Neuron-specific Gene Expression of Synapsin I

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 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. INTRODUCTIONThe 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 Ca2+/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) ( 1The abbreviations used are: NRSEneuron-restrictive silencer elementCREcyclic AMP response elementNRSFneuron-restrictive silencer factorRE-1repressor element 1RESTRE-1 silencing transcription factorRSVRous sarcoma virusGABAAγ-aminobutyric acid ACHOChinese hamster ovary.) 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.EXPERIMENTAL PROCEDURESDNA CloningA genomic clone termed pSyC-1-9a (Rosahl et al., 1993) containing the mouse synapsin I promoter region was kindly provided for us by Thomas Südhof, University of Texas, Dallas.Reporter ConstructsPlasmids OVEC and ICPOref have been described (Westin et al., 1987; Thiel et al., 1994). Plasmids A, B, C, D, E, F, and G containing synapsin I promoter-β-globin reporter genes were generated by inserting the following synapsin I promoter fragments into the SalI site or the SalI-Ecl136II sites of OVEC: plasmid A, −2356 to −22; plasmid B, −422 to −22; plasmid C, −422 to −235; plasmid D, −234 to −22; plasmid E, −199 to −22; plasmid F, −142 to −22; and plasmid G, −115 to −22. The plasmid BΔ contains a deletion of synapsin I promoter sequences from −234 to −200, generated with the restriction enzymes AluI and BstNI. The plasmid pRSVOVEC-1 was constructed by inserting a Bsu36I-EcoRI fragment of the RSV long terminal repeat (sequence from −273 to −49) into the filled in SalI site of OVEC.Electrophoretic Mobility Shift AssayBinding assays were performed as described (Thiel et al., 1994). To generate the probes, the following oligonucleotides were annealed: human synapsin I promoter (−283/-267), 5′-TCGAGAGAGGGGGAGGGGAAAG-3′ and 5′-TCGACTTTCCCCTCCCCCTCTC-3′; consensus sequence of six purine-rich motifs present in the rat GABAA receptor δ subunit gene promoter, 5′- TCGAGAGAGGAGAGGGGAGAGGGGAGAG-3′ and 5′-TCGACTCTCCCCTCTCCCCTCTCCTCTC-3′ (Motejlek et al., 1994); rat Na,K-ATPase α3 subunit gene promoter (−143/-124), 5′-TCGAGGTGAAGGGGGAAGGGGGAG-3′ and 5′-TCGACTCCCCCTTCCCCCTTCACC-3′ (Pathak et al., 1994); mouse neurofilament heavy gene promoter (−107/-70), 5′-TCGAGGGGGAGGAGGTGGAGAGGGTGGGGCCCTCCTCCCCAG-3′ and 5′-TCGACTGGGGAGGAGGGCCCCACCCTCTCCACCTCCTCCCCC-3′ (Schwartz et al., 1994); mouse secretogranin II promoter (−74/-52), 5′-TCGAGAGCCGGTGACGTCAGCGTGGAAG-3′ and 5′-TCGACTTCCACGCTGACGTCACCGGCTC-3′ (Schimmel et al., 1992). All probes contained XhoI and SalI restriction sites for radiolabeling.Miscellaneous TechniquesChinese hamster ovary cells (CHO-K1, ATCC number CCL 61), HeLa cells (ATCC number CCL2), HepG2 cells (ATCC number HB 8065), the neuroblastoma/glioma fusion cell line NG108-15, and the neuroblastoma cell lines NS20Y and NS26 were cultured and transfected as described (Thiel et al., 1991, 1994). Nuclear extracts were prepared as described (Schreiber et al., 1989; Sierra, 1990).RESULTSComparison of the 5′-flanking Region of the Human and Mouse Synapsin I GeneThe sequences of the 5′-flanking regions of the human and mouse synapsin I genes are shown in the upper panel of Fig. 1. The proximal region of the synapsin I promoter sequence is highly conserved between mouse and human including the transcriptional initiation site and the consensus binding sites for the transcription factors CREB (CRE), zif268/egr-1, and NRSF/REST. A dot matrix analysis between the synapsin I promoter of human and mouse, depicted in the lower panel of Fig. 1, revealed that the homology is restricted mainly to the proximal 400 base pairs upstream of the transcriptional start site. We conclude that the proximal region of the synapsin I promoter contains functionally important regulatory motifs due to the high evolutionary conservation of these sequences.The Proximal Region of the Synapsin I Promoter Directs Neuron-specific Gene TranscriptionTo confirm that the proximal region of the synapsin I promoter contains neuron-specific and/or constitutive transcriptional elements, a fragment of the human synapsin I promoter from −422 to −22 was inserted into a β-globin expression vector (plasmid B). Fragments encompassing synapsin I promoter sequences from −2352 to −22 and from −115 to −22 were cloned into the same vector (plasmids A and G) and served as positive and negative controls, respectively, for neuron-specific gene transcription, as determined from previous studies (Thiel et al., 1991). Plasmid pRSVOVEC-1 containing the β-globin gene under control of the RSV long terminal repeat was used to measure the activity of a strong tissue-unspecific promoter. These plasmids, depicted in Fig. 2A, were transiently transfected into the neuronal cell line NG108-15 and the non-neuronal cell line CHO-K1 together with plasmid ICP0ref, a mutated β-globin gene under the control of the herpes simplex virus ICP0 gene promoter, to correct for variations in transfection efficiencies. 48 h post-transfection, cytoplasmic RNA of the transfected cells was isolated, hybridized to a β-globin derived cRNA probe, and analyzed by RNase protection mapping The synapsin I promoter regions present in A and expression of the reporter gene only in NG108-15 cells, indicating that the proximal region of the synapsin I promoter contains neuron-specific The synapsin I promoter region from −115 to −22 showed no transcriptional activity in cell that this region not on own a role in the regulation of transcription of the synapsin I The RSV enhancer was active in both cell These data show that the proximal region of the 5′-flanking region of the human synapsin I gene is sufficient for directing neuron-specific gene proximal region of the synapsin I promoter neuron-specific gene expression. A, reporter Fragments of the proximal region of the human synapsin I promoter as well as the enhancer of the RSV are inserted upstream of the of the reporter plasmid B, reporter and the plasmid were into NG108-15 and CHO-K1 cells. RNA was by RNase protection The β-globin and the were generated by the plasmid transcripts of the shown is an of cRNA and are shown in of the for NRSF/REST Neuron-specific Gene of a has been that a sequence motif homologous to the NRSE/RE-1 sequence present in the SCG10 and type II sodium channel gene a role in regulating neuron-specific expression of synapsin I. However, deletion or mutation of the homologous sequence still preferential expression of the synapsin I promoter-reporter genes in neuronal cells relative to non-neuronal cells. was suggested that the NRSE/RE-1 sequence was not responsible for the neuron-specific expression of synapsin I and that an additional cis-acting sequence was necessary for this regulation (Li et al., 1993). To there are cis-acting elements necessary and to confirm that the NRSE/RE-1 homologous sequence not neuron-specific a deletion from −234 to was into the synapsin I promoter sequence in plasmid B, thus plasmid BΔ This plasmid and plasmid were into the neuronal cell lines NG108-15 and NS20Y as well as in the non-neuronal cell lines and NG108-15 and NS20Y cells been shown to express synapsin I et al., 1995). Fig. that the region from −422 to −22 of the synapsin I promoter transcription of the β-globin gene in NS20Y and NG108-15 cells, no transcripts in and HeLa cells (plasmid B). A deletion of the putative NRSF/REST-binding site no upon transcription in the neuronal cells (plasmid and abolished neuron-specific expression of the reporter gene, allowing β-globin transcripts to in the non-neuronal cell lines and A of the of transcription by analysis to the transcripts revealed that as a of the NRSE/RE-1 transcription in HepG2 and HeLa cells was with that in NG108-15 and NS20Y cells. of the β-globin reporter gene in CHO-K1 cells was a 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 synapsin I and the Na,K-ATPase α3 B, reporter containing (plasmid or lacking (plasmid the NRSE/RE-1 homologous sequence of the synapsin I promoter. C, RNase protection mapping of β-globin mRNA from transfected NG108-15, and HeLa cells. The data are in the same as in Fig. Synapsin I Promoter Region from −199 to as a that the NRSE/RE-1 sequence is for neuron-specific gene expression two is the NRSE/RE-1 the only sequence motif directing neuron-specific gene expression of synapsin this is the a negative regulatory mechanism for the neuron-specific expression of synapsin I. the NRSE/RE-1-binding protein is suggested to shut down a constitutive enhancer in non-neuronal cells, constitutive enhancer elements have to in the proximal region of the synapsin I promoter. To both a deletion analysis of the synapsin I promoter region from −199 to −22 was performed B, D, E, F, and cis-acting element necessary for neuron-specific gene transcription been proposed in this region (Li et al., 1993). Deletion were into NG108-15, and HepG2 cells, and transcription was by mapping of the β-globin transcripts The synapsin I promoter region from −142 to −22 (plasmid not transcription of the reporter gene in both neuronal and non-neuronal cells, indicating that this region on In plasmid that contained the promoter region from −199 to −22 was active in all cell that constitutive cis-acting elements are between −199 and −142 of the synapsin I promoter. of the reporter gene, by synapsin I promoter sequences from −199 to was lower that for synapsin I promoter sequences from −422 to −22 the generated by and This that the region from −199 to is not responsible for constitutive transcription of the synapsin I synapsin I promoter region from −199 to −142 functions as a constitutive enhancer element. A, reporter B, D, E, F, and G containing of the synapsin I promoter The of the NRSE/RE-1 is B, RNase protection mapping of β-globin mRNA from transfected NG108-15, and HepG2 cells. The data are in the same as in Fig. and the synapsin I promoter sequences from −422 to −22 and −234 to respectively, including the motif homologous to the NRSE/RE-1 sequence. transcription of the β-globin gene was restricted to the neuronal cell lines NS20Y and However, transcriptional from the promoter fragment in plasmid was that from the in plasmid D, indicating that the promoter region from −422 to to the transcription of the synapsin I From these we conclude that neuron-specific gene expression is negative regulation by the homologous NRSE/RE-1 sequence and that constitutive cis-acting elements are present a bipartite enhancer consisting of synapsin I promoter region from −199 to and from −422 to Synapsin I Promoter Region from −422 to as or on the synapsin I promoter region from −422 to contains cis-acting elements for transcription, were into NG108-15, and HepG2 cells that contained synapsin I promoter sequences from −422 to −22 and −422 to respectively (plasmids and C, Fig. the synapsin I promoter region from −422 to not show upon transcription on as depicted in Fig. We that this sequence synapsin I promoter sequences downstream from the NRSE/RE-1 site to a upon synapsin I promoter region from −422 to not function as tissue-specific or constitutive enhancer element on A, reporter and containing synapsin I promoter sequences from −422 to −22 and from −422 to B, RNase protection mapping of β-globin mRNA from transfected NG108-15, and HepG2 cells. The data are in the same as in Fig. to the Synapsin I Promoter Region from −422 to synapsin I promoter region from −422 to was analyzed by binding assays in to identify binding sites of transcription This analysis discovered a purine-rich sequence motif this region that is present in the promoter regions of the genes the GABAA receptor δ the Na,K-ATPase α3 and the heavy neurofilament In addition, this region was as a cis-acting element for neuron-specific gene expression (Motejlek et al., et al., et al., 1994). assays were performed with oligonucleotides to the synapsin I promoter region from to and extracts prepared from NG108-15, and cells. major with 1, and in Fig. were with extracts prepared from the neuronal cells as well as the non-neuronal cell line and that the proteins responsible for these are not The were by of a of and In an to with these and To investigate similar proteins bind to the synapsin I promoter and the of the GABAA receptor δ the Na,K-ATPase α3 and the heavy neurofilament gene, oligonucleotides were and Fig. the with of these probes extracts from This that the responsible for the to all four probes, with different protein binds to a purine-rich sequence in the regulatory region of the genes synapsin I, GABAA receptor δ Na,K-ATPase α3 and the heavy A, sequence of the purine-rich motif present in the upstream regions of the synapsin I (−283/-267), GABAA receptor (Motejlek et al., Na,K-ATPase α3 subunit et al., and heavy neurofilament gene et al., 1994). B, extracts derived from NG108-15, and cells. The was derived from the human synapsin I promoter (sequence to A binding is depicted in 1. was to the and to the oligonucleotides containing the of the secretogranin II promoter were used as an and The the 1, and consisting of proteins to the purine-rich motif of the synapsin I promoter. C, with proteins of mouse and probes from the synapsin I, GABAA receptor δ Na,K-ATPase α3 and heavy neurofilament gene All probes generated a with a similar in expression in neurons was analyzed the synapsin I gene as a The 5′-flanking region of the human synapsin I gene was for cis-acting elements regulating transcription by transfection The show that the proximal region of the synapsin I promoter, the sequence from −422 to is necessary and sufficient for neuron-specific gene expression. The of this region is by a sequence between the human and mouse promoter, indicating that this region has been highly conserved in 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 for the neuron-specific expression of synapsin I. a deletion of this element in transcription of a reporter gene in non-neuronal cells a similar to that in neuronal cells. A previous that this element synapsin I transcription in non-neuronal cells (Li et al., 1993). However, this element was or the synapsin I promoter-reporter genes were still expressed in neuronal cells, to the that the NRSE/RE-1 on own was not responsible for the neuron-specific expression of synapsin I and that an additional element was necessary (Li et al., 1993). These data by the of cells as the neuronal system in these We have that in contrast with neuronal cell lines cells an high of transcription following transfection of promoter-reporter gene thus to an of transcription in these cells et al., 1995). that the NRSE/RE-1 is in the cis-acting element for directing neuron-specific expression of synapsin I is upon the that a deletion of this motif in a synapsin I promoter-reporter gene abolished tissue-specific transcription of the reporter and no sequence motif of the proximal synapsin I promoter region neuron-specific expression to a reporter of the NRSE/RE-1 in regulating neuron-specific gene expression has been for the genes type II sodium and Na,K-ATPase α3 subunit (Kraner et al., 1992; Mori et al., 1992; et al., 1994). The gene are of of these are expressed in non-neuronal cells. it has been postulated that a negative regulatory mechanism via the NRSE/RE-1 well for the regulation of gene constitutive enhancer elements are between family and the neuron-specific expression of a gene is accomplished by in non-neuronal cells et al., 1994). in the analysis of synapsin I gene expression that the of genes by a negative regulatory mechanism via the NRSE/RE-1 sequence has to to neuronal In addition, consensus have been discovered in the genes of the and as well as in the neurofilament and neuron-specific a role for the NRSE/RE-1 sequence as negative regulatory element of and Anderson, 1995). show the active regulation of the of neurons is to a upon this negative regulatory a protein termed NRSF/REST has been described that the of a NRSE/RE-1-binding protein, expression in non-neuronal cells and transcriptional repressor activity upon binding (Chong et al., 1995; Schoenherr and Anderson, 1995). The availability of the NRSF/REST as a genes are by this protein in their tissue-specific expression. In addition, it of the same protein the type II sodium the Na,K-ATPase α3 and synapsin I genes or there are homologous of a gene family of different NRSF/REST protein the activity of constitutive (Kraner et al., 1992; Mori et al., 1992). cis-acting element transcriptional in neuronal and non-neuronal cells was between −199 and −142 of the synapsin I promoter consisting of the that was proposed to function in the synapsin I gene as a transcriptional element and not as a enhancer et al., 1994). In addition, the synapsin I promoter region from −422 to was shown to in regulating synapsin I gene expression. However, this region not as a cis-acting element on own in transfection the of downstream the for This is in with studies showing that and upstream promoter elements are of sequence that binding sites for transcription of factors in a of transcription, the enhancer a activity in with the elements in et al., and 1994). In we that the homologous NRSE/RE-1 sequence is the regulatory element necessary and sufficient for neuron-specific expression of the synapsin I INTRODUCTIONThe 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 Ca2+/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) ( 1The abbreviations used are: NRSEneuron-restrictive silencer elementCREcyclic AMP response elementNRSFneuron-restrictive silencer factorRE-1repressor element 1RESTRE-1 silencing transcription factorRSVRous sarcoma virusGABAAγ-aminobutyric acid ACHOChinese hamster ovary.) 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.