From the
Stromelysin-1 (ST-1) is an extracellular matrix
metalloproteinase whose expression is transcriptionally regulated by
nerve growth factor (NGF) in the PC12 rat pheochromocytoma cell line.
In this paper, we define sequences in the proximal ST-1 promoter that
contain a novel NGF-responsive element(s). We show that this
cis-acting promoter element can bind nuclear proteins from
both untreated and NGF-treated PC12 cells in a specific and saturable
manner and is sufficient to confer NGF-inducibility to a heterologous
promoter. At least a portion of this NGF-responsive element lies within
a 12-base pair region between positions -241 and -229 of
the ST-1 promoter and bears no sequence homology to other known
transcriptional elements. In contrast to what has been reported for
fibroblasts, an AP1 site centered around position -68 does not
seem to be involved in the growth factor regulation of ST-1 in PC12
cells. These results suggest that the NGF regulation of ST-1 gene
expression involves different promoter elements, and possibly different
transcription factors, from that described for ST-1 induction by other
growth factors.
Nerve growth factor (NGF)
Unlike NGF, which
triggers neuronal differentiation, epidermal growth factor (EGF)
stimulates proliferation of PC12 cells without differentiation. Both
NGF and EGF are ligands for receptor tyrosine kinases, which can
activate similar intracellular signaling molecules such as
phospholipase C-
Stromelysin-1 (ST-1) is a member of
the matrix metalloproteinase gene family which includes interstitial
collagenase and the gelatinases
(33, 34) . ST-1 itself
is known to degrade various components of the extracellular matrix
associated with basal laminae
(35) and has been implicated in
tissue remodelling events associated with embryonic development, tumor
metastasis, and axonal invasiveness
(36, 37, 38) . In fibroblasts, ST-1 is
transcriptionally induced by EGF, platelet-derived growth factor (PDGF)
and the phorbol ester 12- O-tetradecanoylphorbol-13-acetate
(39, 40, 41) . This induction can be
transcriptionally inhibited by transforming growth factor
In this study, we utilize the ST-1 promoter to
determine the mechanisms responsible for the NGF induction of ST-1 gene
expression in PC12 cells. It was previously shown that a 750-bp
fragment of the proximal ST-1 promoter contained sequences which were
sufficient for the NGF induction of this gene
(28) . Here we
identify a 12-bp region of the ST-1 promoter which contains at least a
portion of a novel NGF-responsive element. We show that the promoter
region containing this element binds nuclear proteins from both
untreated and NGF-treated PC12 cells and is sufficient to confer NGF
responsiveness to a heterologous promoter. This characterization of
cis-acting sequences mediating the effects of NGF may provide
insights into the mechanisms underlying the growth factor-specific
expression of late genes during neuronal development.
DNA binding reactions involved first preincubating the
nuclei in 10 µl containing 20 µg of bovine serum albumin, 15
µg of poly(dI-dC), and unlabeled competitor DNAs for 30 min at room
temperature. End-labeled probes (10,000 cpm; specific activity =
10
To determine whether these
DNA
NGF has been shown to be critical for the normal development
and maintenance of the nervous system in the embryo and for the
survival of neurons in the adult, presumably via its effects on gene
expression
(2, 53) . While many of the initial events in
the NGF signaling pathway have been identified
(9, 10, 11, 14, 54, 55, 56, 57, 58, 59) ,
little is known about nuclear events that regulate growth
factor-specific changes in gene expression.
In this paper, we
identify a 60-bp NGF-responsive region in the ST-1 promoter that is
both necessary and sufficient for NGF responsiveness. When this region
was deleted from the promoter, the remaining portions failed to display
NGF inducibility; when this region was placed next to a heterologous
promoter, it was sufficient to confer NGF inducibility. The orientation
of this 60-bp region was also found to be important for heterologous
gene inducibility, since experiments reversing the orientation of this
region failed to display significant levels of NGF induction. This is
consistent with the fact that this 60-bp region lacks palindromic
sequences of appreciable length, which are often associated with
regulatory elements which have biological activities in either the
forward or reverse orientations. Using various mutants of this region,
we showed that the protein binding site most likely included sequences
within a 12-bp region of the ST-1 promoter. Mobility shift assays
confirmed, moreover, that this region bound a nuclear protein or
proteins expressed by PC12 cells. Interestingly, this protein(s) seemed
to be expressed in both untreated and NGF-treated cells, since the
mobility shift patterns of nuclei isolated from both treatments were
indistinguishable. These similar mobility shift patterns may also
indicate that the binding of accessory proteins to the preformed
complex is not necessary for ST-1 induction. If this were the case, one
might have expected supershifted bands or other differences in the
patterns of bands between untreated and NGF-treated cells.
Alternatively, post-transcriptional modifications of this nuclear
protein(s), such as phosphorylation, may be associated with the
mechanism of ST-1-induction by NGF. This is consistent with
observations that protein kinase A or C activators, such as forskolin
and phorbol esters, augments the NGF induction of ST-1 in PC12 cells
(60) . The notion that phosphorylation is involved in the NGF
induction of ST-1 is also consistent with observations that activation
of the cytoplasmic kinases src, ras, and raf seem to be necessary for the NGF induction of neuronal
differentiation in PC12 cells, including the expression of ST-1
(61, 62, 63, 64, 65) . These
proto-oncogenes are believed to act as upstream regulators of the
microtubule-associated protein kinase cascade leading to the
phosphorylation of critical regulatory proteins in the nucleus
(66, 67, 68) .
Several other NGF-responsive
elements have also been described, but all differ significantly from
sequences present in the ST-1 promoter region described here. These
NGF-responsive elements include a fat-specific element (TH-FSE) in the
tyrosine hydroxylase promoter that binds c- fos as part of the
nucleoprotein complex
(30) and a unique novel negative
regulatory element in the peripherin gene
(31) . In this latter
case, derepression of peripherin gene expression also involved a less
well-defined ``distal positive element'' within a fairly
large 370-bp region of the promoter. These studies did not, however,
test whether these elements conferred NGF responsiveness to a
heterologous promoter. This analysis was included, however, in a recent
report of a 50-bp NGF-responsive region of the neuropeptide Y promoter
(32) . This NGF-responsive region in the neuropeptide Y promoter
also bound a transcription factor present in both untreated and
NGF-treated PC12 cells, and the patterns of shifted bands were similar
in NGF-treated and untreated cultures. Comparison of the neuropeptide Y
and the ST-1 NGF-responsive regions showed no significant sequence
similarities, which may indicate that the transcription factors
recognizing the two NGF-responsive elements also differ. Although the
12-bp sequence in the ST1 promoter contains a relatively high A +
T content, it doesn't seem to correspond to known homeodomain
binding protein consensus sequences, which are typically A +
T-rich
(69) .
We also report here that the AP1 site at
position -71 regulates basal levels of ST-1 expression, at least
in these transient transfection assays. Promoter constructs with point
mutations in the AP1 site show, for example, large reductions in basal
levels of CAT expression in transiently transfected PC12 cells, yet
these cells still displayed statistically significant NGF inducibility.
Although the absolute levels of this induction were attenuated with the
mutated AP1 site, the relative levels of NGF inducibility remained
unaffected in cells transfected with the mutated AP1 site. Further
evidence that the AP1 site is not involved in NGF responsiveness is
that c- fos and c- jun, two transcription factors which
bind to this site, are also induced by EGF in PC12 cells, yet EGF fails
to elicit ST-1 expression
(15, 16, 18, 19, 20) . In any
case, it should be noted that basal levels of endogenous ST-1 gene
expression in PC12 cells are normally nondetectable
(28, 60) , and, therefore, the notion that the AP1 site
regulates basal level of expression may only apply to the truncated
( i.e. 753 bp) ST-1 promoter region used in these studies.
In contrast to PC12 cells, the AP1 site in rat-1 fibroblasts seems
to play a critical role in the induction of ST-1 gene expression by
both EGF and PDGF
(41) . The AP1 site was also implicated in
ST-1 gene expression in a polyomavirus-transformed rat embryonic cell
line, in which the negative regulation of ST-1 mRNA levels by retinoic
acid was shown to be mediated by the same AP1 site
(70) . The
fact that NGF induction of ST-1 gene expression in rat PC12 cells does
not seem to be dependent on the AP1 site would suggest, therefore, that
the tissue-specific mechanisms of regulation differ significantly. On
the other hand, it is interesting to note that phorbol esters, although
they cannot induce ST-1 themselves, can augment the NGF induction of
ST-1 in PC12 cells
(60) . If this phorbol ester augmentation is
mediated via the AP1 site, it would suggest that this element acts to
modulate, rather than activate, gene expression.
The progressive
deletion mutants of the ST-1 promoter also allowed us to determine the
contribution of other potential transcriptional elements to NGF
inducibility. There is, for example, an NGFI-A site at position
-400 in the ST-1 promoter. This site is believed to bind the
NGF-IA protein (also known as zif 268, Egr-1, Krox 24, and Tis 8),
which is a zinc finger DNA binding protein rapidly induced by either
NGF or EGF in PC12 cells
(15, 71, 72) . Removal
of this site, however, was found not to have an appreciable effect on
the NGF inducibility of CAT expression in transient transfection
assays, suggesting that this NGFI-A site is not involved. This
conclusion is consistent with earlier observations in which addition of
the kinase inhibitor staurosporine to PC12 cells was found to have
little effect on NGF induction of the NGFI-A, yet completely blocked
the NGF induction of ST-1 mRNA expression and neurite extension
(60) .
In sum, we have shown that the ST-1 gene regulation by
NGF is mediated by an element present in a 12-bp region of the ST-1
proximal promoter. Future studies characterizing the core element
within this region, as well as studies identifying transcription
factors responsible for mediating activation of the transcription
apparatus, should provide important information about how the
biological effects of NGF are manifested at the level of gene
expression in the nervous system.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) M13012.
(
)
was the first
neurotrophic factor discovered in a class of molecules responsible for
the development, differentiation, and growth of the nervous system. NGF
supports the survival and maintenance of sympathetic and sensory
neurons of the peripheral nervous system and promotes the
differentiation of selected cholinergic and adrenergic neurons of the
central nervous system
(1, 2) . NGF can also induce
neural crest-derived adrenal medullary cells to transdifferentiate into
sympathetic neurons
(3) , a phenomenon recapitulated in
vitro by the PC12 rat pheochromocytoma cell line
(4) .
Following several days of exposure to NGF, PC12 cells undergo
transcriptionally dependent transformation into a neuronal phenotype
characterized by the extension of neurites, the development of
electrical excitability, and the expression of genes encoding neuronal
cell-specific proteins
(4, 5) .
, phosphoinositol 3-kinase, and ras (6, 7, 8, 9, 10, 11, 12, 13, 14) .
Within minutes, both NGF and EGF induce several immediate-early genes
encoding transcriptional regulatory proteins, including c- fos,
c- jun, and NGF-IA/ egr-1 in PC12 cells
(15, 16, 17, 18, 19, 20) .
Despite similarities in the signaling pathways, these growth factors
have very different effects on the physiology and morphology of PC12
cells. Neuronal differentiation by NGF is accompanied by expression of
a subset of genes which encodes proteins that are important for the
differentiated phenotype. Most of these genes are transcriptionally
active hours after NGF addition, including those encoding tyrosine
hydroxylase
(21) , neuropeptide Y
(22) , peripherin
(23) and other neurofilament components
(24) , brain type
II Na
channels
(25) , SCG10
(26) , VGF
(27) , and stromelysin (also known as transin)
(28) .
None of these late genes are activated by EGF, suggesting that some
critical aspects of these two signaling pathways differ. In order to
better understand the mechanisms underlying the development and
expression of the neuronal phenotype in PC12 cells, several groups,
including ours, have examined the transcriptional regulation of
NGF-induced genes
(29, 30, 31, 32) . In
this study, we utilize the stromelysin gene as a model for NGF-induced
regulation of gene expression.
-1
through a 10-bp sequence in the ST-1 proximal promoter region known as
the transforming growth factor
-1 inhibitory element
(42, 43, 44) . Recently, PDGF has been shown to
induce ST-1 gene expression in NIH 3T3 cells via a 6-bp palindromic
sequence in the distal ST-1 promoter
(45) . In PC12 cells,
however, we found that NGF, but not EGF nor PDGF, increased the levels
of ST-1 mRNA
(28) . This increase in ST-1 mRNA is at least a
hundredfold over initially undetectable levels, making ST-1 one of the
most highly induced NGF-responsive late gene products described
(21) .
Oligonucleotides and Plasmids
5` promoter
deletion mutants of the p750TRCAT plasmid were generated using an
Erase-a-Basekit (Promega), as described
(44) .
Deletions were analyzed by DNA sequencing with Sequenase (Version 2.0,
U. S. Biochemical Corp.). The pCATbasicTK vectors (pCBTK) was the gift
of Dr. Bruce Magun (Oregon Health Sciences University)
(46) . In
some experiments, a modified form of the pCBTK vector was used in which
an AP1 site was inserted upstream of the thymidine kinase (TK)
promoter. This vector is referred to as ``pAS.'' The NRR-ABC
fragment was obtained by polymerase chain reaction (PCR) amplification
of the p715TRCAT vector using appropriate oligonucleotides with
BglII and ClaI restriction sites placed at the
termini. These oligonucleotides were:
5`-GCACAGATCTCTTCTGGAAGTTCTTTGTAC-3` (upstream) and
5`-GCACATCGATAAATGCTTCCTGCCTTAG-3` (downstream). The PCR product was
then subcloned into the BglII and ClaI sites of the
pCBTK vector. The NRR-A, NRR-B, and NRR-C fragments were obtained by
restriction digestion of the NRR-ABC fragment using MaeIII and
HaeIII, followed by gel isolation. The NRR-C fragment was
treated with Klenow polymerase to fill in the overhangs and then
blunt-end-ligated into the SalI site of the pAS vector.
Cell Culture and Transient Transfection
Assays
Stock cultures of PC12 cells were maintained in
Dulbecco's modified Eagle's medium containing 5% fetal
bovine serum and 5% horse serum at 37 °C in a 95% air, 5% COatmosphere. The original PC12 cell line (subclone GR-5) was
obtained from Dr. Rae Nishi (Oregon Health Sciences University). For
transient transfections, PC12 cells were plated at an initial density
of 2
10
cells per 10-cm Primaria
plate
(Falcon Plastics) 2 days before transfections. Calcium phosphate-DNA
precipitates containing 15 µg of DNA were added to the cultures for
4 h
(47) . The cells were then ``shocked'' for 2.0 min
with 15% glycerol in HEPES-buffered (25 m
M; pH 7.0) saline
solution. The cultures were washed twice with phosphate-buffered
saline, allowed to recover overnight in serum-containing medium, and
then cultured for 24 h in ``N2-supplemented'' medium
(48) . The next morning, NGF (50 ng/ml) or EGF (5 ng/ml) was
added directly to the cultures for an additional 24 h prior to harvest.
Protein levels were assayed
(49) and 50-µg aliquots were
used in a kinetic chloramphenicol acetyltransferase (CAT) assay using
[
H]acetyl coenzyme A, as described
(50) .
Gel Mobility Shift Assays
Nuclei from PC12 cells
were prepared using a modified version of the method of
Hagenbüchle and Wellauer
(51) in which intact nuclei are
used instead of extracts. PC12 cells were first dissociated by
treatment with PBS, pelleted by centrifugation, and then resuspended in
0.3
M sucrose in Buffer A (60 m
M KCl, 15 m
M NaCl, 0.15 m
M spermine, 0.5 m
M spermidine, 15
m
M HEPES, pH 7.8, 14 m
M mercaptoethanol, 0.5 m
M phenylmethylsulfonyl fluoride and 8 µl/ml aprotinin). Nonidet
P-40 was then added to this nuclear preparation to a final
concentration of 0.1% (v/v). Cells were lysed in a Dounce homogenizer
using 30 strokes with a type B pestle, and then the suspension was
centrifuged atop a 0.9
M sucrose cushion in buffer A. The
nuclei pellet was resuspended and treated again to Dounce
homogenization in buffer A (without Nonidet P-40) with 5 strokes with
the pestle. After the second centrifugation, the pellet was resuspended
in a small volume of buffer B (75 m
M NaCl, 0.5 m
M EDTA, 20 m
M Tris, pH 7.9, 0.8 m
M dithiothreitol,
0.1 m
M PMSF, and 50% glycerol) and the number of nuclei in
each extraction was determined. Nuclei from untreated and NGF-treated
PC12 cells were then adjusted to a final concentration of
10/ml, and 50-µl aliquots were stored at -80
°C.
cpm/µg) were added to the nuclei in a final buffer
concentration of 15 m
M HEPES (PH 7.5), 60 M
M KCL, 5 M
M MGCL
, 2 M
M EDTA, AND 12% GLYCEROL. BINDING
REACTIONS WERE ALLOWED TO INCUBATE FOR 30 MIN ON ICE. SAMPLES WERE THEN
SUBJECTED TO ELECTROPHORESIS IN 6% SDS-POLYACRYLAMIDE GELS
(ACRYLAMIDE:BIS RATIO OF 37:1) AT 4 °C IN 0.5
TBE (50
M
M TRIS-HCL, PH 8.3, 41 M
M BORIC ACID, 0.5 M
M EDTA). GELS WERE DRIED, AND RADIOLABELED BANDS WERE VISUALIZED BY
AUTORADIOGRAPHY.
Site-directed Mutagenesis of the NGF-responsive
Region
Five versions of the ST-1 promoter were generated using
the Altered Sitesin vitro mutagenesis system
(Promega). These 5 plasmids contained contiguous 6-bp nested mutations
spanning the 30-bp NRR-C` region (see Fig. 6 A). The
EcoRI fragment of p715TRCAT (containing the promoter) was
subcloned into pAlter-1 in the sense orientation and used as a template
for the mutagenesis reaction with the following oligonucleotides:
mutant 1 (m1), 5`-CAGCTTCTGAAGGATATAGTACTTTTCCAAAGTAG-3`; mutant 2
(m2), 5`-GAAGGATAGTTACAAGACTGAAAGTAGAAAAAAATGCC-3`; mutant 3 (m3),
5`-GATAGTTACATTTTCCG-TATCTGAAAAAAATGCCCC-3`; mutant 4 (m4),
5`-CATTTTCCAAAGTATTACTGAATGCCCCAGTTTTC-3`; and mutant 5 (m5),
5`-CCAA-AGTAGAAAAAGCCTATGCAGTTTTCTCTTTTGC-3`. Annealing, extension, and
screening of mutations were carried out using protocols supplied by the
manufacturer. Mutated forms of the promoter were then reintroduced into
the pCBTK vector by PCR amplification of the mutant NRR-ABC region and
subsequently subcloned into the BglII and ClaI sites
of pCBTK.
Figure 6:
Competition gel mobility shift assay with
DNA containing nested mutations, showing that the 3`-end of the 30-bp
sequence is necessary for nuclear protein binding. A, DNA
sequences of the wild type and 5 mutated forms of the 30-bp NRR-C`
region which were used as competitors in the experiment represented in
B. B, P-labeled NRR-C was incubated with nuclei
from NGF-treated PC12 cells and subjected to electrophoresis.
Competitor unlabeled NRR-ABC fragments from either wild type
( wt) or mutant plasmids were included in the binding reactions
at 25- or 100-fold molar excesses over labeled probe, as indicated by
the ramps at the top of the gel. The arrow on the right points to the shifted protein
DNA
complex. Note that wild type and m1, m2, and m3 were able to compete
for nuclear protein binding, but that m4 and m5 were
not.
Transient Transfection Studies Using a Mutated AP1 Site
in the ST-1 Promoter
In previous work, Kerr et al. (41) showed that the AP1 site at position -65 of the ST-1
promoter mediated the EGF induction of ST-1 gene expression in NIH 3T3
cells. To determine whether this AP1 site was necessary for NGF
induction of ST-1, PC12 cells were transiently transfected with either
the parental p750TRCAT vector containing a 753-base pair region of the
ST-1 promoter, or a site-directed mutant of this vector in which two
base substitutions were introduced into the AP1 site, rendering it
functionally inactive
(41) . Fig. 1shows that PC12 cells
transfected with the wild type p750TRCAT plasmid showed a 5- to 7-fold
NGF-induced increase in CAT activity as compared with untreated control
cells. Basal levels of CAT activity were significantly lower in cells
transfected with the plasmid containing the mutated AP1 site as
compared with those transfected with the wild type AP1 site ( p < 0.001), whereas NGF still caused a significant increase in
CAT levels ( p < 0.050). In neither case was there a
statistically significant change in CAT activities following exposure
to EGF. Since the ability of the mutated plasmid to respond to NGF was
not lost, we infer that the AP1 site is not directly responsible for
the NGF responsiveness of the ST-1 promoter. This experiment was
performed three times with qualitatively similar results.
Figure 1:
The AP1 site is not directly involved
in the NGF responsiveness of the ST-1 promoter. PC12 cells were
transiently transfected with a plasmid containing 750 bp of the ST-1
promoter driving expression of the reporter gene, chloramphenicol
acetyltransferase (CAT) containing the native AP1 site or with a
plasmid in which two base changes were made in the AP1 site (indicated
by asterisks). CAT enzymatic activity was assayed in PC12
cells that were either untreated ( C; open bars),
treated with 50 ng/ml NGF ( N; black bars), or treated
with 5 ng/ml EGF ( E; stippled bars). Results from
Student's t test analysis between bracketed samples is indicated. N.S., not statistically
significant.
5`-Deletion Analysis of the ST-1 Promoter
The
observation that a mutated AP1 site failed to abolish the NGF induction
of CAT indicates that a different element must confer NGF
responsiveness to the ST-1 promoter. To localize this element,
5`-deletion mutants of the parental p750TRCAT plasmid were generated
(see Fig. 2 A; the arrows indicate the various
deletion mutants). PC12 cells were then transiently transfected with
these plasmids and treated for 24 h with either control culture medium
or medium supplemented with NGF or EGF. As a control for transfection
efficiencies, all of the cultures were co-transfected with a second
plasmid containing the luciferase reporter gene driven by the Rous
sarcoma virus constitutive promoter. Fig. 2 B shows that
removal of the terminal 38 bases from the 5` end of the parental
plasmid resulted in an increase in NGF-responsive CAT expression from
approximately 5-fold to 11-fold above basal levels, indicating the
possible presence of a negative regulatory element in this region. In
contrast, there was no statistically significant change in basal or
EGF-induced levels of CAT expression. Deletion of the subsequent 400
base pairs from the 5` end of the ST-1 promoter, however, produced no
consistent or statistically significant changes in the levels of NGF
responsiveness. Although basal levels of CAT expression remained
unchanged following deletions of this region, the levels of EGF-induced
gene expression were slightly higher with the p573TRCAT and p578TRCAT
plasmids. This observation, however, was not pursued further.
Figure 2:
The region between positions -247
and -315 in the ST-1 promoter is necessary for NGF
responsiveness. A, sequence of the ST-1 promoter (39) numbered
with respect to the transcription start site. Putative regulatory
elements are bracketed and include a transforming growth
factor -1 inhibitory element, NGF-IA (also known as zif 268, Tis
8), CAAT box, TATA box, and AP1 site. The narrow underlined
sequence corresponds to a 9-bp palindromic sequence. The thick
underlined sequence corresponds to the 12-bp sequence that
contains at least a portion of the NGF-responsive element. The
arrows and numbers below the sequence refer to the
various 5`-deletions analyzed for promoter activity. B,
analysis of ST-1 promoter activity in transient transfection assays.
PC12 cells were incubated with medium in the absence (control; open
squares) or presence of either NGF (50 ng/ml; closed
circles) or EGF (5 ng/ml; closed squares) for 24 h. All
cultures were co-transfected with a plasmid containing the luciferase
gene driven by the Rous sarcoma virus promoter. The results presented
here have been normalized against luciferase levels. Note that there is
a significant decrease in NGF-induced CAT activities between positions
-315 and -247. This experiment has been repeated three
times with qualitatively similar results.
The
largest change in NGF responsiveness occurred following the deletion of
a 68-base pair region between positions -315 and -247, in
which the fold NGF induction fell from approximately 8-fold to about
1.5-fold (averaging the results of three experiments). In contrast,
this deletion produced no significant differences in either basal
levels or EGF-induced levels of CAT expression. Further deletions in
the ST-1 promoter had only a slight effect on NGF responsiveness.
However, removal of the region between -247 and -228
resulted in a small but significant increase in EGF-responsive CAT
expression, indicating the possible presence of a growth
factor-specific silencer region. This experiment was performed three
times with qualitatively similar results. These results indicate that
the region between -315 and -247 bp upstream of the ST-1
transcription start site contains sequences that are required for
NGF-responsiveness.
Gel Mobility Shift Experiments Using Various Regions of
the ST-1 Promoter
DNA sequences which function as regulatory
elements are likely to bind nuclear proteins
(52) . To determine
whether the region between positions -247 and -315 would
bind such a protein, gel mobility shift assays were performed. As a
probe for these experiments, we first generated a polymerase chain
reaction (PCR) fragment which included the region between -247
and -315 plus significant portions of 5`- and 3`-flanking DNA
(labeled in Fig. 3 A, NRR-ABC for
``NGF-responsive region'') to ensure that this probe would
contain the entire protein binding site. PC12 cells were then treated
for 2 h in the presence or absence of NGF, and then nuclei were
isolated and incubated with P-end-labeled probe, allowing
protein
DNA complexes to form. Fig. 3 B is an
autoradiograph of a nondenaturing gel loaded with these binding
reactions showing that, in the absence of nuclear proteins, no shifted
complexes were present ( lane 1). When nuclei from either
control or NGF-treated PC12 cells were used, several shifted bands were
visible ( lanes 2 and 3). The arrow at the
left indicates the most heavily labeled band, which was
subsequently found to be the only one which displayed specific and
saturable DNA-protein binding.
Figure 3:
Nuclear protein(s) bind to a 60-bp NRR-C
region of the ST-1 promoter. A, schematic of the NRR-ABC
fragment containing the region between positions -315 and
-247 of the ST-1 promoter, plus flanking DNA. Also diagrammed are
the 45-bp NRR-A, 43-bp NRR-B, and 60-bp NRR-C restriction fragments.
B, gel mobility shift assay using different regions of the
ST-1 promoter. DNA fragments were first end-labeled with
P, incubated with nuclei from either untreated
( C) or 2-h NGF-treated ( N) PC12 cells, and then
subjected to electrophoresis in a 6% acrylamide gel under nondenaturing
conditions, as described under ``Materials and Methods.''
Note that, in the absence of nuclei, all of the label is present at a
single band ( asterisks), but in the presence of nuclei from
either untreated or NGF-treated cells, the NRR-ABC ( lanes
1-3) and NRR-C fragments ( lanes 10-12)
produce shifted complexes containing multiple bands
( arrows).
To dissect this protein binding
region further, the NRR-ABC fragment was digested with HaeIII
and MaeIII restriction enzymes, which cut the fragment into 3
similarly sized pieces (termed NRR-A, NRR-B, and NRR-C; see
Fig. 3A). Each of these pieces was then end-labeled and
used in gel mobility shift assays, as described above. Lanes
4-9 of Fig. 3 B show that neither the NRR-A
nor NRR-B fragments produced shifted proteinDNA complexes, whereas
the NRR-C fragment did with nuclei from either control or NGF-treated
PC12 cells (Fig. 3 B, lanes 10-12). This
experiment was performed three times with qualitatively similar
results. These results suggest, therefore, that a region within the
NRR-C fragment binds DNA-binding protein(s), and that this protein(s)
is present constitutively.
protein interactions were specific and saturable, competition
gel shift assays were performed. In these experiments, molar excesses
of unlabeled DNA corresponding either to the full-length NRR-ABC or
each of the three fragments was included in the incubation mixture
along with radiolabeled probe. Figs. 4 A and 4 B show
competition mobility shift assays using the full-length NRR-ABC with
nuclei isolated from untreated and NGF-treated cells. In either case,
only the full-length NRR-ABC and the NRR-C could compete for the
shifted complex, and this competition was apparent when a molar excess
of as little as 25
unlabeled-to-labeled DNA was used ( lanes
3-6 and 15-18). Fig. 4, C and
D, shows the converse experiment and that the NRR-C
radiolabeled probe was effectively competed for binding with both
NRR-ABC and NRR-C unlabeled DNA. Again, this competition was seen when
using nuclei from either NGF-treated or control PC12 cell cultures.
This experiment has been performed three times with qualitatively
similar results. Several other nonspecific oligonucleotides were also
used as competitors, and none were able to disrupt binding of nuclear
protein(s) to the NRR-ABC (data not shown). These results are
consistent, therefore, with the notion that a specific and saturable
protein binding element can be found within region NRR-C, and that the
protein recognizing this element is present in the nuclei of both
NGF-treated and untreated PC12 cells. Interestingly, the NRR-B fragment
contains a 9-base pair palindromic sequence centered around position
-268 (Fig. 2 A, single underlined
sequence). When an oligomer corresponding to this region was used
in competition gel mobility shift assays, however, it was also unable
to compete with the NRR-ABC probe for binding (data not shown),
consistent with the notion that the NRR-B fragment does not contain
this regulatory element.
Figure 4:
Binding of nuclear protein(s) from both
untreated and NGF-treated PC12 cells to the NRR-C is specific and
saturable.P-labeled DNA fragments corresponding to
different regions of the ST-1 promoter were incubated with nuclei from
either NGF-treated or untreated PC12 cells and electrophoresed as
described under ``Materials and Methods.'' Competitor
nonlabeled DNA ( NRR-ABC, NRR-A, NRR-B, and
NRR-C) was included in the binding reactions at increasing
amounts of 25-, 50-, 100-, and 200-fold molar excess over labeled probe
(represented by the ramps at the top of each gel).
The arrows at the right identify the shifted
complexes which showed specific and saturable DNA-protein binding.
A, the probe used was the NRR-ABC fragment (see Fig.
3 A), and nuclei were from untreated PC12 cells. B,
the probe used was the NRR-ABC fragment, and nuclei were from
NGF-treated (50 ng/ml) cells. C, the probe used was the NRR-C
fragment (see Fig. 3 A), and nuclei were from untreated cells.
D, the probe used was the NRR-C fragment, and nuclei were from
NGF-treated cells. Essentially identical results were obtained in two
other experiments.
Since the NRR-C contains a large segment of
DNA flanking the original deletion at position -247 (see
Fig. 2A), it seems likely that the protein binding
element would be found near this position in the 5`-half of the NRR-C
fragment. To test this assumption, additional competition mobility
shift assays were performed using a 30-bp synthetic oligonucleotide
that corresponded to the first 30 bp of NRR-C. Fig. 5 A shows that this oligonucleotide (termed NRR-C`) competed with
radiolabeled NRR-C for binding to nuclear proteins in both control
( lanes 3-7) and NGF-treated ( lanes 9- 13) PC12
cells. The arrow points to the shifted complex that was
specifically competed by NRR-C`, and the asterisk indicates
another complex which is not competed with unlabeled DNA.
Fig. 5B shows that the NRR-C` oligonucleotide, when used
as a labeled probe, could also bind nuclear proteins in both
NGF-treated and untreated PC12 cells, indicating that this 30-bp region
of the ST-1 promoter contains the binding site(s) for nuclear proteins
from PC12 cells. These experiments were performed three times with
qualitatively similar results.
Figure 5:
The region between -229 and
-259 of the ST-1 promoter contains the nuclear protein binding
site. A, P-labeled NRR-C was incubated with
nuclei from either untreated ( C; lanes 2-7) or
NGF-treated ( N; lanes 8-13) PC12 cells and
electrophoresed, as described under ``Materials and
Methods.'' Competitor unlabeled synthetic oligonucleotides
corresponding to the NRR-C` region of the ST-1 promoter was included in
the binding reactions at increasing amounts of 25-, 50-, 100-, 200-,
and 400-fold molar excess over labeled probe as indicated by the
ramps on the top of the gel. The arrow on
the right points to the shifted protein-DNA complex that is
specifically competed out by the NRR-C`. The asterisk (*)
indicates a shifted complex which is not competed out by NRR-C`.
B,
P-labeled NRR-C ( lanes 1-3) or
radiolabeled NRR-C` ( lanes 4-6) was mixed with nuclei
from either untreated ( C) or NGF-treated ( N) PC12
cells and electrophoresed as described under ``Materials and
Methods.''
To further localize the
NGF-responsive element within the 30-bp region, we performed
site-directed mutagenesis. For these studies, 5 mutant plasmids were
first generated using the Altered Sitessystem (Promega)
containing contiguous 6-bp mutations spanning the NRR-C` region (see
Fig. 6A). The NRR-ABC region of each of these mutant
plasmids was then isolated and used in gel mobility shift assays as
competitors. Fig. 6 B shows that m1, m2, and m3 fragments
were able to compete for protein binding with radiolabeled wild type
probe ( lanes 5-10), whereas m4 and m5 fragments were not
( lanes 11-14), suggesting that the region of the
promoter necessary for protein binding is present within a 12-base pair
region at the 3`-end of the NRR-C` fragment (see the bold
underlined region between positions -241 and -229 of
Fig. 2A). Qualitatively similar results were seen in
assays using nuclei from non-NGF-treated PC12 cells (data not shown).
Interestingly, gel mobility shift assays using radiolabeled probes
corresponding to each of these mutated fragments showed that all five
were able to bind nuclear proteins to some extent in the absence of any
competitor DNA (data not shown). This suggests that the mutations in m4
and m5 reduced, rather than abolished, protein binding affinity.
Heterologous Promoter Experiments Using the NRR-C
Fragment
The mobility shift assays and 5` deletion analysis show
that an element within the NRR-C region is necessary for NGF
responsiveness. To show that this element is also sufficient for NGF
responsiveness, the NRR-C was cloned into a vector containing the basal
thymidine kinase promoter with an added AP1 site driving expression of
the CAT reporter gene. The NRR-C was inserted in either the forward
``AS{NRR-C(F)}'' or reverse
``AS{NRR-C(R)}'' alignment in order to determine
whether it was equally active in either orientation. PC12 cells were
then transiently transfected with these plasmids or the parental AS
plasmid and treated for 24 h with culture medium containing either NGF
or EGF, and then the cultures were harvested for CAT enzymatic
activity. Fig. 7shows that a single copy of the NRR-C region in
the forward orientation was sufficient to induce a 2-fold increase of
CAT activity by NGF, but not EGF. In the reverse orientation, however,
this region failed to induce a statistically significant increase in
CAT activity by either NGF or EGF in the three times this experiment
was performed. Similar results were obtained using the NRR-ABC fragment
in either the forward or reverse orientation (data not shown).
Together, these results indicate that the NRR-C region is both
necessary and sufficient to function as an orientation-specific
NGF-responsive element.
Figure 7:
The
NRR-C region of the ST-1 promoter confers NGF responsiveness to a
heterologous promoter. PC12 cells were transiently transfected with a
plasmid containing the NRR-C in either the forward
AS{NRR-C(F)} or reverse AS{NRR-C(R)} orientation
upstream of the basal thymidine kinase (TK) promoter driving expression
of the reporter gene CAT. As a negative control, cells were transfected
with the pAS vector alone. The p715 vector was used as a positive
control. CAT enzymatic activity was assayed for PC12 cells that were
either untreated ( C; open bars), treated with 50
ng/ml NGF ( N; black bars), or 5 ng/ml EGF
( E; stippled bars). Results from Student's
t test analysis between bracketed samples is
indicated. NS, not statistically significant ( i.e. p < 0.05).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.