/48
constructs. Sp1 and Egr-1 binding elements are identified. X
designates site directed mutation of binding element as described
under "Experimental Procedures." B, relative luciferase
activity expressed from wild-type and mutant PNMT promoter-luciferase
reporter gene constructs in the absence (white bars) and
presence (shaded bars) of exogenous Sp1. **, significantly
different from the respective controls, p 0.01; ***,
significantly different from control, p 0.001.
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The effect of Sp1 over expression was also compared in the Neuro2A and
COS1 cells to examine whether increasing Sp1 alone in the COS1 cells
would increase PNMT promoter activity. Luciferase activity
progressively rose as increasing amounts of the pCGNSp1 expression
plasmid were cotransfected into the Neuro2A cells, whereas no
significant induction of the wild-type expression construct pGL3RP392
was observed at all concentrations of Sp1 in the COS1 cells (Fig.
7).

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Fig. 7.
Sp1 responsiveness of PNMT
promoter-luciferase reporter gene construct. The wild-type
(pGL3RP392) construct was cotransfected with increasing amounts of the
Sp1 expression plasmid pCGNSp1 into Neuro2A and COS1 cells. The control
plasmid pCGN was included to ensure that the total plasmid DNA
transfected into the cells was equivalent. Luciferase activity was
determined as described under "Experimental Procedures." ***,
significantly different from control, p 0.001.
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Thus, the functionality of the Sp1 sites seems to depend on both the
relative affinity of Sp1 for each of the Sp1 target sequences and the
concentration of Sp1 protein available for occupancy of the sites. In
addition, another transcription factor must be expressed by the Neuro2A
cells that interacts with Sp1 to stimulate the PNMT promoter or a
factor may preclude Sp1 activation of the PNMT promoter in the COS1 cells.
Sp1 Competition at the Proximal Sp1 Binding Element--
As
described above, another transcriptional protein present in the Neuro2A
cell nuclear extract has a recognition site 3' to the proximal -48 bp
Sp1 site. It appears to form less protein-DNA complex with the WTB
oligonucleotide (proximal Sp1/Egr-1 site) by comparison to the amount
of Sp1-DNA complex formed with the WTA oligonucleotide (distal
Sp1/Egr-1 site) when equivalent amounts of Neuro2A cell nuclear extract
and probes (approximately equivalent specific activity) are used. The
latter would suggest that the factor is less abundant, with apparent
higher affinity for its recognition site than Sp1 for the proximal Sp1
site. In addition, when complexed to the WTB oligonucleotide probe, the
factor has a faster electrophoretic mobility than Sp1 bound to the same
probe, indicating that its relative molecular weight is less than that of Sp1. Examination of the DNA sequences 3' to the -48 bp Sp1 site
suggests that the factor may be MAZ (13) given the 11-bp match to the
MAZ binding element (Fig. 8A).
It also appears that the MAZ and Sp1 binding sites overlap by 1 bp.
Southwestern analysis was performed to see if the binding protein was
of the appropriate molecular mass for MAZ. As shown in Fig.
8B, two proteins form complexes with 32P-labeled
WTB. The larger, predominant protein has an apparent molecular mass of
105 kDa (lane 1), identical to the relative molecular mass
of authentic human Sp1 protein (lane 2). The smaller, less
abundant protein has a molecular mass of 60 kDa, which is the
anticipated molecular mass for MAZ. Gel mobility shift assays with
antibodies were used to confirm that MAZ was the protein in the Neuro2A
cell nuclear extracts binding to the proximal Sp1/Egr-1 site (Fig.
8C). The 32P-labeled WTB oligonucleotide probe
was combined with Neuro2A cell nuclear extract in the presence of
bovine serum albumin, IgG, anti-Egr-1, anti-Sp1, or anti-MAZ antibody.
No supershifts were apparent with any of the antibodies, but the
anti-MAZ antibody either disrupted or prevented DNA-complex formation,
as evidenced by the marked reduction in the major radiolabeled
protein-DNA complex. A low molecular mass complex was also apparent,
and it was reduced in the presence of anti-MAZ antibody as well. Gel mobility shift assays with Sp1 protein are consistent, demonstrating that anti-Sp1 antibody supershifts the Sp1-WTB DNA complex, whereas anti-MAZ antibody has no effect whatsoever.

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Fig. 8.
MAZ binding proximal to the Sp1 binding
site. Consensus sequence match using the Transfac version 3.2 data
base indicated that the transcription factor MAZ might be the protein
binding to the DNA sequences proximal to the Sp1 site. Southwestern
analysis was performed to determine whether the molecular mass of the
protein was that expected for MAZ, and gel mobility shift assays with
antibodies were used to confirm the identity of MAZ protein.
A, schematic of the Sp1 and MAZ sequences in the proximal
PNMT promoter. The nucleotide sequence from -61 to -37 in the PNMT
promoter is shown. The Sp1 binding element spans from -57 to -48,
whereas the MAZ binding element spans sequences from -48 to -38.
B, Southwestern analysis of Neuro2A cell nuclear extracts.
The nuclear proteins contained in a 20-µg sample of Neuro2A cell
nuclear extract were separated on 10% SDS-polyacrylamide gels as
described under "Experimental Procedures." Following transfer to
nitrocellulose, the proteins were visualized by forming protein-DNA
complexes with 32P-labeled WTB probe. Prestained molecular
mass markers were included in order to determine the molecular mass of
the proteins forming complexes with the WTB probe. Human Sp1 protein
was run as a control. Lane 1, Neuro 2A cell nuclear extract;
lane 2, purified human Sp1 (Santa Cruz Biotechnology).
C, gel mobility shift assays with antibodies. Gel mobility
shift assays were performed as described under "Experimental
Procedures" using the 21-bp WTB probe end-labeled with
32P and nuclear extract from Neuro2A cells or purified
human Sp1 protein (Santa Cruz Biotechnology). Antibodies were used to
identify the proteins constituting the protein-DNA complexes. Neuro 2A
cell nuclear extract (lanes 1-5), bovine serum albumin
(BSA), IgG, anti-Egr-1 antibody, anti-Sp1 antibody, and
anti-MAZ antibody, respectively. Sp1 protein (lanes 1-3),
Sp1 alone, anti-Sp1antibody, and anti-MAZ antibody, respectively.
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Thus, the binding of the transcription factor MAZ, a nuclear protein
present in the Neuro2A cells, to its consensus element (3' to the -48
bp Sp1 site with a 1-bp overlap) in the rat PNMT promoter, prevents Sp1
binding and thereby inhibits Sp1 induction of the PNMT promoter through
its proximal binding site. Although Sp1 seems to be the transcription
factor responsible for the higher PNMT promoter activity observed in
the Neuro2A cells, blockade of Sp1 activation at the -48 bp Sp1 site
may limit Sp1 stimulation of the PNMT promoter and, hence, PNMT gene expression.
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DISCUSSION |
Although several transcriptional activators, including the
glucocorticoid receptor, Egr-1, Sp1, and AP-2 (7, 8, 18, 19), and
negative regulatory factors (20) have been shown to control the rat
PNMT gene promoter and the endogenous PNMT gene, transcriptional
regulatory proteins participating in the developmental-specific,
tissue-specific, or stimulus-specific expression of PNMT remain to be
determined. The present studies suggest that Sp1 and MAZ may be two
transcriptional regulators participating in tissue-specific expression.
Luciferase activity produced from PNMT promoter-luciferase reporter
gene constructs containing 863, 444, and 392 bp of 5' upstream PNMT
promoter/regulatory sequence was high in cell lines derived from
tissues likely to express PNMT, i.e. Neuro2A, PC12 and RS1
cells. In contrast, it was barely detectable in cell lines derived from
tissues unlikely to express PNMT, COS1 and NIH/3T3 cells. DNase I
footprinting further showed that the high basal luciferase levels in
the Neuro2A cells might be due to nuclear proteins interacting with the
Sp1/Egr-1 binding elements at -168/-165 and -48/-45 bp in the PNMT
promoter. However, gel mobility shift assays demonstrated that Sp1, but not Egr-1, was present within the nuclei of Neuro2A cells. Consistent with these findings, mutation of the Sp1 sites in the PNMT promoter reduced basal luciferase activity expressed from PNMT promoter-reporter gene constructs. In addition, another Neuro2A cell nuclear protein appeared to bind to a consensus element 3' to the proximal Sp1 site
(one bp overlap), preventing Sp1 binding at this site and activation of
the PNMT promoter by Sp1. DNA consensus sequence identity, Southwestern
analysis, and gel mobility shift assays with antibodies indicate that
this factor is the 60-kDa protein MAZ, an activator of the c-myc (13,
21, 22), insulin (23), and 5HT1A receptor (24) genes. Thus,
the exclusion of Sp1 binding by MAZ may provide another mechanism
contributing to tissue-specific PNMT gene regulation.
Previous studies showed that both Sp1 binding elements within the
proximal 863 bp of 5' PNMT promoter sequences likely participated in
Sp1 activation of the PNMT gene. However, the -48 bp Sp1 site had
higher affinity for Sp1 (8). We now demonstrate that Sp1 activation of
the PNMT promoter depends on intracellular Sp1 concentrations as well.
When intracellular Sp1 concentrations were increased by cotransfection
of an Sp1 expression plasmid (pCGNSp1) along with the wild-type PNMT
promoter-luciferase reporter gene construct into Neuro2A cells,
luciferase activity rose 2.5-fold above basal values. Similar increases
in luciferase expression were observed in PNMT promoter-luciferase
reporter gene constructs with mutations in either the -168 or -48 bp
Sp1 site. Only if both Sp1 binding elements were mutated was Sp1 unable
to stimulate PNMT promoter induction. These results confirm that intact
Sp1 binding elements are required for Sp1-mediated PNMT promoter
activity and underscore the importance of considering both the relative
abundance of transcriptional regulators and their affinities for their
respective binding elements when investigating the actions of
transcriptional regulatory proteins in vitro.
Gel mobility shift assays identified two Sp1 protein-DNA binding
complexes arising from either the Neuro2A cell nuclear extract or Sp1
protein. Whereas the complex with the faster electrophoretic mobility
predominated, both complexes were supershifted by anti-Sp1 antibody.
Two forms of Sp1 protein have been identified with relative molecular
masses of 95 and 105 kDa (25, 26). Both molecular mass forms of the
protein are equivalently glycosylated, but the 105-kDa form is
phosphorylated as well, and the latter accounts for the molecular mass
difference (27). In Fig. 5, however, the slower migrating Sp1-DNA
complex observed probably represents dimeric Sp1 bound to the -48 bp
binding element, because the 5% polyacrylamide gels used for the gel
mobility shift assays would not have resolved two Sp1-DNA complexes so
similar in molecular mass. Consistent with this interpretation, Sp1, as
other zinc finger proteins, reportedly forms functionally active dimers
(28).
Finally, Sp1 binding at the proximal -48 bp Sp1 consensus element
seems to be prevented by MAZ, another nuclear protein expressed in the
Neuro2A cells, at an adjacent 3' binding element. Both the Sp1 and MAZ
binding elements consist predominantly of guanine and cytosine
nucleotides, and many transcription proteins bind to GC-rich regions of
DNA (29). In the case of the PNMT gene, we have previously shown that
the immediate early gene transcription factor Egr-1 binds to sequences
3' to the -48 bp Sp1 binding element; these sequences overlap the MAZ
recognition site. However, we had further suggested that this proximal
-48/-45 bp Sp1/Egr1 site likely functioned as an Sp1 activation
element (7, 8). The DNA consensus element for MAZ has been defined as
5'-G(G/C)GG(C/A)GGGG(C/A)(G/T)-3', whereas that for Sp1 is represented
by 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3'. In the rat PNMT gene, the promoter
sequences spanning the nucleotides to which Sp1 and MAZ appear to bind
include 5'-CTGGCGGGGGGGAGGGGACC-3' (24). Based on the Sp1 and MAZ
consensus sequences defined above, a core motif, 5'-GGGG(C/A)GGGG-3',
is common to both binding elements, indicating that either site may
interact with Sp1 or MAZ. The 5' flanking region of the
5HT1A receptor gene has been shown to possess four MAZ
binding sites that share this motif as well, and in the case of that
gene, Sp1 binds to three of the four MAZ sites (24). However, Sp1 does
not appear to bind to the nucleotides designated as constituting the
MAZ site (-38 to -48 bp) and MAZ does not appear to bind to the
nucleotides designated as the Sp1 site (-548 to -56 bp) in the PNMT
gene. Evidence in support of distinct but overlapping binding elements
for MAZ and Sp1 is provided by the finding that a MAZ-DNA complex forms
in lieu of an Sp1-DNA complex if the MAZ binding sequences are intact,
but only an Sp1 complex forms when the 3' MAZ consensus sequence is mutated.
The exclusionary competition between MAZ and Sp1 once again underscores
the importance of considering both binding affinities and relative
abundance of transcription factors in gene activation. From
Southwestern analysis, we see that in the Neuro2A nuclear extracts, MAZ
is present at approximately 4-fold lower concentrations than Sp1. MAZ
must therefore have a higher affinity for its binding site than Sp1
does for the -48 bp binding site. However, when Sp1 levels are raised,
it is possible to drive PNMT promoter activity through Sp1 induction.
Thus, increasing Sp1 concentrations disproportionately to MAZ seems to
offset the lower affinity of Sp1 for its recognition site.
Both Sp1 and MAZ have been shown to facilitate transcription in
TATA-less gene promoters by interacting with TATA-binding protein
associated factors to facilitate TFIID binding to the promoter (24). We
know, however, that the rat PNMT gene possesses an ATAAA box from which
transcription initiation can occur (6). Recently, it has been
demonstrated that Sp1 and MAZ can facilitate transcription of TATA box
containing genes as well (30). Although less is known about the
transcriptional regulatory properties of MAZ, Sp1 is known to be
regulated developmentally and in response to specific stimuli (27, 31).
Taken together, these various findings suggest a potential mechanism
whereby MAZ and Sp1 may participate in the orchestration of
tissue-specific expression of the PNMT gene. Present studies are
further defining the independent and exclusionary control of the PNMT
gene by MAZ and Sp1.