(Received for publication, October 16, 1995; and in revised form, November 28, 1995)
From the
The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The human synapsin I gene was functionally analyzed to identify control elements directing the neuron-specific expression of synapsin I. By directly measuring the mRNA transcripts of a reporter gene, we demonstrate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between mouse and human. Deletion of a putative binding site for the zinc finger protein, neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), abolished neuron-specific expression of the reporter gene almost entirely, allowing constitutively acting elements of the promoter to direct expression in a non-tissue-specific manner. These constitutive transcriptional elements are present as a bipartite enhancer, consisting of the region upstream (nucleotides -422 to -235) and downstream (nucleotides -199 to -143) of the putative NRSF/REST-binding site. The latter contains a motif identical to the cAMP response element. Both regions are not active or are only weakly active in promoting transcription on their own and show no tissue-specific preference. From these data we conclude that neuron-specific expression of synapsin I is accomplished by a negative regulatory mechanism via the NRSF/REST binding motif.
The synapsins are a family of neuronal phosphoproteins that coat
the cytoplasmic surface of small synaptic vesicles (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). Synapsins I and II 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 lies in the C-terminal domain of
the synapsin I isoforms. This domain 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 (Greengard et al., 1993). In addition, a role for synapsin I in the regulation of short term plasticity has been suggested (Rosahl et al., 1993). Mice lacking synapsin I or both synapsins I and II are viable and fertile with no gross anatomical abnormalities. These mice, however, frequently experience seizures, indicating the essential functions of the synapsins in synaptic vesicle regulation (Rosahl et al., 1995).
Virtually all neurons express the synapsins
(Südhof et al., 1989) and there are no
non-neuronal counterparts known for the synapsins, in contrast to the
synaptic vesicle proteins synaptobrevin, synaptophysin, and
synaptotagmin (Zhong et al., 1992; McMahon et al.,
1993; Li et al., 1995). The restricted expression of synapsins
I and II in the nervous system establishes the synapsin genes as good
candidates for an investigation of neuron-specific gene expression. The
rat and human synapsin I genes have been analyzed, and it was shown
that the 5`-flanking region is sufficient for neuron-specific
expression (Sauerwald et al., 1990; Thiel et al.,
1991). The synapsin I gene promoter contains a sequence motif similar
to the neuron-restrictive silencer element/repressor element-1
(NRSE/RE-1) ()of the SCG10 and type II sodium channel gene,
respectively (Kraner et al., 1992; Mori et al.,
1992). This element was suggested to function as a binding site for a
protein that is expressed only in non-neuronal cells. This
NRSE/RE-1-binding protein was proposed to shut down the activity of a
constitutive enhancer upon binding to the NRSE/RE-1 sequence. A zinc
finger protein termed neuron-restrictive silencer factor (NRSF)/RE-1
silencing transcription factor (REST) was recently discovered that
binds to this motif and functions as a transcriptional repressor (Chong et al., 1995; Schoenherr and Anderson, 1995). A homologous
motif present in the synapsin I promoter was shown to function in
silencing synapsin I gene expression in non-neuronal cells (Li et
al., 1993). However, because deletion or mutation of this motif
still showed preferential expression of synapsin I promoter-reporter
genes in neuronal cells relative to non-neuronal cells, it was
suggested that an additional cis-acting element in the synapsin I
promoter is necessary for neuron-specific gene expression (Li et
al., 1993). To further investigate the mechanisms involved in the
neuron-specific expression of synapsin I, the proximal part of the
5`-flanking region was functionally analyzed. Here, we present data
showing that the negative regulatory mechanism via the NRSE/RE-1
sequence is soley responsible for restricting the expression of
synapsin I to neuronal cells.
Figure 1: Comparison of the nucleotide sequence of the 5`-flanking region of the human and mouse synapsin I gene. Upper panel, the partial nucleotide sequence of the mouse genomic clone pSyC-1-9a encompassing the proximal region of the synapsin I promoter is depicted together with the human counterpart (Südhof, 1990). The initiator element containing the transcriptional start site is marked by an arrow. Consensus sites for the transcription factors CREB (CRE), zif268/egr-1, and NRSF/REST (NRSE) are boxed. Lower panel, dot blot comparison of the mouse and human synapsin I promoter, performed with the program ``seqapp alpha'' with a window size of 21 and a stringency level of 14. The results are displayed with the program ``Dotplot''.
Figure 2:
The proximal region of the synapsin I
promoter directs neuron-specific gene expression. A, reporter
plasmids. Fragments of the proximal region of the human synapsin I
promoter as well as the enhancer of the RSV are inserted upstream of
the TATA box of the reporter plasmid OVEC. B, reporter
plasmids and the ICP0ref internal standard plasmid were introduced into
NG108-15 and CHO-K1 cells. Cytoplasmic RNA was measured by RNase
protection assay. The bands labeled test indicate correctly
initiated -globin transcripts, and the bands labeled ref were generated by the internal standard plasmid ICP0ref. 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 lane M.
Figure 3:
The sequence homologous to the NRSE/RE-1
is necessary and sufficient for directing neuron-specific expression of
synapsin I. A, sequence of the NRSE/RE-1 derived from the
SCG10 and sodium channel genes and homologous sequences from the genes
encoding synapsin I and the Na,K-ATPase 3 subunit, respectively. B, reporter plasmids containing (plasmid B) or lacking
(plasmid B
) the NRSE/RE-1 homologous sequence of the synapsin I
promoter. C, RNase protection mapping of
-globin mRNA
isolated from transfected NG108-15, NS20Y, HepG2, CHO-K1, and
HeLa cells. The data are presented in the same manner as in Fig. 2.
Figure 4:
The synapsin I promoter region from
-199 to -142 functions as a weak constitutive enhancer
element. A, reporter plasmids B, D, E, F, and G containing
progressive 5` deletions of the synapsin I promoter region. The
location of the NRSE/RE-1 is indicated. B, RNase protection
mapping of -globin mRNA isolated from transfected NG108-15,
NS20Y, CHO-K1, and HepG2 cells. The data are presented in the same
manner as in Fig. 2.
Plasmids B and D contain the
synapsin I promoter sequences from -422 to -22 and
-234 to -22, respectively, including the motif homologous
to the NRSE/RE-1 sequence. As expected, transcription of the
-globin gene was restricted to the neuronal cell lines NS20Y and
NG108-15. However, transcriptional induction directed from the
longer promoter fragment in plasmid B was much stronger than that from
the shorter one in plasmid D, indicating that the promoter region from
-422 to -235 contributes to the transcription of the
synapsin I gene. From these data, we conclude that neuron-specific gene
expression is mediated solely through negative regulation by the
homologous NRSE/RE-1 sequence and that constitutive cis-acting elements
are present within a bipartite enhancer structure consisting of
synapsin I promoter region from nucleotides -199 to -143
and from nucleotides -422 to -235.
Figure 5:
The synapsin I promoter region from
-422 to -235 does not function as either tissue-specific or
constitutive enhancer element on its own. A, reporter plasmids
B and C containing synapsin I promoter sequences from -422 to
-22 and from -422 to -235, respectively. B,
RNase protection mapping of -globin mRNA isolated from transfected
NG108-15, NS20Y, CHO-K1, and HepG2 cells. The data are presented
in the same manner as in Fig. 2.
Figure 6:
A common protein binds to a purine-rich
sequence in the regulatory region of the genes encoding synapsin I,
GABA receptor
subunit, Na,K-ATPase
3 subunit,
and the heavy neurofilament. A, sequence of the purine-rich
motif present in the upstream regions of the synapsin I
(-283/-267), GABA
receptor (Motejlek et
al., 1994), Na,K-ATPase
3 subunit (-143/-124,
Pathak et al., 1994), and heavy neurofilament gene
(-107/-70, Schwartz et al., 1994). B,
electrophoretic mobility shift assay using nuclear extracts derived
from NS20Y, NG108-15, and NIH3T3 cells. The radiolabeled probe
was derived from the human synapsin I promoter (sequence -283 to
-267). A binding experiment without extract is depicted in lane 1. Competitor DNA was added to the reaction at 100-fold (lanes 3, 4, 6, 7, 9, and 10) molar excess to the probe. Synthetic oligonucleotides
containing the CRE of the secretogranin II promoter (denoted
``CRE'') were used as an unrelated competitor (lanes
4, 7, and 10). The arrowheads indicate
the DNA-protein complexes 1, 2, and 3 consisting of nuclear proteins
bound to the purine-rich motif of the synapsin I promoter. C,
gel retardation assay with nuclear proteins of mouse brain and
radiolabled DNA probes originating from the synapsin I, GABA
receptor
subunit, Na,K-ATPase
3 subunit, and heavy
neurofilament gene promoters. All probes generated a DNA-protein
complex with a similar mobility than complex 3 in B.
Gene expression in neurons was analyzed using the synapsin I gene as a model. The 5`-flanking region of the human synapsin I gene was studied for cis-acting elements regulating transcription by transient transfection experiments. The results presented here show that the proximal region of the synapsin I promoter, i.e. the sequence from -422 to -22, is necessary and sufficient for neuron-specific gene expression. The importance of this region is supported by a sequence comparison between the human and mouse promoter, indicating that this region has been highly conserved in evolution.
Deletion mutagenesis of the proximal synapsin I promoter revealed that a sequence element homologous to the NRSE/RE-1 of the SCG10 and type II sodium channel genes is critical for the neuron-specific expression of synapsin I. Strikingly, a deletion of this element resulted in transcription of a reporter gene in non-neuronal cells at a level similar to that in neuronal cells. A previous report noted that this element represses synapsin I transcription in non-neuronal cells (Li et al., 1993). However, although this element was deleted or mutated, the synapsin I promoter-reporter genes were still preferentially expressed in neuronal cells, leading to the suggestion that the NRSE/RE-1 on its own was not fully responsible for the neuron-specific expression of synapsin I and that an additional element was necessary (Li et al., 1993). These data can be explained by the use of PC12 cells as the sole neuronal model system in these experiments. We have found that in contrast with other neuronal cell lines tested, PC12 cells give an exceptionally high level of transcription following transfection of promoter-reporter gene constructs, thus leading to an overestimation of transcription in these cells (Petersohn et al., 1995). Our conclusion that the NRSE/RE-1 is in fact the sole cis-acting element for directing neuron-specific expression of synapsin I is based upon the observations that 1) a deletion of this motif in a synapsin I promoter-reporter gene abolished tissue-specific transcription of the reporter and 2) no other sequence motif of the proximal synapsin I promoter region could confer neuron-specific expression to a reporter gene.
The involvement of the NRSE/RE-1 in regulating neuron-specific
gene expression has been demonstrated for the genes encoding SCG10,
type II sodium channel, and Na,K-ATPase 3 subunit (Kraner et
al., 1992; Mori et al., 1992; Pathak et al.,
1994). The gene products are members of multigene families where some
of these members are expressed in non-neuronal cells. Thus, it has been
postulated that a negative regulatory mechanism via the NRSE/RE-1 fits
very well for the regulation of those gene families where constitutive
enhancer elements are shared between single family members and the
unique, neuron-specific expression of a particular gene is accomplished
by repression in non-neuronal cells (Hoyle et al., 1994). Our
results obtained in the analysis of synapsin I gene expression indicate
that the group of genes regulated by a negative regulatory mechanism
via the NRSE/RE-1 sequence has to be extended to bona fide neuronal
genes. In addition, consensus NRSEs have been discovered in the genes
encoding subunits of the glycine, acetylcholine, and N-methyl-D-aspartate receptors as well as in the
neurofilament and neuron-specific tubulin genes, suggesting a role for
the NRSE/RE-1 sequence as master negative regulatory element of
neurogenesis (Schoenherr and Anderson, 1995). Future work will show
whether the active regulation of the differentiated state of neurons is
based to a large extent upon this negative regulatory mechanism.
Recently a protein termed NRSF/REST has been described that fulfills
the expected properties of a NRSE/RE-1-binding protein, i.e. expression in non-neuronal cells and transcriptional repressor
activity upon sequence-specific binding (Chong et al., 1995;
Schoenherr and Anderson, 1995). The availability of the NRSF/REST cDNA
as a tool will certainly help clarify which genes are regulated by this
protein in their tissue-specific expression. In addition, it will be of
interest whether the same protein regulates the SCG10, type II sodium
channel, the Na,K-ATPase
3 subunit, and synapsin I genes or if
there are homologous members of a larger gene family of repressors
displaying different specificities.
The NRSF/REST protein represses the activity of constitutive enhancers (Kraner et al., 1992; Mori et al., 1992). One cis-acting element mediating transcriptional induction in neuronal and non-neuronal cells was mapped between -199 and -142 of the synapsin I promoter consisting most likely of the CRE, that was proposed to function in the synapsin I gene as a basal transcriptional element and not as a cAMP-inducible enhancer (Jüngling et al., 1994). In addition, the synapsin I promoter region from -422 to -235 was shown to participate in regulating synapsin I gene expression. However, this region could not serve as a cis-acting element on its own in transfection experiments but rather required the presence of downstream element, most likely the CRE, for its function. This is in agreement with other studies showing that enhancers and upstream promoter elements are typically composed of various sequence modules that provide binding sites for transcription factors. Binding of those factors results in a synergistic activation of transcription, i.e. the intact enhancer displays a distinct activity in comparison with the individual elements (reviewed in Müller et al., 1988; Tjian and Maniatis, 1994). In conclusion, we find that the homologous NRSE/RE-1 sequence is the sole regulatory element necessary and sufficient for neuron-specific expression of the synapsin I gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X91238[GenBank].