(Received for publication, April 7, 1995; and in revised form, August 4, 1995)
From the
Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5`-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box. Two major transcriptional start sites were mapped. A hybrid gene consisting of the Escherichia coli chloramphenicol acetyltransferase gene under the control of 837 base pairs of the synapsin II 5`-upstream region was transfected into neuronal and non-neuronal cells. While reporter gene expression was low in neuroblastoma and non-neuronal cells, high chloramphenicol acetyltransferase activities were monitored in PC12 pheochromocytoma cells. However, there was no correlation between reporter gene expression in the transfected cells and endogenous synapsin II immunoreactivity. Using DNA-protein binding assays we showed that the transcription factors zif268/egr-1, polyoma enhancer activator 3 (PEA3), and AP2 specifically contact the synapsin II promoter DNA in vitro. Moreover, the zif268/egr-1 protein as well as PEA3 were shown to stimulate transcription of a reporter gene containing synapsin II promoter sequences. In the nervous system, zif268/egr-1 functions as a ``third messenger'' with a potential role in synaptic plasticity. PEA3 is expressed in the brain and its activity is regulated by proteins encoded from non-nuclear oncogenes. We postulate that zif268/egr-1 and PEA3 couple extracellular signals to long-term responses by regulating synapsin II gene expression.
The synapsins are a family of neuronal phosphoproteins that are
localized on the cytoplasmic surface of small synaptic vesicles (for
review, see Greengard et al.(1993) and Thiel(1993)). This
family consists of four proteins, synapsin Ia and synapsin Ib
(collectively termed synapsin I) and synapsin IIa and synapsin IIb
(collectively termed synapsin II). These isoforms are generated via
alternative splicing from two different genes
(Südhof et al., 1989). Molecular cloning
of bovine, human, and rat synapsins revealed striking homologies in the
amino-terminal 420 amino acids of all four synapsins. The major
difference between synapsins I and II is the presence of a very
proline-rich COOH-terminal domain in the synapsin I isoforms that
contains clusters of basic amino acids as well as two recognition sites
for Ca/calmodulin-dependent protein kinase II
(Südhof et al., 1989).
Synapsin I has been postulated to link synaptic vesicles to the cytoskeleton, thus regulating the availability of synaptic vesicles for exocytosis. In addition, a role for synapsin I in the regulation of short-term plasticity has recently been suggested (Rosahl et al., 1993). The functional role of synapsin II is not yet clear. However, the fact that synapsin II interacts with both synaptic vesicles (Thiel et al., 1990; Siow et al., 1992) and actin filaments (Chilcote et al., 1994) in a similar fashion as synapsin I suggests that all synapsin isoforms share basic functional properties. This hypothesis was supported by results obtained with mice lacking synapsin I (Rosahl et al., 1993). These mice seem to have no serious impairment of brain function indicating that synapsin II might be compensating the loss of synapsin I.
Synapsins I and II have no
non-neuronal counterpart in contrast to the synaptic vesicle proteins
synaptobrevin and synaptophysin (Zhong et al., 1992; McMahon et al., 1993). The synapsin genes are therefore good
candidates for an investigation of neuron-specific gene expression.
Virtually all neurons express the synapsins, but not every isoform of
synapsin is expressed in every nerve cell (Südhof et al., 1989). The human and rat synapsin I genes have been
analyzed and it was shown that the 5`-flanking region contains
neuron-specific enhancer elements (Sauerwald et al., 1990;
Thiel et al., 1991). Here, we report the sequence of the human
synapsin II promoter. Our studies have revealed similarities as well as
distinct differences in comparison with the synapsin I promoter.
Moreover, we present data indicating that the transcription factors
zif268/egr-1, PEA3, ()and AP2 regulate synapsin II gene
expression.
Figure 1: Nucleotide sequence and GC profile of the 5`-flanking region of the human synapsin II gene. A, the nucleotide sequence of clone pSyC58-10 is depicted. The two transcriptional start sites are marked by arrows. Nucleotides are numbered with respect to the most 3` start site. Consensus sites for the transcription factors PEA3, AP2, and zif268/egr-1 are boxed. The ATG codon is shown as well as the start of the human synapsin II gene open reading frame. B, GC-content of the promoter and 5`-untranslated region of the synapsin II gene.
Figure 2: Comparison of human synapsin I and synapsin II promoter regions. The analysis was performed with the program ``compare'' with a window size of 21 and a stringency level of 14. The results are displayed with the program ``Dotplot.''
Figure 3: Mapping of transcriptional initiation sites by S1 nuclease protection assay. A, the cartoon shows the strategy in synthesizing a human synapsin II specific DNA probe by primer extension. B, S1 nuclease protection mapping using human brain total RNA or as controls yeast tRNA and total RNA from HeLa cells. An aliquot of undigested probe is depicted as well as molecular weight markers (HaeIII cut pBR322). The arrowheads indicate the two transcriptional initiation sites. On the right-hand site, a sequencing reaction is depicted that was generated with the primer used to synthesize the S1 probe.
Figure 4:
Transfection analysis of synapsin II
promoter-CAT and synapsin I promoter-CAT fusion genes. The constructs
pSyIICAT (A) and pSyCAT-10 (B) were introduced into
the neuronal cell lines NS20Y, NS26, NG108-15, PC12/NY, PC12/HD,
and PC12/SF. To control for the presence of tissue-specific elements,
both plasmids were also transfected into CHO-KI and HeLa cells. CAT
activity was normalized for variations in transfection efficiency by
dividing CAT activity by -galactosidase activity. At least four
experiments were done with each cell type and the mean ± S.E. is
depicted. C, Western blot analysis of endogenous synapsins I
and II. Cell homogenates (70 µg/lane) were subjected to 10%
SDS-poyacrylamide gel electrophoresis and immunoblotted. As a positive
control 3 µg of forebrain homogenate was loaded into the left
lane.
Immunoblot analysis of the endogenous synapsins in neuronal cells used here revealed that both synapsins I and II are present in NS20Y, NS26, and NG108-15 cells (Fig. 4C). The expression of synapsins I and II in PC12 cells is in general much lower. A quantitative estimation of the bound iodinated protein A by PhosphorImager analysis revealed that PC12/HD and PC12/SF cells contain only 20 or 13% of the synapsin IIb expressed in NG108-15 cells. There was no correlation between reporter gene expression and endogenous synapsin II immunoreactivity. The synapsin II promoter-CAT gene is transcribed in PC12/NY cells 10 times more efficiently than in NS26 or NG108-15 cells although the endogenous synapsin II levels are comparable. Furthermore, transfection experiments of PC12/HD cells revealed 8 times higher CAT activities than in NS26 or NG108-15 cells despite the fact that endogenous synapsin II levels are much lower in the PC12 clone. Based on the low CAT activities in neuroblastoma cells as well as the lack of correlation between endogenous synapsin II expression and CAT activity following transfection experiments of a synapsin II promoter-CAT construct, we conclude that the elements directing neuron-specific expression of synapsin II are located outside the analyzed region encompassing the sequence -837 to +110.
The 5`-flanking region also contains a repetitive sequence element consisting of multiple GT repeats (nucleotides -595 to -540). Similar sequences were found in other neuronally expressed genes, i.e. the genes encoding neuron-specific enolase (Sakimura et al., 1987), synapsin I (Südhof, 1990), and GAP-43 (Nedivi et al., 1992). It has been suggested that the GT element activates transcription together with the adenovirus E1A protein (Berg et al., 1989). We could not confirm this observation in transfection experiments of synapsin II-CAT constructs into EIA containing 293 cells (data not shown). Alternatively, the GT element has been suggested to be a structural element in helping to form left-handed or Z-form DNA (Sakimura et al., 1995).
Additionally, consensus sites for the transcription factors zif268/egr-1, PEA3, and AP2 were detected in the synapsin II 5`-flanking region. These factors were therefore analyzed for their possible functional roles in synapsin II gene regulation.
Figure 5:
Zif268/egr-1 binds to the synapsin II
promoter in vitro and transactivates a reporter gene
containing zif268/egr-1 binding sites derived from the synapsin II
promoter in its upstream regulatory region. A, sequence of
zif268/egr-1 consensus sites in the upstream regions of the synapsin I
and synapsin II gene; B, electrophoretic mobility shift assay
using nuclear extract derived from human 293 cells transfected with
expression vectors for zif268/egr-1 (lanes 2-11 and 14-23) or PEA3 (lanes 12 and 24). The
radiolabeled probes were the zif268/egr-1 site of the synapsin I
promoter, termed EBS-2 (left panel, lanes 1-12), and the
putative zif268/egr-1 site of the synapsin II promoter (-174 to
-153), termed EBS-3 (right panel, lanes 13-24).
Binding experiments without extract are depicted in lanes 1 and 13. Competitor DNA was added to the reaction at
10-fold (lanes 3, 6, 9, 15, 18, and 21), 100-fold (lanes 4, 7, 10, 16, 19, and 22), and 500-fold (lanes 5, 8, 11, 17, 20, and 23) molar excess to the
probe. Synthetic oligonucleotides containing the PEA3 binding motif
(denoted ``PEA3'') were used as an unrelated competitor (lanes 9-11 and 21-23). The arrowheads indicate the protein-DNA complexes originating from
zif268/egr-1 binding to its cognate binding site. C, reporter
plasmids containing rabbit -globin as reporter gene, a TATA box
(plasmid OVEC), and 2 copies of the zif268/egr-1 binding site of the
synapsin I promoter (EBS-2
/OVEC) and synapsin II promoter
(EBS-3
/OVEC). The zif268/egr-1 cDNA is expressed under
control of the cytomegalovirus IE gene promoter (pCMVzif). D,
reporter plasmids, expression plasmids, and the JH514ref internal
standard plasmid were introduced into CHO-KI cells. Cytoplasmic RNA was
analyzed for
-globin mRNA by RNase protection mapping. The bands
labeled test indicate correctly initiated
-globin
transcripts, and the bands labeled ref were generated by the
internal standard plasmid JH514ref. RT indicates incorrectly
initiated read-through transcripts of the test templates. Also shown is
an aliquot of undigested cRNA (riboprobe). Size markers, BstEII cut
DNA (left) and HaeIII-digested pBR322, are shown in lanes
M.
To test whether zif268/egr-1 binds to
this motif we performed electrophoretic mobility shift assays. As a
source for zif268/egr-1, 293 human embryonic kidney cells were
transfected with an expression vector encoding zif268/egr-1 and nuclear
extracts were prepared. As a control, 293 cells were transfected with
an expression vector for PEA3. Fig. 5B shows in the left panel the results obtained with the zif268/egr-1 site
derived from the synapsin I promoter. Using nuclear extract prepared
from 293 cells containing zif268/egr-1, we observed a specific
DNA-protein complex that was absent in the reaction with nuclear
extracts from 293 cells transfected with an expression vector for PEA3
instead of zif268/egr-1 (compare lanes 2 and 12,
marked by an arrowhead). When unlabeled EBS-2 DNA was added to
the reaction, this complex disappeared (lanes 3-5).
Unlabeled oligonucleotides encompassing the EBS-3 site from the
synapsin II promoter also competed efficiently (lanes
6-8), whereas an unrelated oligonucleotide containing the
PEA3 site from the synapsin II promoter did not reduce the intensity of
the observed DNA-protein complex (lanes 9-11). On the right panel of Fig. 5B, the results obtained
with the probe containing the EBS-3 site are depicted. Nuclear extracts
of 293 cells containing zif268/egr-1 revealed a similar DNA-protein
complex (lane 14, marked by an arrowhead) that was
competed efficiently by an excess of EBS-3 (lanes 15-17)
or EBS-2 DNA (lanes 18-20). The control probe containing
the PEA3 binding site failed to compete (lanes 21-23),
suggesting that zif268/egr-1 recognizes the EBS-3 site in a
sequence-specific manner. Moreover, the EBS-3zif268/egr-1 complex
was not detectable in nuclear extracts from 293 cells transfected with
a PEA3 expression vector (lane 24). The binding experiments
depicted in Fig. 5B show, in addition, unspecific
complexes that are present in 293 extracts transfected with either
zif268/egr-1 or PEA3 expression vectors. However, the retarded band
originating from the presence of zif268/egr-1 is clearly defined. We
conclude from these results that zif268/egr-1 binds specifically to the
promoter region of the synapsin II gene.
To analyze whether
zif268/egr-1 functions as a transcriptional activator for synapsin II
gene expression, two copies of the EBS-3 sequence were cloned
immediately upstream of the TATA box of the OVEC plasmid that contains
the rabbit -globin gene as a reporter (Fig. 5C).
As a positive control, a plasmid containing two copies of the
zif268/egr-1 binding site derived from the synapsin I promoter was
used. These plasmids were transfected into CHO-KI cells together with
pCMV5 DNA or an expression vector for zif268/egr-1 (Fig. 5C). Plasmid JH514ref containing a mutated
-globin gene under the control of the SV40 promoter/enhancer was
included in the transfection to control for variability in transfection
efficiency (Thiel et al., 1994). 48 h post-transfection,
cytoplasmic RNA of the transfected cells was isolated, hybridized to a
-globin derived cRNA probe, and analyzed by RNase protection
mapping (Fig. 5D). When zif268/egr-1 was overexpressed
in cells transfected with EBS-2
/OVEC and
EBS-3
/OVEC plasmids, transactivation was observed. A
quantitative estimation of the transactivation properties of
zif268/egr-1 by PhosphorImager analysis revealed a 5.8- and 3.6-fold
increase in transcription for EBS-2 and EBS-3 binding sites,
respectively. These results indicate that zif268/egr-1 functions as a
transcriptional activator after binding to the zif268/egr-1 motif of
the synapsin II promoter.
Figure 7: PEA3 binds to the synapsin II and synaptophysin promoters in vitro.A, sequence of the PEA3 consensus site (PEA3 cons.) (Xin et al., 1993). PEA3 (SyII) and PEA3 (p38) denotes sequences of the human synapsin II and synaptophysin promoter that contain the PEA3 binding core motif. B, electrophoretic mobility shift assay using recombinant GST-PEA3 (lanes 2-10, 12-20, and 22-30). The assay was performed with radiolabeled double-stranded, synthetic oligonucleotides listed under A. Binding experiments without fusion protein are depicted in lanes 1, 11, and 21. Competitor DNA was added to the reaction at 10-fold (lanes 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, and 29) or 100-fold (lanes 4, 6, 8, 10, 14, 16, 18, 20, 24, 26, 28, and 30) molar excess to the probe. Synthetic oligonucleotides containing the zif268/egr-1-like motif of the synapsin I promoter (denoted zif268/egr-1) were used as an unrelated competitor (lanes 9, 10, 19, 20, 29, and 30). The arrowheads indicate the protein-DNA complexes originating from GST-PEA3 binding to its cognate binding site. C, DNase I protection analysis was performed with a radiolabeled DNA probe from the human synapsin II promoter (sequence -657 to -302). The lower strand was radiolabeled with the Klenow fragment of E. coli polymerase I including radioactive dCTP. The lane marked with control are the result of a DNase I reaction without adding protein. The other reactions contained either 25 µg of GST or GST-PEA3 fusion protein. The region protected by GST-PEA3 is indicated on the right-hand side.
Figure 6:
Expression of PEA3 in E. coli. A, diagram showing the construction of a GST-PEA3 fusion
protein; the ets domain of PEA3 is marked. B, total
homogenate of E. coli harboring plasmid pGEX-PEA3 before (lane 1) or after
isopropyl--D-thiogalactopyranoside induction (lane
2) were separated by SDS-polyacrylamide gel electrophoresis and
the gel stained with Coomassie Blue. The
isopropyl-
-D-thiogalactopyranoside-induced cells were
lysed by sonication and the expressed fusion protein was
affinity-purified using a glutathione affinity matrix (lane
3). The purified GST control protein is also shown (lane
4).
The members of the family of ets- transcription factors bind to the core sequence 5`-(A/C)GGAA-3`. The flanking sequences are variable and there is evidence that these sequences are necessary to determine which ets protein will bind (Wasylyk et al., 1992, 1993). We performed DNase I footprinting analysis to identify the sequence of the synapsin II promoter that is protected by PEA3 from DNase I digestion. Fig. 7C shows that the GST-PEA3 protein protects the synapsin II promoter sequence -628 to -647 from DNase I digestion, including the core sequence 5`-AGGAAG-3`. No nuclease protection was observed by adding no protein (control) or GST protein. These data confirm the electrophoretic mobility shift analysis that PEA3 binds specifically to the synapsin II promoter through sequences containing the PEA3 binding motif.
We next asked if PEA3 functions
as a transcriptional activator following binding to the synapsin II or
the synaptophysin promoter. Using the CAT gene as a reporter, four
binding sites for PEA3 from the synapsin II promoter or the
synaptophysin promoter were cloned immediately upstream of a minimal
promoter, thus generating plasmids SyIIPEA3CAT and
p38PEA3
CAT, respectively (Fig. 8A). As a
negative control, four copies of the zif268/egr-1 site from the
synapsin I promoter were inserted into the same vector (plasmid
EBS-2
CAT). These plasmids were transfected in CHO-KI cells
together with the expression vector pCMV5 or an expression vector
encoding PEA3. Also transfected in all experiments was a plasmid
containing the
-galactosidase gene under control of the CMV
promoter/enhancer to correct for differences in transfection
efficiencies. The results of this experiment are depicted in Fig. 8B. Overexpression of PEA3 increased transcription
of the CAT gene 7.5- and 15-fold, respectively, when PEA3 binding sites
derived from the synapsin II or the synaptophysin gene promoter were
present in the regulatory region. There was no increase in
transcription when zif268/egr-1 binding sites were inserted upstream of
the CAT gene. These data indicate that PEA3 functions as a
transcriptional activator after binding to the synapsin II or
synaptophysin promoter.
Figure 8:
Transactivation of synapsin II
promoter-CAT and synaptophysin promoter-CAT constructs in CHO-KI cells
by PEA3. A, reporter plasmids containing the E. coli CAT gene as reporter, a minimal promoter consisting of TATA box
and an Inr element, and 4 copies of the PEA3 binding site of the
synapsin II promoter (SyIIPEA3CAT) or synaptophysin
promoter (p38PEA3
CAT). As a control, 4 copies of the
zif268/egr-1 binding site of the synapsin I promoter were inserted
upstream of the TATA box (plasmid EBS-2
CAT). PEA3 cDNA is
expressed under control of the CMV promoter (pCMVPEA3). B,
relative CAT activities obtained after transfection of reporter and
expression constructs into CHO-KI cells. Cells were cotransfected with
plasmid pCMV
to correct for variations in transfection
efficiencies. CAT activity was normalized by dividing CAT activity by
-galactosidase activity. ``-'' denotes the
transfections with the ``empty'' expression vector pCMV5,
whereas ``+'' indicates transfections with the PEA3
expression vector pCMVPEA3. At least four experiments were done and the
mean ± S.E. is depicted.
Figure 9:
AP2 binds to the synapsin II promoter in vitro.A, sequence of the AP2 consensus site (AP2 cons.). AP2 (hMT-II), AP2 (SyII prox.), and AP2 (SyII dist.) are sequence
motifs of the human metallothionein and the synapsin II promoter,
respectively, that matched the AP2 consensus site. B, gel
retardation assay using recombinant AP2 (lanes 2-10,
12-20, and 22-30). The assay was performed
with radiolabeled double-stranded, synthetic oligonucleotides listed
under A. Binding experiments without AP2 are depicted in lanes 1, 11, and 21. Competitor DNA was added to the
reaction at 10-fold (lanes 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, and 29) or 100-fold (lanes 4, 6, 8, 10, 14, 16, 18,
20, 24, 26, 28, and 30) molar excess to the probe.
Synthetic oligonucleotides containing a zif268/egr-1 motif of the
synapsin I promoter (denoted zif268/egr-1) were used as
unrelated competitor (lanes 9, 10, 19, 20, 29, and 30). The arrowheads indicate the protein-DNA
complexes originated from binding of AP2 to the AP2 motifs of the
metallothionein and synapsin II promoters.
We have analyzed a genomic clone containing the human synapsin II 5`-flanking region. A comparison with the genes encoding the synaptic vesicle proteins synapsin I (Südhof et al., 1990), synaptophysin (Özcelik et al., 1990), and synaptobrevin II (Archer et al., 1990) revealed that the promoter regions of these genes are very GC-rich and lack TATA as well as CAAT boxes. We also observed that there is no extensive promoter sequence homology between the human genes encoding synapsin I and synapsin II. This lack of homology has also been reported for the mouse synapsin promoters (Chin et al., 1994). Transcription factors interact in general with short consensus sequences, thus a lack in overall homology between the regulatory region of the synapsin genes does not necessarily implicate different regulatory mechanisms.
Neuron-specific control elements have been identified in the proximal region of the human, and rat synapsin I promoter (Sauerwald et al., 1990; Thiel et al., 1991). Recently, an analysis of the mouse synapsin II promoter showed that a reporter gene under the control of 5 kilobase pairs of the 5`-upstream region is highly transcribed in the neuronal cell lines PC12, NS20Y, NS26, and NG108-15, but not in the non-neuronal cells HeLa, L6, 3T3, or HepG2 (Chin et al., 1994). In addition, transfection experiments of deletion constructs into HeLa and PC12 cells implicated a neuron-specific core promoter between -79 and +153 of the mouse synapsin II gene (Chin et al., 1994). Our own transfection experiment in PC12 and HeLa cells using the 5`-proximal region of the human synapsin II promoter yielded results similar to those of Chin et al.(1994). There was, however, a striking lack of correlation between the endogenous synapsin II gene expression in PC12 cells and the high transcriptional activity of a transfected synapsin II promoter-CAT construct. We had noted this phenomenon before as problematic in respect to synapsin I gene expression as measured in transfected PC12 cells (Thiel et al., 1991). We therefore found it necessary to test other neuronal cell lines as model systems for measuring synapsin II gene expression. The data we obtained from these neuronal cell lines, along with the non-neuronal cell lines tested, indicated that the 5`-proximal region of the synapsin II promoter does not confer tissue specificity of transcription. Thus, we believe that, as was the case with synapsin I promoter transfection experiments (Thiel et al., 1991), the use of PC12 cells as the sole neuronal model system to define the neuron-specificity of a promoter sequence is ill advised since the high level of transcriptional activity from the transfected synapsins I and II promoter-CAT constructs in these cells, 1) show no correlation with endogenous synapsin levels in these cells and 2) is at variance with the other neuronal cell lines tested.
The synapsin II gene promoter contains binding sites for the transcription factors zif268/egr-1, PEA3, and AP2, transcription factors that have been suggested to be involved in connecting extracellular signals to gene expression. A cartoon depicting the binding sites of these proteins in the synapsin II promoter is shown in Fig. 10.
Figure 10: Landmarks of the synapsin II promoter. The cartoon summarizes the protein binding sites of the synapsin II promoter, including the binding of zif268/egr-1, PEA3, and AP2. In addition, the major start site of transcription is depicted.
The zinc finger transcription factor zif268/egr-1 (Christy et al., 1988; Sukhatme et al. 1988), also known as NGFI-A (Milbrandt, 1987) and Krox24 (Lemaire et al., 1988) belongs to a group of genes termed the cellular immediate early genes (Sheng and Greenberg, 1990). Zif268/egr-1 gene expression is highly responsive to neuronal stimulation. In the hippocampal model of long-term potentiation (LTP), the zif268/egr-1 gene is induced by high-frequency stimulation and this induction is inhibited by N-methyl-D-aspartic acid-receptor antagonists (Cole et al., 1989; Wisden et al., 1990). These findings suggest a potential role for zif268/egr-1 in synaptic plasticity. Recently we presented data indicating that the synapsin I gene is a target for zif268/egr-1 (Thiel et al., 1994). Here, our results suggest that zif268/egr-1 might also regulate synapsin II gene expression. LTP is known to cause an increase in synaptic contact area as revealed by morphological studies (Desmond and Levy, 1988; Schuster et al., 1990; Geinisman et al., 1991). In addition, LTP-dependent changes in the number of vesicles in the synapse have been reported (Applegate et al., 1987; Meshul and Hopkins, 1990). It seems therefore likely that the concentrations of synaptic vesicle proteins are subject to regulation depending on the actual ``need'' of a particular neuron. In support of this view, Lynch et al.(1994) showed that LTP induced an increase of synapsin I, synaptotagmin, and synaptophysin immunoreactivity in the dentate gyrus. Thus, we propose a signaling cascade where induction of LTP is accompanied by an increase in zif268/egr-1 mRNA and protein (Abraham et al., 1993). The zif268/egr-1 protein translocates to the nucleus and activates synapsin I gene expression that can be monitored 3 h later by immunoblotting with specific antibodies to synapsin I. This cascade can be blocked at early stage by administration of an N-methyl-D-aspartic acid-receptor antagonist prior to induction of LTP. It will be of interest to determine whether synapsin II immunoreactivity also increases following LTP.
PEA3 was recently
cloned and analyzed for tissue-specific expression. Detectable amounts
of PEA3 mRNA were found only in brain and epididymis (Xin et
al., 1992). Here, using DNA-protein binding experiments and
transfection analysis, it was demonstrated that PEA3 binds to the
synapsin II promoter and transactivates a reporter gene that has
synapsin II promoter sequences in its regulatory region. Similar
results were obtained for a PEA3-binding site in the synaptophysin
promoter. It has been published that the DNA binding and
transcriptional activity of PEA3 is influenced by serum and tumor
promoters as well as by the gene products of several non-nuclear
oncogenes including Ha-ras, polyoma middle T-antigen, v-raf,
and v-src (Wasylyk et al., 1989; Gutman and Wasylyk,
1990; Bruder et al., 1992). Interestingly, many of these
proteins are involved in a signal transduction cascade that activates
mitogen-activated protein kinase. Upon activation this kinase is
translocated to the nucleus where it activates transcription by
phosphorylating transcription factors such as p62,
c-myc, c-jun, c-fos, NF-IL6, and ATF-2 (for
review, see Davis(1993)). PEA3 was shown to be a substrate of
mitogen-activated protein kinase
in vitro suggesting that phosphorylation of PEA3 by mitogen-activated
protein kinase might result from the various stimuli known to activate
PEA3. Taken together we postulate that synapsin II and synaptophysin
gene expression are regulated by PEA3 through extracellular signal
molecules via the mitogen-activated protein kinase pathway.
Finally, we showed in DNA-protein binding experiments that AP2 interacts in a sequence-specific manner with the synapsin II 5`-flanking region. The sequence encompassing the synapsin II promoter from -238 to -218 contains two AP2 consensus sites. These sites, however, are very close together making it unlikely that both are occupied by AP2 molecules. It has been proposed that AP2 mediates the effect of protein kinase C and cAMP upon transcription (Imagawa et al., 1987; Hyman et al., 1989). This effect is not executed by an increase in AP2 mRNA but rather by post-translational modifications of AP2 (Lüscher et al., 1989). It will be of interest to determine whether cAMP or tumor promoters increase synapsin II transcription via AP2.
The findings that zif268/egr-1, PEA3, and AP2 bind to the synapsin II promoter and activate transcription implicates the possibility that various signaling pathways may play a role in synapsin II gene expression and indicates possible mechanisms for which future studies should be directed.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89851[GenBank].