(Received for publication, March 20, 1995; and in revised form, May 12, 1995)
From the
The rat phenylethanolamine N-methyltransferase (PNMT)
gene contains overlapping consensus elements for the Sp1 and Egr-1
transcription factors located at -45 bp and -165 bp in the
PNMT promoter. In the present study, we show that Sp1 and Egr-1 can
specifically bind to these overlapping elements, that this binding
appears to be mutually exclusive, and that binding site occupancy is
dependent upon the concentration of each factor and its binding
affinity for each site. Egr-1 binds to the -165 bp site with
relatively high affinity (IC
Phenylethanolamine N-methyltransferase (PNMT)
In an effort to identify
the factors that regulate PNMT gene expression, we have recently shown
that the immediate early gene transcription factor Egr-1 (Ebert et
al., 1994), also known as NGFI-A (Milbrandt, 1987), Krox 24
(Lemaire et al., 1988), zif 268 (Christy et al.,
1988) and TIS8 (Lim et al., 1987), can stimulate transcription
of the rat PNMT gene. Stimulation appears to occur through direct
binding of Egr-1 protein to PNMT promoter elements located at
-165 bp and to a lesser extent, at -45 bp, relative to the
transcriptional initiation site at +1 bp (Ebert et al.,
1994). A perfect (9/9) match to the Egr-1 consensus DNA binding
sequence (CGCCCCCGC) (Cao et al., 1990) is located at the
-165 bp site, while an 8/9 consensus match (GCGGGGGGG) is located
at the -45 bp site. Overlapping the 5` end of these Egr-1
consensus elements are consensus binding sites for another
transcription factor, Sp1 (consensus =
(G/T)(G/A)GGC(G/T)(G/A)(G/A)(G/T) (Faisst and Meyer, 1992), indicating
that Sp1 may also influence PNMT gene expression.
Sp1 is a
ubiquitous mammalian transcription factor thought to be involved in the
regulation of a large number of genes (Kadonaga et al., 1986;
Kriwacki et al., 1992). For housekeeping genes that lack TATA
and CAAT boxes, it has been suggested as being the signal initiating
transcriptional activation (Blake et al., 1990; Joliff et
al., 1991; Bosclair et al., 1993). Sp1 binding sites have
also been shown to overlap the binding sites for other transcription
factors, and it is thought that competition between Sp1 and these
factors for binding site occupancy may be an important mechanism for
controlling the expression of specific genes (Lemaigre et al.,
1990; Ackerman et al., 1991; Cao et al., 1993).
Using gel mobility shift assays, we have previously shown that Sp1,
present in nuclear extracts from a PC12-derived cell line (RS1 cells),
can bind to DNA sequences representing the overlapping Sp1/Egr-1
consensus elements from the rat PNMT promoter (Ebert et al.,
1994). Furthermore, mutations designed to disrupt Egr-1 binding to its
cognate consensus elements in the PNMT promoter also decreased
base-line (uninduced) PNMT promoter activity. We speculated that this
decline might be due to a reduction in Sp1 binding.
The present
study was undertaken to determine the role Sp1 may play in the
regulation of PNMT gene expression and to distinguish its action from
that of Egr-1. We demonstrate that Sp1 can bind to both overlapping
consensus elements (-45 bp and -165 bp) with similar
affinities and that this binding appears to stimulate transcriptional
activity from the PNMT promoter. Egr-1, which also binds to both
consensus elements, has
In general, wild-type DNA sequences are
designated with the prefix ``wt'' while mutants have the
prefix ``mut'' followed by an ``A'' or
``B'' to indicate which of the overlapping elements from the
rat PNMT promoter are represented by each duplex oligomer (A,
-159 to -179 bp; B, -39 to -59 bp) (see Fig. 2). Each mutation carries an additional annotation
indicating whether the mutation is specific for the Sp1 consensus
sequence (designated by the number 1) or the Egr-1 consensus sequence
(designated by the number 2). Thus, ``wtA'' refers to the
wild-type overlapping consensus elements for Sp1 and Egr-1 located
between -159 and -179 bp in the rat PNMT promoter, while
``mutA1'' and ``mutA2'' indicate that this same DNA
fragment harbors a mutation that specifically targets the Sp1 and the
Egr-1 consensus elements, respectively. The DNA sequences for the
various oligonucleotides are as follows (mutant nucleotides are
distinguished by lowercase letters): wtA, 5`-CCTCCCCGCCCCCGCGCGTCC-3`;
mutA1, 5`-CCTCaaCGCCCCCGCGCGTCC-3`; mutA2, 5`-CCTCCCCGCCCCataGCGTCC-3`;
wtB, 5`-GTCTGGGCGGGGGGGAGGGGA-3`; mutB1, 5`-GTCTaaGCGGGGGGGAGGGGA-3`;
and mutB2, 5`-GTCTGGGCGGGGaaaAGGGGA-3`.
Figure 2:
Footprinting analysis of Sp1 binding to
the rat PNMT promoter sequences. A, purified Sp1 protein was
incubated with a DNA probe (+20 to +391 bp) generated as a HindIII-NheI restriction fragment from the PNMT
promoter. For footprinting, two gel loadings were used. For the 1stload samples, electrophoresis was executed for 6.5 h.
After 4 h, an identical set of samples was loaded (2ndload), so that the latter were subjected to
electrophoresis for 2.5 h. Lanes1-6, probe
labeled at the NheI site plus 0, 0.1, 0.5, 1.0, 2.0, and 5.0
fpu of Sp1, respectively. Lanes7-12, probe
labeled at the HindIII site plus 0, 0.1, 0.5, 1.0, 2.0, and
5.0 fpu of Sp1, respectively. Protected regions after DNase I digestion
are indicated by solidverticallines. B, diagrammatic depiction of Sp1 binding sites (shadedovals) designated A (-165 bp) and B (-45 bp). C, protected nucleotide sequences. Numbers in parentheses designate the 5` and 3` ends of the protected
sequences.
Finally, for site-directed mutagenesis, the following mutagenic
33-mer oligonucleotide primers were synthesized: mutA1P,
5`-CCACATCTCCCCCCTCaaCGCCCCCGCGCGTCC-3`; mutA2P,
5`-CCCCCTCCCCGCCCCataGCGTCCGTCCGGCGC-3`; mutB1P,
5`-GTGATGGAGGGGTCTaaGCGGGGGGGGAGGGG-3`; and mutB2P,
5`-GGGGTCTGGGCGGGGaaaAGGGGACCCAGTAG-3`.
All oligonucleotides were obtained from DNA International, Inc. (Lake
Oswego, OR).
The Egr-1
expression vector, pCMVEgr-1, and the corresponding nonfunctional
control plasmid, pCMVETTL, have been described elsewhere (Gupta et
al., 1991). All plasmids were amplified in the XL1-Blue strain of E. coli (Stratagene, Inc., La Jolla, CA) and purified through
two successive CsCl density gradients. Following purification, plasmid
integrity and identity was again verified by restriction digestion,
agarose gel electrophoresis, and DNA sequence analysis.
Approximately 7.5
For
DNA competition experiments, excess concentrations of specific
unlabeled duplex competitor 21-mers were included in the binding
reactions. Relative binding was quantitated from the intensity of the
protein-DNA complexes as determined densitometrically from the
autoradiographic images using Image 1.52 software (National Institutes
of Health) on a MacIntosh FX computer. The concentration of competitor
oligomer that effectively inhibited maximum binding by 50%
(IC
Transient transfection assays were performed
by calcium-phosphate co-precipitation as described previously (Ebert et al., 1994). Total protein and luciferase and
Figure 1:
Overlapping Sp1 and Egr-1
consensus elements in the PNMT promoter. The rat PNMT promoter contains
two overlapping Sp1 and Egr-1 consensus elements at -165 and
-45 bp. Six core nucleotides are shared at each overlapping site. Lowercaseletters designate mismatches between the
PNMT DNA sequences and the published consensus DNA-binding sequences
for Sp1 and Egr-1. Also shown are the ATA box (-30 bp) and
glucocorticoid response element (GRE, -513
bp).
To determine the relative binding
affinity of Sp1 for these sites, competitive gel mobility shift assays
were performed. A representative experiment, utilizing a probe
corresponding to the wild-type A site (wtA), is shown in Fig. 3.
The results demonstrated that Sp1 binding to the wtA probe was
effectively competed by either wtA itself or the wild-type B oligomer
sequence (wtB) with similar relative affinities (IC
Figure 3:
Relative Sp1 binding affinity for PNMT
promoter elements. Gel mobility shift assays were performed using a
21-bp oligomeric probe (wtA) containing the overlapping Sp1/Egr-1
consensus elements at the A site in the absence and presence of
increasing concentrations of competitor duplex oligonucleotides. A, constant amounts of Sp1 protein (0.5 fpu) and probe DNA (1
ng) with varying amounts of unlabeled DNA. Lane1, no
competitor; lanes2-5, 1, 10, 100, and 1000 ng
of wtA competitor, respectively; lanes6-9, 1,
10, 100, and 1000 ng of wtB competitor, respectively; lanes10-13, 1, 10, 100, and 1000 ng of Sp1 (a high
affinity Sp1 binding site) competitor, respectively; lanes14-17, 1, 10, 100, and 1000 ng of GRE competitor,
respectively. B, competition curves for the competitors shown
in A.
Figure 4:
Sp1 displacement of Egr-1 binding at
overlapping consensus elements in the rat PNMT promoter. Gel mobility
shift assays were performed with either the wtA (lanes1-8) or the wtB (lanes9-16) probes in the presence of increasing Sp1
protein concentrations and the presence or absence of purified Egr-1
DNA-binding domain (V5 protein, 40 ng/reaction) (Gashler et
al., 1993). Sp1 concentrations were titrated as follows: lanes1-4, wtA probe plus 0, 0.5, 2.0, and 5.0 fpu of
Sp1, respectively; lanes5-8, wtA probe and V5
protein plus 0, 0.5, 2.0, and 5.0 fpu of Sp1, respectively; lanes9-12, wtB probe plus 0, 0.5, 2.0, and 5.0 fpu of
Sp1 respectively; lanes13-16, wtB probe and V5
protein plus 0, 0.5, 2.0, and 5.0 fpu of Sp1,
respectively.
Figure 5:
V5 displacement of Sp1 binding at the A
site. Gel mobility shift assays were performed using the 21 bp wtA
probe in the presence of a constant amount of Sp1 protein (0.5
fpu/reaction) and increasing quantities of V5 protein. Lane1, probe alone; lanes2-6, probe
and Sp1 protein plus 100, 200, 400, and 1000 ng of V5, respectively.
The specific complexes are identified to the left of the
autoradiogram.
Figure 6:
Effects of selective mutagenesis of Sp1
and Egr-1 consensus sequences on respective binding affinities. The
effects of selective mutations to the Sp1 and Egr-1 binding elements
were analyzed by gel mobility shift competition assays. Only that
portion of the autoradiogram containing the protein-DNA binding
complexes is shown. A, Sp1 plus the wtA probe in the presence
of increasing concentrations of unlabeled competitor DNA representing
wtA (self-competition), mutA1 (Sp1 consensus mutant), or mutA2 (Egr-1
consensus mutant). The lowercaseletters denote the
base substitutions for each of the mutant binding sites. B, V5
plus the wtA probe in the presence of increasing concentrations of
unlabeled wtA, mutA1, or mutA2. C, Sp1 plus the wtB probe in
the presence of increasing concentrations of unlabeled wtB, mutB1, or
mutB2. D, V5 was incubated with the wtB probe in the presence
of increasing concentrations of unlabeled competitor DNA representing
wtB, mutB1, or mutB2. IC
Similarly, the mutB1 mutation effectively reduced Sp1
binding, but its ability to compete for Egr-1 binding was relatively
unchanged (Fig. 6, C and D, Table 1). As
expected, the mutB2 mutation, designed to reduce Egr-1 binding
affinity, substantially reduced Egr-1 binding compared with its
wild-type counterpart (Fig. 6D). Surprisingly, however,
the mutB2 sequence enhanced Sp1 binding 3.5-fold compared with the
wild-type B sequence. Thus, the mutations appeared to behave
predictably with respect to their ability to interfere with either Sp1
or Egr-1 binding, with the exception of mutB2, which displayed
increased affinity for Sp1.
To evaluate the effects of these same
mutations on PNMT promoter activity, the mutA1, mutA2, mutB1, and mutB2
mutations were incorporated into PNMT-luciferase reporter plasmid
constructs, and reporter gene expression was evaluated by transient
transfection assays using RS1 cells. Reporter gene constructs with
mutations reducing relative Sp1 binding affinity (mutA1, mutA2, and
mutB1) produced 30-50% less luciferase activity in comparison
with the wild-type control (Fig. 7). In contrast, the reporter
gene construct containing the mutation that increased Sp1 DNA-binding
affinity, mutB2, also increased luciferase activity approximately
4-fold. Thus, the changes in luciferase expression appeared to reflect
the changes in the relative binding affinities for Sp1.
Figure 7:
Basal luciferase activity of wild-type and
mutant rat PNMT promoter-reporter gene constructs. Wild-type (WT) and mutant PNMT-luciferase plasmid constructs were
transfected into RS1 cells, and basal PNMT promoter activity was
evaluated by measuring luciferase production as described under
``Experimental Procedures.''
Figure 8:
Egr-1-stimulated luciferase activity of
wild-type and mutant rat PNMT-luciferase reporter constructs. Wild-type (WT) and mutant PNMT-luciferase plasmid constructs were
co-transfected with either pCMVEgr-1 (hatchedbars)
or the control plasmid, pCMVETTL (solidbars), into
RS1 cells, and luciferase activity was quantitated as described under
``Experimental Procedures.''***, significantly different from
control, p < 0.001.
The present study extends our previous findings demonstrating
that Egr-1 activates the expression of the rat PNMT gene and that Sp1
is a potential transcriptional activator as well. We show that Sp1
binds to both overlapping Sp1/Egr-1 consensus elements (-45 and
-165 bp) while Egr-1 binds preferentially to the A site
(-165 bp). The integrity of the Egr-1 consensus element at the A
site is essential for Egr-1-stimulated PNMT promoter activity, while
the B site (-45 bp) does not appear to influence Egr-1-mediated
PNMT promoter activation. Mutations that decrease Sp1 binding to its
consensus elements in the PNMT promoter reduce basal promoter activity
in the RS1 cells, while a mutation that increases Sp1 binding
stimulates PNMT promoter activity. Finally, Sp1 displaces Egr-1 binding
at the overlapping consensus elements in a concentration-dependent
manner.
While the evidence suggests that both Egr-1 and Sp1 function
as transcriptional activators of the PNMT gene, their specific
transcriptional roles remain to be elucidated. Egr-1, a member of the
immediate early gene family, is usually present at low levels
endogenously but can be rapidly and transiently induced in response to
a variety of external stimuli (Milbrandt, 1987; Cao et al.,
1990; Iwaki et al., 1990; Bhat et al., 1992; Mailleux et al., 1992; Katayama et al., 1993). Egr-1 is
therefore considered to be a transcriptional factor that acts as a
modulator of basal gene expression. Consistent with this role, we have
recently shown that Egr-1 expression in the rat adrenal medulla is
rapidly activated neurally by reserpine treatment, which also causes
significant increases in PNMT mRNA (Wong et al., 1993; Ebert et al., 1994). We have further shown that the phorbol ester,
phorbol 12-myristate 13-acetate, can induce PNMT promoter activity via
an Egr-1-dependent mechanism in RS1 cells (Morita et al.,
1995). In contrast, Sp1 is a ubiquitous protein, constitutively
expressed at very high levels, and it appears to act as a transcription
initiator for many housekeeping genes that lack TATA and CAAT boxes
(Blake et al., 1990; Joliff et al., 1991; Boisclair et al., 1993). Recently, however, Sp1 protein has been
demonstrated to be regulated developmentally and in response to
specific stimuli (Jackson et al., 1990; Saffer et
al., 1991), thereby suggesting that this factor may be critical
during differentiation and has the potential for regulated expression
as well. If the latter is indeed the case, then under appropriate
circumstances, Sp1 might also function as a modulator of basal gene
transcription.
Based on our studies to date, the PNMT promoter
functions as a weak promoter in RS1 cells in comparison with other
promoters, particularly the viral promoters (Ebert et al.,
1994). It does possess sequences consistent with an ATA (-30 bp)
box. Hence, if Sp1 participates in constitutive PNMT expression, we
would predict that the observed low levels of basal PNMT promoter
activity may be due to the fact that the Sp1 consensus binding sites in
the PNMT promoter are not high affinity sites. However, when we
compared the Sp1 sites within the PNMT promoter with the
nine-nucleotide Sp1 sequence identified by Faisst and Meyer(1992),
their perfect match suggested that these sites should be very effective
Sp1 sites (Table 2). Yet, gel mobility shift competition assays
showed that the Sp1 sites were only of moderate binding affinity. A
10-nucleotide Sp1 consensus sequence has been identified as an
alternative to the nine-bp Sp1 consensus element and has been confirmed
as a high affinity site (Kadonaga et al., 1986; Kriwacki et al., 1992). If we consider the PNMT Sp1 sequences, the 10th
nucleotide, located at the 3` end, is mismatched and reduces Sp1
binding 10-fold in comparison with the perfect match 10-bp sequence,
suggesting that this nucleotide is critical in specifying the binding
affinity for Sp1. In fact, the 10-bp Sp1 element at the B site in the
PNMT promoter is identical to an Sp1 element in the herpes simplex
virus type 1 IE3 gene promoter (Kadonaga et al., 1986;
Kriwacki et al., 1992), which also has intermediate affinity
for Sp1 protein. Thus, the wobble in the distal 3` nucleotide of the
PNMT Sp1 consensus elements is consistent with the observed low basal
PNMT promoter activity and moderate binding affinity as predicted from
the 10-bp Sp1 high affinity consensus sequence.
Several other genes have also been identified with
overlapping or closely adjacent Sp1/Egr-1 binding motifs (Ackerman et al., 1991; Li et al., 1993; Molnar et
al., 1994). In the case of the murine adenosine deaminase gene,
Sp1 stimulates, while Egr-1 represses, adenosine deaminase promoter
activity (Ackerman et al., 1991). The inhibitory action of
Egr-1 is opposite to its effects on the PNMT promoter reported here.
However, both the stimulatory and inhibitory actions of Egr-1 on
transcriptional activity are consistent with the functional domains of
Egr-1 protein (Gashler et al., 1993). Egr-1 has been shown to
have three activating and one inhibitory domain, although the specific
biological roles and activities of these domains remain to be
identified.
In summary, we have shown that Sp1 and Egr-1 can
differentially stimulate PNMT gene expression through interactions with
overlapping consensus elements in the PNMT promoter. Sp1 seems to
interact with both the A and B sites with moderate binding affinities
to induce PNMT promoter activity. In contrast, Egr-1 acts predominantly
through the A site, where it appears to effectively exclude the binding
of Sp1, thereby increasing PNMT promoter activity. The differences in
binding affinities of Sp1 and Egr-1 for their overlapping consensus
sequences in the PNMT promoter, their apparent exclusionary binding,
and differences in their abilities to activate transcriptional activity
from the PNMT promoter suggest that these two factors may play unique
roles in the control of PNMT gene expression in response to specific
biological stimuli. Further definition of their biological roles will
therefore be an important focus for future studies.
This paper is dedicated to the memory of Dr. Roland D. Ciaranello,
the Nancy Pritzker Professor, Laboratory of Developmental and Molecular
Neurobiology, whose guiding spirit, encouragement, and scientific
counsel through the years were unwavering.
= 14 nM) and
to the -45 bp site with relatively low affinity (IC
= 1360 nM), whereas Sp1 binds to both sites with
intermediate affinities (IC
= 210 and 140
nM, respectively). Consistent with the DNA-binding data, Egr-1
stimulates PNMT promoter activity primarily through interaction with
the -165 bp site, while Sp1 stimulates PNMT promoter activity by
interacting with both the -45 bp and the -165 bp sites.
These results show that Sp1 and Egr-1 are capable of differentially
activating PNMT gene expression, thereby suggesting that different
stimuli may control the activity of the PNMT gene by selectively
regulating Sp1 and/or Egr-1.
(
)catalyzes the final step in the catecholamine
biosynthetic pathway, converting norepinephrine to epinephrine. During
periods of stress, epinephrine is secreted into the bloodstream from
the adrenal medulla, where it is produced. Following epinephrine
release, PNMT levels increase (Ciaranello et al., 1971;
Kvetnansky et al., 1971; Ciaranello, 1980; Weisberg et
al., 1989; Wong et al., 1992; Viskupic et al.,
1994), suggesting that control of PNMT expression may be an important
component of the mammalian stress response.
100-fold higher affinity for the distal
site (-165 bp), and the integrity of this site is critical for
Egr-1 activation of PNMT gene expression. These findings suggest that
both Sp1 and Egr-1 can activate PNMT gene expression at these
overlapping consensus elements, raising the possibility that selective
activation of Sp1 or Egr-1 in response to specific stimuli may be
biologically important for the activation and responsiveness of the
PNMT gene.
Oligonucleotides
Eight duplex 21-mer
oligonucleotides were prepared by annealing individually synthesized
complimentary DNA fragments as described previously (Ebert et
al., 1994). These duplex oligonucleotides represent wild-type and
mutant forms of the overlapping Sp1/Egr-1 DNA-binding elements from the
rat PNMT promoter.
As a positive control for
Sp1 binding, a sequence previously shown to be a high affinity Sp1
binding site (Christy and Nathans, 1989), ``Sp1''
(5`-TAGAGGGGCGGGGCTCTAGAC-3`) was generated. As a negative control, an
oligomer containing the PNMT glucocorticoid response element,
``GRE'' (5`-GCCAGAACAGAGTGTCCTTTC-3`), was also prepared.
Plasmids
The wild-type PNMT-luciferase plasmid
construct, pRP863LUC, contains the region of the rat PNMT promoter
extending from -863 bp to +20 bp fused to the firefly
luciferase reporter gene (Ebert et al., 1994). Mutant
PNMT-luciferase constructs were created by incorporating each of the
mutagenic primers independently into pRP863LUC using the
Transformer site-directed mutagenesis kit from Clontech
(Palo Alto, CA) as described previously (Ebert et al., 1994).
The identity and integrity of the resulting plasmids, pRPmutA1LUC,
pRPmutA2LUC, pRPmutB1LUC, and pRPmutB2LUC, were verified by DNA
sequence analysis (Sequenase 2.0, U.S. Biochemical Corp.).
DNase I Footprinting
Using
[-
P]ATP (DuPont NEN, Wilmington, DE) and T4
polynucleotide kinase (Life Technologies, Inc.), a DNA fragment from
the rat PNMT promoter was labeled at either the HindIII site
(+20 bp) or the NheI site (-391 bp, Sambrook et
al., 1989). Fragments labeled at the HindIII site were
digested further with NheI, while those labeled at the NheI site were digested with HindIII, and
radiolabeled DNA fragments (+20 bp to -391 bp) isolated by
polyacrylamide gel electrophoresis as described previously (Muller,
1987).
10
dpm of each DNA
fragment was incubated with 0-5 footprinting units (fpu) of
purified Sp1 (1 fpu,
50 ng of Sp1 protein; Promega, Madison, WI)
in 50 µl on ice using the binding buffer supplied in the Core
Footprinting System
(Promega). After incubation for 1 h,
DNase I footprinting was performed according to the
manufacturer's recommendations. The optimal RQ1 DNase I (Promega)
concentration (0.1 unit/reaction) was determined empirically in
preliminary experiments. DNase I cleavage patterns were identified by
polyacrylamide gel electrophoresis (7% sequencing gel), and protected
sequences were mapped by comparison to Maxam and Gilbert G and G/A
chemical sequencing ladders generated from these same DNA probes and
run in adjacent lanes on the gels (Sambrook et al., 1989). The
banding patterns were visualized by autoradiography.
Gel Mobility Shift Assays
Gel mobility shift
assays were performed as described previously (Ebert et al.,
1994) using 1 ng of radiolabeled probe (3 nM, specific
activity = 2.5
10
dpm/µg) and purified
proteins in place of crude nuclear extracts. Typically, 0.5 fpu of
purified Sp1 protein (Promega) was used per binding reaction, while 100
ng of the Egr-1 DNA binding domain protein, V5 (Gashler et
al., 1993), kindly provided by Dr. Vikas P. Sukhatme (Department
of Medicine, Harvard Medical School), was utilized per reaction.
) was calculated by plotting the concentration of each
competitor versus the intensity of radiolabeled DNA-protein
complexes.
Cell Cultures and Transient Transfection
Assays
RS1 cells, a subclone of the PC12 cell line (Ebert et
al., 1994), were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 5% defined iron-supplemented
bovine calf serum (Hyclone, Inc., Logan, UT), 5% donor equine serum
(Hyclone, Inc.), gentamycin sulfate (50 µg/ml; U.S. Biochemical
Corp.), and hygromycin B (200 units/ml; Calbiochem) in a 37 °C
incubator (7% CO, 93% air) as described previously (Ebert et al., 1994).
-galactosidase activities were also determined as described
previously (Ebert et al., 1994). Experiments were repeated at
least twice with six replicates/sample for each experiment. Statistical
significance was determined by one way analysis of variance.
Sp1 Binding to PNMT Promoter DNA
The rat PNMT
promoter contains two overlapping Sp1/Egr-1 consensus binding elements (Fig. 1). Both consensus sequences share six base pairs, with
the Sp1 consensus sequences extending an additional three bp in the 5`
direction and the Egr-1 consensus sequences extending three bp in the
3` direction. The distal overlapping site, located at -165 bp,
contains a perfect match to both the Egr-1 (Cao et al., 1990)
and Sp1 consensus sequences (Faisst and Meyer, 1992). The proximal
site, located at -45 bp, contains an 8/9 match to the Egr-1
consensus DNA binding sequence and a perfect match to the Sp1 consensus
sequence. We have previously shown that Egr-1 can bind to these two
sites in the rat PNMT promoter, and Sp1 appeared to bind as well (Ebert et al., 1994).
To identify the DNA sequences involved in
Sp1 binding to the rat PNMT promoter, DNase I footprinting analysis was
performed. As shown in Fig. 2, Sp1 binds at two sites, both of
which were predicted from the overlapping Sp1/Egr-1 consensus elements.
The distal or ``A'' site spans the region between -176
and -162 bp, while the proximal or ``B'' site includes
the nucleotide sequences between -55 and -39 bp. No other
protected regions were observed.
for
wtA = 210 nM; IC
for wtB = 140
nM). However, the wtA and wtB sequences appeared to represent
only moderate affinity Sp1 binding sites, since the high affinity Sp1
competitor sequence (Christy and Nathans, 1989) more effectively
competed for Sp1 binding than either the wtA or wtB sequences
(IC
for Sp1 = 12 nM). In contrast, the GRE
oligonucleotide, representing the glucocorticoid response element, did
not compete for Sp1 binding (IC
for GRE > 2000
nM). Similar results were obtained when wtB was used as the
probe instead of wtA (data not shown). Thus, while Sp1 bound
specifically to the A and B sites in the PNMT promoter, it did so with
only moderate affinity.
Sp1 and Egr-1 Competition at Overlapping
Sites
Since Sp1 binding in the PNMT promoter overlaps those
regions bound by Egr-1, Sp1 and Egr-1 may potentially compete for
binding at a given site. To examine this possibility, the wtA and wtB
probes were incubated with increasing concentrations of Sp1 in the
presence of a constant amount of the purified Egr-1 DNA binding domain
protein (V5) (Fig. 4). V5 bound to the probe and formed a
complex that migrated between the unbound probe and the Sp1-DNA
complex. As the concentration of Sp1 was increased, Sp1-DNA complex
formation correspondingly increased while V5-DNA complex formation
diminished. Conversely, when the Sp1 concentration was kept constant
and the V5 concentration increased, Sp1-DNA complex formation appeared
to be displaced in favor of V5-DNA complex formation (Fig. 5).
Since no additional or intermediate protein-DNA complexes were
observed, Sp1 and V5 binding to either the A or the B site appeared to
be mutually exclusive, with occupancy dependent upon the relative
concentration of these two factors.
In addition, occupancy of these
promoter elements by Sp1 or Egr-1 appeared to be dependent upon the
relative binding affinities of these two factors for each site. For
example, V5 binding to the A site was much stronger than its binding to
the B site (compare V5-DNA complex intensities in Fig. 4, lanes5 and 13). Consistent with this
observation is the fact that equivalent concentrations of Sp1 could
displace V5 binding at the B site much more effectively than they could
at the A site (Fig. 4, compare lanes14-16 with lanes6-8). Similarly, Sp1 binding at
the A site was readily displaced by V5 (Fig. 5), whereas
equivalent concentrations of V5 only partially interfered with Sp1
binding at the B site (data not shown). Hence, binding site occupancy
was dependent upon both the relative concentration of Sp1 and Egr-1 and
the relative binding affinity of each factor for the two overlapping
sites.
Functional Dissection of Sp1 and Egr-1 Binding Sites in
the PNMT Promoter
To discriminate between the actions of Sp1 and
Egr-1 in the PNMT promoter, we attempted to selectively interfere with
the binding of either factor by incorporating specific mutations into
the nonoverlapping region of each binding site (Fig. 6). The
mutations were then evaluated for their ability to bind to Sp1 and
Egr-1 using competitive gel mobility shift assays (Fig. 6). The
mutA1 mutation targets the Sp1 consensus binding element at the A site
by changing the second and third cytosine bases to adenosine (Fig. 6, A and B), while the mutA2 mutation
converts the 3` CGC triplet of the Egr-1 binding site into ATA (Fig. 6, A and B). A similar set of mutations
was generated for the overlapping consensus elements at the B site. The
mutB1 mutation targets Sp1 binding by converting the second and third
guanosine residues to adenosine (Fig. 6, C and D), whereas the mutB2 mutation specifically alters the Egr-1
consensus element by replacing the 3` terminal GGG triplet with AAA (Fig. 6, C and D). IC values for
these competitors are shown in Table 1.
values were calculated for the
wild-type and mutant Sp1 and Egr-1 competitor DNA sequences and are
shown in Table 1.
As is apparent from Fig. 6A and Table 1, the mutA1 mutation reduced
the binding affinity for Sp1 approximately 5-fold compared with the wtA
sequence, yet this mutation had very little influence on Egr-1 binding (Fig. 6B). In contrast, the mutA2 mutation eliminated
Egr-1 binding (Fig. 6B). In addition, although its
ability to compete for Sp1 binding was also compromised, the reduction
in Sp1 binding affinity was relatively small compared with the decrease
in Egr-1 binding affinity (2.5-fold compared with 140-fold,
respectively; Table 1). Thus, mutA2 preferentially reduced Egr-1
DNA-binding affinity, while mutA1 selectively reduced Sp1 binding
affinity.
Finally, the
ability of Egr-1 to stimulate PNMT promoter activity was examined in
co-transfection assays using the same complement of PNMT-luciferase
plasmids and either a functional or a nonfunctional Egr-1 expression
construct. As shown in Fig. 8, Egr-1 significantly increased (p < 0.001) luciferase expression from the wild-type,
mutA1, mutB1, and mutB2 PNMT-luciferase plasmids but not from the mutA2
PNMT-luciferase construct. Moreover, the responsiveness of the mutA1
and mutB1 PNMT-luciferase plasmids to Egr-1 was not significantly
different from that of the wild-type construct (Fig. 8). As
indicated earlier, neither of these mutations substantially changed the
relative affinity for Egr-1 binding (Table 1), consistent with
their inability to influence Egr-1 activation of luciferase activity.
In contrast, the mutB2 mutation reduced Egr-1 binding to the B site (Fig. 6, Table 1), yet Egr-1 still significantly
stimulated luciferase activity from the mutB2 PNMT promoter, likely due
to the integrity of the A site. These results, therefore, suggest that
Egr-1 stimulation of PNMT promoter activity may primarily depend upon
Egr-1 binding to PNMT promoter DNA at the A site.
The relative
affinities of the overlapping Sp1/Egr-1 binding sites for their
respective proteins and the ability of Sp1 to compete with Egr-1 for
binding at the overlapping Sp1/Egr-1 sites in a mutually exclusive,
concentration-dependent fashion also suggest important biological
ramifications. Since Egr-1 can preferentially displace Sp1 binding at
the A site, and in so doing, enhance PNMT promoter activity, the A site
may normally function as a high affinity Egr-1 site in the presence of
sufficient amounts of Egr-1 protein. However, when Egr-1 levels are
low, the A site may alternatively function as an Sp1 site, albeit of
moderate affinity. In contrast, the B site where Egr-1 binding seems to
have little effect on Egr-1-mediated PNMT promoter activity, may be a
biologically active Sp1 site under most circumstances. Consistent with
this interpretation is a marked reduction in basal PNMT promoter
activity when the B site is mutated. A similar reduction is observed
with mutation of the A site, but this occurs at low basal Egr-1 levels,
a circumstance where the A site may function as an Sp1 site as
predicted above.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.