Báo cáo khoa học: RE-1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence-specific DNA-binding site Michael Lietz, Mathias Hohl and Gerald Thiel Department of Medical Biochemistry and Molecular

doi:10.1046/j.1432-1033.2003.03360.x

Eur. J. Biochem. 270, 2–9 (2003) (cid:1) FEBS 2003

RE-1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence-specific DNA-binding site

Michael Lietz, Mathias Hohl and Gerald Thiel

Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, Homburg, Germany

transcription. Furthermore, REST has been shown to be the major regulator of neuronal expression of synapsin I. Here, we have identified a functional binding site for REST in the first intron of the human synaptophysin gene indicating that REST blocks human synaptophysin gene transcription through an intronic neuron-specific silencer element. The synaptophysin promoter is, however, devoid of neuron- specific genetic elements and directs transcription in both neuronal and non-neuronal cells. Using a dominant-negat- ive approach we have identified the transcription factor Sp1 as one of the regulators responsible for constitutive tran- scription of the human synaptophysin gene.

Keywords: neuronal genes; REST; Sp1; synapsin I; synapto- physin. Synaptophysin, one of the major proteins on synaptic vesi- cles, is ubiquitously expressed throughout the brain. Syn- aptophysin and synapsin I, another synaptic vesicle protein, are also expressed by retinoic acid-induced neuronally dif- ferentiated P19 teratocarcinoma cells. Here, we show that inhibition of histone deacetylase activity in P19 cells is suf- ficient to activate transcription of the synaptophysin and synapsin I genes, indicating that neuronal differentiation and impairment of histone deacetylases results in a similar gene expression pattern. The transcription factor REST, a repressor of neuronal genes in non-neuronal tissues, has been shown to function via recruitment of histone deacety- lases to the transcription unit, indicating that modulation of the chromatin structure via histone deacetylation is of major importance for REST function and neuron-specific gene

that synaptophysin and synaptogyrin I perform essential, redundant functions in synaptic plasticity [7]. Recently, a further role of synaptophysin in regulating activity-depend- ent synapse formation has been proposed [8].

Synaptophysin is ubiquitously expressed in neurons throughout the brain and also in neuroendocrine cells [9]. During neuronal development, synaptophysin expression is correlated with synaptogenesis [10] and synaptophysin has been widely used as marker for neurons and nerve terminal differentiation, due to its abundance and pan-neuronal expression.

Synaptophysin is a major integral membrane protein of small synaptic vesicles [1]. Synaptophysin forms a hetero- multimeric complex in the vesicle membrane, consisting of at least four synaptophysin molecules and the synaptic vesicle protein synaptobrevin [2]. Many functions have been attributed to synaptophysin in the past: synaptophysin has been proposed to form a channel/fusion pore [3], to function as a Ca2+ sensor in the synapse [4], and to be essential for neurotransmitter release [5]. Gene targeting experiments in transgenic mice revealed, however, that synaptophysin is not essential for neurotransmitter release. No difference between wild-type and mutant mice in synaptic transmission and short-term and long-term plasticity was observed [6] suggesting that other synaptic vesicle proteins may com- pensate for the lack of synaptophysin. Double knockout mice lacking synaptophysin and the structurally related synaptic vesicle protein synaptogyrin I showed a severe reduction in short-term and long-term plasticity, indicating

Here, we have analyzed the regulation of the human synaptophysin gene, in comparison with the regulation of the synapsin I gene. We show that expression of both genes is sensitive to histone deacetylase inhibition, indicating that chromatin structure governs synaptophysin and synapsin I gene transcription. Furthermore, two transcription factors, Sp1 and the RE-1 silencing transcription factor (REST) were identified that are responsible for either constitutive or neuron-specific transcription of the synaptophysin gene.

Experimental procedures

Reporter constructs

Correspondence to G. Thiel, Department of Medical Biochemistry and Molecular Biology, Building 44, University of Saarland Medical Center, D-66421 Homburg, Germany. Fax: + 49 6841 1626500, Tel.: + 49 6841 1622606, E-mail: bcgthi@uniklinik-saarland.de Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; NRSE, neural-restrictive silencer element; REST, RE-1 silencing transcription factor; Syp, synaptophysin; TSA, trichostatin A. (Received 8 August 2002, revised 6 November 2002, accepted 11 November 2002)

The reporter constructs are derivatives of pGL3-Basic and pGL3-Promoter (Promega). To construct plasmid pSyI-2309/+47luc we cut plasmid pSyCAT10 [11] with SalI and ligated the fragment into the XhoI site of pGL3-Basic. The genomic clone p10C-6 containing the promoter, exons I– III and introns I and II of the human synaptophysin gene

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and 0.5–1 lg of pRSVb internal standard plasmid. 293T cells were transfected as described previously [22] with 1 lg of luciferase reporter plasmid, 1–5 lg of GST expression vector and 0.2 lg of the internal standard plasmid pSV40lacZ. The titration experiments with NS20Y cells were performed with 1 lg of luciferase reporter plasmid, 1–5 lg of GST-expres- sion vector and 0.2 lg of the internal standard plasmid pSV40lacZ. P19 cells were grown as described previously [23]. For neuronal differentiation, cells were aggregated and treated with 0.5 lM all-trans-retinoid acid for 4 days. Cell aggregates were then lightly trypsinized, plated onto tissue culture plates and cultured for 5 days in the absence of retinoid acid, but in the presence of 5 lgÆmL)1 of cytosine b-D-arabinofuranoside (Sigma C6645). Trichostatin A (TSA) was purchased from Wako Chemicals GmbH (Neuss, Germany) and used at a concentration of 100 ngÆmL)1 dissolved in dimethylsulfoxide.

RNase protection mapping

[12], was a kind gift of T. Su¨ dhof, HHMI, Dallas, TX, USA. To construct a synaptophysin promoter/luciferase reporter gene, we first subcloned a KpnI fragment of the genomic clone p10C-6 into pGEM7 (Promega). This plasmid was modified by inserting the annealed oligonucleotides 5¢-CTAGCCGAGCCTCCCGCCCCCTGCATTGCTGG TCGACGCATG-3¢ and 5¢-CGTCGACCAGCAATGCA GGGGGCGGGAGGCTCGG-3¢ into the NheI and SphI sites. This plasmid was subsequently cut with XbaI, filled in with the Klenow fragment of Escherichia coli DNA poly- merase I, and recut with SalI. The fragment encompassing the 5¢-flanking region of the human synaptophysin gene (nucle- otides 29255–31636, accession no. U93305) was isolated and inserted into plasmid pGL3-Basic, generating plasmid pSyp- 2356/+27luc. Plasmid pSypIntronluc, containing nucleotides 3– 427 of the first intron of the human synaptophysin gene (nucleotides 28788–29216, accession no. U93305) upstream of the SV40 promoter, was constructed by inserting an RsaI fragment derived from plasmid p10C-6 into Ecl136II-cut pGL3-Promoter. Plasmids pSypNRSE2 SV40luc and pSyI- NRSE2SV40luc, containing two copies of the intronic NRSE (neural-restrictive silencer element) derived from the synapt- ophysin gene or two copies of the NRSE derived from the human synapsin I promoter 5¢ of the SV40 promoter, were generated by subcloning of the synthetic oligonucleotides 5¢-TCGAGTCCAGCACCGTGGACAGAG CCG-3¢ and 5¢-TCGACGGCTCTGTCCACGGTGCTG GAC-3¢ (syn- aptophysin gene) or 5¢-TCGAGCTTCAG CACCGCGG ACAGTGCCTTG-3¢ and 5¢-TCGACAA GGCACTGTC CGCGGTGCTGAAGC-3¢ (synapsin I gene) into the XhoI and SalI sites of plasmid pHIVTATA CAT [13]. The sequences were subsequently multimerized as described previously [13], excised with XhoI and SalI and cloned into the XhoI site of plasmid pGL3-Promoter.

Expression constructs

Cytoplasmic RNA of undifferentiated, differentiated, dimethylsulfoxide and TSA-treated P19 cells was prepared as described previously [23]. For RNase protection mapping, 20 lg RNA was used for the detection of synapsin I and synaptophysin mRNA, and 2.5 lg RNA for the detection of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and b-actin mRNA. The template for mouse synapsin I cRNA synthesis (plasmid pSP6-mSyI-2) has been described previously [23]. To synthesize a synaptophysin-specific riboprobe, a cDNA fragment encompassing nucleotides 1730–2011 of the mouse synaptophysin gene (accession no. X95818) was amplified by PCR using the primers 5¢- GGTTCTGGTCAGGATTGC-3¢ and 5¢-TCAGTAAGG GACATTTCG-3¢ and cloned into the SmaI site of pBlue- script to generate plasmid pT3mp38. Hybridization with synapsin I and synaptophysin mRNA protected fragments of 194 and 282 nucleotides, respectively, from RNase digestion. Plasmids SP6-b-actin and pTRI-GAPDH-Rat, used to synthesize b-actin and GAPDH-specific riboprobes, were purchased from Ambion. Hybridization with b-actin and GAPDH mRNA protected fragments of 250 and 316 nucleotides, respectively, from RNase digestion.

Reporter gene assays

The REST expression vector pCMVFLAG-REST is iden- tical to the previously described plasmid pCMVmycREST [14], except that the myc epitope has been exchanged for a triple FLAG epitope (sequence: MDYKDHDG DYKDHDLDYKDDDDK). The expression vector enco- ding a positive-dominant mutant of REST, FLAG-DP- REST, is identical to the previously described plasmid pCMVDP-REST [15,16], except that the hemagglutinin epitope has been exchanged for a triple FLAG epitope. The expression vector encoding myc-tagged REST4 [16] and mammalian glutathione S-transferase (GST) encoding expression vectors pEBGN and pEBGN-Sp1 [17,18] have been described previously. Plasmids pRSVb and pSV40lacZ encodes for b-galactosidase of E. coli [19,20]. Cell lysates of transfected cells were prepared 48 h post- transfection using the cell culture lysis buffer (Promega), and b-galactosidase and luciferase activities were determined as described [20,21]. Each experiment included four separate transfections for each experimental setting, and the experi- ments were repeated at least twice giving consistent results.

Cell culture and transfections Expression and purification of recombinant GST fusion proteins

The murine neuroblastoma cell line NS20Y, the immortal- ized septal cell line SN56, the murine teratocarcinoma cell line P19 and human 293T cells were maintained as described previously [11,19,21,22]. NS20Y cells were transfected using the calcium phosphate coprecipitation method with 0.5–2 lg of luciferase reporter plasmid, 100 ng of expression vector encoding FLAG-REST, FLAG-DP-REST or myc-REST4, 293T cells were transfected with plasmids pEBGN or pEBGN-Sp1, encoding a nuclear targeted GST or a GST- Sp1 fusion protein. Forty-eight hours post-transfection, cells were harvested, washed with NaCl/Pi and lyzed in RIPA buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% NP-40, 0.5% deoxycholate, 0.1% SDS) for

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30 min at 4 (cid:3)C and centrifuged for 10 min. The supernatant was incubated with 50 lL glutathione-agaraose beads (Pharmacia Biotech) for 30 min. The glutathione-agarose- GST protein complexes were isolated by centrifugation, washed twice with RIPA buffer and dissolved in 100 lM of SDS-stop solution (125 mM Tris/HCl, pH 6.8, 3 mM EDTA, 20% glycerol, 9% SDS, 0.05% bromophenol blue).

Results

Induction of neuronal gene transcription in P19 teratocarcinoma cells by retinoid acid or TSA

in non-neuronal cells. REST contains two (cid:2)active(cid:3) transcrip- tional repression domains on its N- and C-termini. The N-terminal repression domain of REST recruits histone deacetylases to the target genes of REST [26,27]. Likewise, repression mediated by this domain was shown to be sensitive to inhibitors of histone deacetylases such as TSA. Moreover, there are indications that the C-terminal repression domain also functions via recruitment of histone deacetylases [28]. Histone deacetylation generates a compact chromatin struc- ture that is not as accessible to the transcriptional machinery. Thus, alterations of the chromatin structure are essential for transcriptional repression via REST. Therefore, we tested whether an inhibition of histone deacetylases in P19 cells is sufficient to induce neuronal gene transcription. P19 cells were treated for 24 h with TSA and cytoplasmic RNA was prepared and analyzed by RNase protection mapping. Figure 1B shows that inhibition of histone deacetylases by TSA was sufficient to induce synapsin I gene transcription. Moreover, TSA treatment also induced transcription of the synaptophysin gene, indicating that expression of both genes is controlled by alterations of the chromatin structure.

The human synaptophysin promoter is devoid of neuron-specific genetic elements

The P19 teratocarcinoma cell line is frequently used as an in vitro model system for neuronal differentiation, neurite outgrowth and neuronal gene expression. We differentiated P19 cells on bacterial plates in the presence of retinoid acid. Four days later, the cell aggregates were lightly trypsinized and plated onto tissue culture plates for 5 days in the presence of cytosine b-D-arabinofuranoside to suppress growth of non-neuronal cells. Cytoplasmic RNA was prepared and analyzed by RNase protection mapping using specific riboprobes for the detection of synapsin I, synapto- physin, b-actin and GAPDH mRNA, respectively. Synap- sin I and synaptophysin are synaptic vesicle proteins and serve as marker proteins for neuronal differentiation. b-Actin and GAPDH are constitutively expressed in P19 cells. Figure 1A shows that neuronal differentiation of P19 cells induced the expression of the synapsin I and synaptophysin genes, confirming previous results [23,24]. Neuronal expres- sion of the synapsin I gene has been shown to be controlled by REST [25], a transcriptional repressor of neuronal genes

The synapsin I promoter contains a neuron-specific control element, the REST binding site termed neuron-restrictive silencer element (NRSE). Transfection of a human synap- sin I promoter/luciferase reporter gene depicted in Fig. 2A in neuronal and non-neuronal cells revealed that the promoter mainly directs luciferase expression in the neur- onal cell lines NS20Y and SN56, but not in the human embryonic kidney cell line 293T (Fig. 2B, upper panel). Both NS20Y and SN56 cells have been shown by RNase protection mapping to express synapsin I and synaptophy- sin (data not shown). A human synaptophysin promoter/ luciferase reporter gene, however, showed constitutive transcriptional activity in NS20Y, SN56 and 293T cells, indicating that neuron-specific expression of synaptophysin is not regulated by genetic elements located in the 5¢-flanking region of the synaptophysin gene.

Identification of a REST consensus binding site within the first intron of the human synaptophysin gene

A data base search revealed the presence of an NRSE in the first intron of the synaptophysin gene of the rat [29]. We analyzed the database of the human genome and found an NRSE in the first intron of the synaptophysin gene at an identical position as in synaptophysin gene of the rat (Fig. 3A). A comparison of the sequence revealed that the the synaptophysin gene is 100% intronic NRSE of conserved between the rat and human gene and has only three mismatches in comparison to the NRSE found in the human synapsin I promoter (Fig. 3B).

Fig. 1. Activation of synapsin I and synaptophysin gene transcription in P19 teratocarcinoma cells. (A) P19 teratocarcinoma cells were differ- entiated via aggregation and treatment with retinoic acid for 4 days, than plated and cultured for a further 5 days in the presence of cytosine b-D-arabinofuranoside. (B) P19 cells were treated for 24 h with the histone deactylase inhibitor TSA or with the vehicle dimethylsulfoxide. Cytoplasmic RNA from undifferentiated (A, denoted (cid:2)–(cid:3)), neuronally differentiated (A, denoted (cid:2)+(cid:3)), dimethylsulfoxide-treated (B, denoted (cid:2)–(cid:3)) and TSA-treated (B, denoted (cid:2)+(cid:3)) P19 cells were isolated and analyzed by RNase protection mapping using cRNAs specific for synapsin I, synaptophysin, b-actin and GAPDH, respectively.

The intronic REST binding site confers REST regulation to reporter genes

REST has been shown to repress transcription despite the location or orientation of its binding site within a gene [14].

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Fig. 3. Localization of an NRSE in the human synaptophysin gene. (A) Schematic representation of part of the human synaptophysin gene containing the promoter region, exons I to III and introns I and II. The location of the NRSE within the first intron of the gene is indicated. The accession number for the human synaptophysin gene is U93305. The NRSE encompassed nucleotides 29190–29210. (B) Sequence of the NRSEs derived from the human synapsin I gene and the human and rat synaptophysin genes.

Fig. 2. Activity of the human synapsin I and synaptophysin promoter in neuronal and non-neuronal cells. (A) Schematic representation of the synapsin I promoter/luciferase and synaptophysin promoter/luciferase reporter genes pSyI-2309/+47luc and pSyp-2356/+27luc. (B) One of the reporter plasmids pSyI-2309/+47luc or pSyp-2356/+27luc, was transfected into NS20Y, SN56 (1 lg per plate) or 293T cells (0.5 lg per plate) together with the reference plasmid pRSVb (NS20Y, SN56 cells: 0.5 lg per plate; 293T cells: 0.25 lg per plate) that encoded b-galac- tosidase under control of the Rous sarcoma virus long-terminal repeat. Forty-eight hours post-transfection cell extracts were prepared and the b-galactosidase and luciferase activities of these extracts determined. The data are presented as the ratio of luciferase activity (light units) to b-galactosidase units (A units) measured in the cell extracts. At least two experiments in quadruplicate were performed and the mean ± SEM is depicted.

DNA-binding domain [16]. REST4 binds only weakly to the NRSE, due to the lack of zinc finger 7 that is important for DNA binding [30]. REST4 was used as a negative control in the experiment. Transient tranfections were performed with NS20Y neuroblastoma cells. One of the reporter plasmids, pSypIntronluc or pGL3-Promoter, was transfected into NS20Y cells together with plasmid pRSVb, encoding b-galactosidase under the control of the Rous sarcoma virus long-terminal repeat, to correct for variations in transfection efficiencies. In addition, the (cid:2)empty(cid:3) expres- sion vector pCMV5 (control) or expression vectors enco- ding FLAG-REST, FLAG-DP-REST or myc-REST4 were transfected. Forty-eight hours post-transfection, cells were harvested, cell extracts prepared and the relative luciferase activities determined. The results show that FLAG-REST repressed transcription of the transcription unit containing part of the first intron of the human synaptophysin gene (plasmid pSypIntronluc). Likewise, expression of FLAG-DP- REST increased reporter gene expression over the level already obtained by the strong SV40 promoter (Fig. 4C, left panel). In contrast, myc-REST4 did not show any tran- scriptional activity as expected from previous experiments [16]. Naturally, the SV40 promoter was not regulated by either FLAG-REST, FLAG-DP-REST or myc-REST4 (Fig. 4C, right panel). These data indicate that the NRSE derived from the human synaptophysin gene is biological active and functions as a REST-regulated silencer.

The REST binding sites derived from the human synaptophysin and synapsin I gene are functionally indistinguisable

Thus, a functional NRSE of the synaptophysin gene should operate from any position within the gene. We therefore generated the reporter plasmid, pSypIntronluc, consisting of the luciferase open reading frame and the SV40 promoter. To the 5¢ of the SV40 promoter, we inserted 424 nucleotides from the first intron of the human synaptophysin gene including the NRSE. As a control, we used plasmid pGL3- Promoter, containing the luciferase gene under control of the SV40 promoter (Fig. 4A). As expression vectors, we transfected plasmids encoding either a FLAG-tagged REST, a FLAG-tagged DP-REST or a myc-tagged REST4. The modular structure of FLAG-DP-REST and myc-REST4, is depicted in in comparison to REST, Fig. 4B. DP-REST contains the DNA-binding domain of REST fused to the activation domain of the herpes simplex virus protein VP16. Following binding to the REST cognate site, DP-REST strongly activates transcription, due to the presence of a transcriptional activation domain [15,16]. REST4 contains the N-terminal repression domain of REST and five of the eight zinc fingers that constitutes the To compare the biological activity of the NRSEs derived from the synapsin I promoter and the first intron of the human synaptophysin gene, we used model promoters containing the luciferase gene as reporter, the SV40 promoter, and two copies of the NRSE derived from the synapsin I and synaptophysin gene, respectively (reporter

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Fig. 4. REST regulates the transcription activity of a strong viral pro- moter via the intronic NRSE derived from the human synaptophysin gene. (A) Reporter plasmid pSypIntronluc and pGL3-Promoter containing the luciferase reporter gene and the SV40 promoter. Plasmid pSypIntronluc contains a fragment of the first intron of the human synaptophysin gene, including the NRSE, 5¢ of the SV40 promoter. (B) Modular structure of FLAG-REST, FLAG-DP-REST, and myc-REST4. REST contains a cluster of eight zinc fingers that function as DNA-binding domain, and two repressor domains on the N- and C-termini of the molecule. FLAG-DP-REST, a positive-dominant mutant of REST, retains the DNA-binding domain, but lacks the repression domains and has instead a transcriptional activation domain derived from the VP16 protein of herpex simplex virus. REST4 is a neuron-specific splice variant of REST that contains the N-terminal repression domain and five of the eight zinc finger motifs of the DNA-binding domain. In addition, FLAG-REST, FLAG-DP-REST, and myc-REST4 contain recognition sequences (triple FLAG tag or myc-tag) on the N-termini. (C) One of the reporter plasmids pSypIntronluc or pGL3-Promoter (1 lg per plate), 0.5 lg per plate of the pRSVb internal standard plasmid and either 100 ng per plate of the (cid:2)empty(cid:3) expression vector pCMV5 or one of the expression vectors encoding FLAG-REST, FLAG-DP-REST, or myc-REST4 were introduced into NS20Y cells. Transcription was analyzed by determination of the b-galactosidase and luciferase activ- ities of the cell extracts.

The zinc finger transcription factor Sp1 is responsible for constitutive transcription via the human synaptophysin promoter

the reporter plasmids,

plasmids pSyINRSE2SV40luc and pSypNRSE2SV40luc, Fig. 5A). One of the internal reference plasmid pRSVb and expression vectors encoding FLAG-REST, FLAG-DP-REST or myc-REST4 were transfected into NS20Y neuroblastoma cells. Forty-eight hours post-transfection, cells were harvested, cell extracts prepared and the relative luciferase activities determined. The results show that the presence of a REST binding site, either from the synapsin I or synaptophysin gene, together with expression of FLAG-REST, caused a striking decrease in transcription (Fig. 5B, upper panels). Likewise, expres- sion of FLAG-DP-REST increased reporter gene transcrip- tion significantly (Fig. 5B, middle panels). The splice variant of REST, REST4, however, did not activate or repress transcription of NRSE-containing reporter genes (Fig. 5B, lower panels), confirming previous results [16]. Taken together, no major differences were detected between the NRSE derived from the synapsin I or synaptophysin gene, indicating that the NRSE functions in both genes as a neuron-restrictive silencer element. The presence of a functional NRSE in the first intron of the human synaptophysin gene indicates that REST blocks human synaptophysin gene transcription through this intronic neuron-specific silencer element. In contrast, no neuron-specific genetic elements were found in the synapto- physin 5¢-flanking region. Rather, this part of the transcrip- tion unit contained constitutive transcriptional elements that are active in neuronal as well as in non-neuronal cells. The 5¢-flanking region of the human synaptophysin gene is GC-rich, and potential binding sites for the transcription factor Sp1 have been proposed [31]. To test whether Sp1 is responsible for the constitutive transcriptional activity of the human synaptophysin promoter, we used a dominant- negative Sp1 mutant [17], consisting of the GST fused to the DNA-binding domain of Sp1. Both domains were separ- ated by a nuclear localization sequence, to ensure nuclear targeting. As a control, an expression vector encoding a nuclear-targeted GST was used (Fig. 6A). The expression vectors were first transiently transfected into 293T cells. The recombinant proteins were purified by glutathione affinity chromatography and separated by SDS/PAGE. Both proteins migrated on SDS/PAGE as expected. Next, 293T and NS20Y cells were transfected with the human synaptophysin promoter/luciferase reporter plasmid pSyp-2356/+27luc, the GST-N encoding expression vector as control, or increasing amounts of the expression vector encoding GST-Sp1. The results show that the dominant- negative Sp1 mutant decreased the constitutive transcrip- tional activity of the synaptophysin promoter (Fig. 6C), indicating that Sp1 is, at least in part, responsible for the constitutive, tissue-unspecific activity of the 5¢-flanking region of the human synaptophysin gene.

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Fig. 6. A dominant-negative form of Sp1 interfered with constitutive transcription of the human synaptophysin promoter/luciferase reporter gene. (A) Modular structure of GST-N and the fusion protein GST-Sp1. Both proteins are expressed in the nucleus due to the presence of a nuclear localization signal derived from the SV40 large T antigen. The zinc finger domain of Sp1 is indicated. (B) Expression of GST-N and GST-Sp1 in human 293T cells. Cells were transfected with plasmids pEGBN and pEBGN-Sp1, respectively, and incubated for 2 days. The recombinant proteins were purified by glutathione affinity chromato- graphy. An aliquot of the proteins were separated via SDS/PAGE and the gel stained with Coomassie Blue. (C) Titration experiment using different amounts of plasmid pEBGN-Sp1 for transfection (0, 1, 2, 3, 4, 5 lg per plate). As reporter gene, we transfected 1 lg per plate of the synaptophysin promoter luciferase reporter plasmid pSyp-2356/+27luc into NS20Y and 293T cells. As an internal standard plasmid, we used 2 lg per plate (NS20Y) or 0.2 lg per plate (293T cells) of plasmid pSV40lacZ. Luciferase activities were normalized for transfection effi- ciency by dividing luciferase light units by b-galactosidase activities.

Fig. 5. Biological activity of the NRSEs derived from the synapsin I promoter and the first intron of the synaptophysin gene. (A) Reporter plasmid pSyINRSE2SV40luc and pSypNRSE2SV40luc. The plasmids contain the luciferase reporter gene and the SV40 promoter. In addi- tion, two REST binding motifs (NRSEs), derived from the human synapsin I or synaptophysin gene were inserted 5¢ to the SV40 pro- moter. (B) One of the reporter plasmids pSyINRSE2SV40luc or pSypNRSE2SV40luc (2 lg per plate), 1 lg per plate of the internal reference plasmid pRSVb and 100 ng per plate of one of the expression vectors encoding FLAG-REST, FLAG-DP-REST, or myc-REST4 were transfected into NS20Y neuroblastoma cells. Luciferase activities were normalized for transfection efficiency by dividing luciferase light units by b-galactosidase activities.

Discussion

respect to the enhancer and the promoter. Accordingly, REST binding sites (NRSEs) were identified not only in promoter and enhancer positions but also in the 5¢- untranslated region of neuronal genes as well as in intronic positions. The genes encoding the adhesion molecules NgCAM and L1, for example, both contain functional binding sites for REST in the first or second intron, respectively [35,36]. Here, we identified a REST binding site in the first intron of the human synaptophysin gene. This sequence is identical in the human and rat genes and differs only on three positions from the NRSE found in the human synapsin I promoter. Moreover, the NRSE derived from the synaptophysin gene fits with the proposed NRSE consensus sequence NNCAGCACCNNGCACAGNNNC [29].

The NRSE functions as a neuron-specific silencer element. Thus, the biological activity is not dependent on the location within a transcription unit, as we have previously demonstrated [14]. We therefore transferred a part of the first intron of the human synaptophysin gene to the promoter region of a reporter gene, 5¢ of the SV40 promoter. Transfection experiments revealed that the activity of this newly generated transcription unit was under the control of REST. This result indicates that the intronic REST binding site of the synaptophysin gene is functional. Moreover, in further transfection experiments, no signifi- cant differences in the activities of the NRSEs derived from the human synapsin I or synaptophysin genes were observed. Likewise, a previous comparison of five NRSEs REST, also known as neuron-restrictive silencer factor (NRSF), functions as a transcriptional repressor of neur- onal genes in non-neuronal tissues [32,33]. Target genes of REST are the genes encoding synapsin I, brain-derived neurotrophic factor, choline acetyltransferase, the type II sodium channel, SCG10, the m4 muscarinic acetylcholine receptor, the N-methyl-D-aspartate receptor subunit NR2C, the adhesion proteins L1 and NgCAM and others. A recent BLAST search of the CELERA mouse database identified at least 324 potential REST-regulated genes [34], including genes encoding synaptic vesicle proteins, neuronal receptors, channels, and molecules involved in adhesion and signaling. Recently, we have shown that REST represses transcrip- tion independent of the location or orientation of its binding site within a gene [14]. REST fulfils the criterion of a transcriptional silencer binding protein that blocks tran- scription regardless of its location or orientation with

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the interplay between a genes(cid:3). Here, we show that constitutive transcription factor (Sp1) and a transcriptional repressor (REST) finally leads to a very distinct, tissue- specific gene expression pattern, as demonstrated for the synaptophysin gene.

Acknowledgements

We thank Thomas Su¨ dhof for providing the genomic clone p10C-6, Karl Bach and Angela Magin for cloning work, and Libby Guethlein for critical reading of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (TH 377/6-3).

References

1. Su¨ dhof, T.C., Lottspeich, F., Greengard, P., Mehl, E. & Jahn, R. (1987) A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions. Science 238, 1142–1144.

2. Johnston, P.A. & Su¨ dhof, T.C. (1990) The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization. J. Biol. Chem. 265, 8869–8873.

3. Thomas, L., Hartung, K., Langosch, D., Rehm, H., Mamberg, E., Franke, W.W. & Betz, H. (1988) Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane. Science 242, 1050–1053.

4. Rehm, H., Wiedenmann, B. & Betz, H. (1986) Molecular char- acterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane. EMBO J. 5, 535–541.

5. Alder, J., Lu, B., Valtorta, F., Greengard, P. & Poo, M.-M. (1992) Calcium-dependent transmitter secretion reconstituted in Xenopus oocytes: requirement for synaptophysin. Science 257, 657–661. 6. McMahon, H.T., Bolshakov, V.Y., Janz, R., Hammer, R.E., Siegelbaum, S.A. & Su¨ dhof, T.C. (1996) Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release. Proc. Natl Acad. Sci. USA 93, 4760–4764.

7. Janz, R., Su¨ dhof, T.C., Hammer, R.E., Unni, V., Siegelbaum, S.A. & Bolshakov, V.Y. (1999) Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I. Neuron 24, 687–700. 8. Tarsa, L. & Goda, Y. (2002) Synaptophysin regulates activity- dependent synapse formation in cultured hippocampal neurons. Proc. Natl Acad. Sci. USA 99, 1012–1016.

derived from genes encoding synapsin I, SCG10, a1-glycine receptor, the b2-subunit of the neuronal nicotinic acetyl- choline receptor and the m4-subunit of the muscarinic acetylcholine receptor did not show any differences in REST-mediated reporter gene silencing, although – in their natural context – they differ in their orientation and location. These data show that the intronic NRSE of the synaptophysin gene functions as a REST-binding site and directs neuron-specific gene expression via the binding of REST. A recent search of the CELERA mouse database has proposed the genes encoding synaptotagmin IV, 6, and 13 as potential REST-responsive genes [34], suggesting that REST may control neuron-specific expression of a set of synaptic vesicle encoding genes. The conclusion that REST is responsible for the neuronal expression of synaptophysin is mainly based on transient transfections of reporter constructs with or without the NRSE derived from the synaptophysin gene. The use of different reporters together with expression vectors encoding REST, a positive-domin- ant mutant of REST or a biologically inactive splice variant of REST convincingly demonstrated that the NRSE is required for REST or DP-REST to either repress or activate reporter gene transcription. Moreover, the fact that both the synapsin I and synaptophysin genes are inducible in P19 cells by histone deacetylase inhibitors suggests that both genes are regulated by a similar mechanism. Cell lines overexpressing REST or DP-REST will be very helpful to confirm these data in living cells.

9. Navone, F., Jahn, R., Di Gioia, G., Stukenbrok, H., Greengard, P. & De Camilli, P. (1986) Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendrocine cells. J. Cell Biol. 103, 2511–2527.

10. Knaus, P., Betz, H. & Rehm, H. (1986) Expression of synapto- the mouse brain.

physin during postnatal development of J. Neurochem. 47, 1302–1304.

11. Petersohn, D., Schoch, S., Brinkmann, D.R. & Thiel, G. (1995) The human synapsin II gene promoter – possible role for the transcription factors zif268/egr-1, polyoma enhancer activator 3, and AP2. J. Biol. Chem. 270, 24361–24369.

12. O¨ zcelik, T., Lafreniere, R.G., Archer, B.T., Johnston, P.A., Willard, H.F., Francke, U. & Su¨ dhof, T.C. (1990) Synaptophysin: Structure of the human gene and assignment to the X chromo- some in man and mouse. Am. J. Hum. Genet. 47, 551–561. 13. Thiel, G., Petersohn, D. & Schoch, S. (1996) pHIVTATA-CAT, a versatile vector to study transcriptional regulatory elements in mammalian cells. Gene 168, 173–176.

14. Thiel, G., Lietz, M. & Cramer, M. (1998) Biological activity and modular structure of RE-1 silencing transcription factor (REST), a repressor of neuronal genes. J. Biol. Chem. 273, 26891–26899.

15. Lietz, M., Bach, K. & Thiel, G. (2001) Biological activity of RE-1 silencing transcription factor (REST) towards distinct transcrip- tional activators. Eur. J. Neurosci. 14, 1303–1312.

Synaptophysin is not only expressed in neurons, but also in neuroendocrine cells [37]. Pancreatic b-cells, for example, are known to express several neuronal genes including synaptic vesicle proteins, neurotransmitters and neurotransmitter synthesizing enzymes such as glutamic Interestingly, neuronal gene acid decarboxylase [38]. expression in pancreatic b cells has been explained by an absence of REST expression [39], indicating that impair- ment of REST function is the basis for the transcription of neuronal genes in neurons as well as in endocrine pancreatic cells.

The 5¢-flanking region of the human synaptophysin gene directs constitutive transcription of the gene. We did not observe significant differences in the transcriptional activity of a synaptophysin promoter/luciferase reporter gene transfected in neuronal or non-neuronal cells. Like- wise, 1.2 kb of the rat synaptophysin promoter have been shown to be insufficient to confer cell-type specific gene transcription [31]. In contrast, clear cell-type specific differences were seen following transfection of a synapsin I promoter/luciferase transcription unit, due to the NRSE in the synapsin I promoter. The promoter region of the synaptophysin gene is GC-rich and contains four potential [31]. Using a dominant-negative Sp1-binding motifs approach we show that the constitutive transcriptional activity of the synaptophysin promoter is generated, at least in part, by the transcriptional activity of Sp1, or Sp1- releated transcription factors that bind to the recognition site of Sp1. Sp1 has been reported to contribute to the regulation of transcription of other neuron-specific genes, including the gene encoding aldolase C, synaptobrevin II or the cyclin-dependent protein kinase 5 regulator p35 [17,40,41]. Sp1 is ubiquitously expressed and is involved in the constitutive transcription of many (cid:2)housekeeping

Regulation of synaptophysin gene transcription (Eur. J. Biochem. 270) 9

(cid:1) FEBS 2003

16. Magin, A., Lietz, M., Cibelli, G. & Thiel, G. (2002) RE-1 silencing is neither a transcriptional

29. Schoenherr, C.J., Paquette, A.J. & Anderson, D.J. (1996) Identi- fication of potential target genes for the neuron-restrictive silencer factor. Proc. Natl Acad. Sci. USA 93, 9881–9886.

transcription factor-4 (REST4) repressor nor a de-repressor. Neurochem. Int. 40, 193–200.

17. Petersohn, D. & Thiel, G. (1996) Role of zinc finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human syna- ptobrevin II gene. Eur. J. Biochem. 239, 827–834.

30. Shimojo, M., Lee, J.-H., Lee & Hersh, L.B. (2001) Role of zinc fingers of the transcription factor NRSF/REST in DNA binding and nuclear localization. J. Biol. Chem. 276, 13121–13126. 31. Bargou, R.C.E.F. & Leube, R.E. (1991) The synaptophysin- encoding gene in rat and man is specifically transcribed in neuroendocrine cells. Gene 99, 197–204.

18. Thiel, G. & Cibelli, G. (1999) Corticotropin-releasing factor and vasoactive intestinal polypeptide activate gene transcription through the cAMP signaling pathway in a catecholaminergic immortalized neuron. Neurochem. Int. 34, 183–191.

32. Chong, J.A., Tapia-Ramı´ rez, J., Kim, S., Toledo-Aral, J.J., Zheng, Y., Boutros, M.C., Altshuller, Y.M., Frohman, M.A., Kraner, S.D. & Mandel, G. (1995) REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80, 949–957.

19. Ju¨ ngling, S., Cibelli, G., Czardybon, M., Gerdes, H.-H. & Thiel, G. (1994) Differential regulation of chromogranin B and synapsin I gene promoter activity by cAMP and cAMP-dependent protein kinase. Eur. J. Biochem. 226, 925–935.

33. Schoenherr, C.J. & Anderson, D.J. (1995) The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron- specific genes. Science 267, 1360–1363.

20. Thiel, G., Kaufmann, K., Magin, A., Lietz, M., Bach, K. & Cramer, M. (2000) The human transcriptional repressor protein NAB1: expression and biological activity. Biochim. Biophys. Acta 1493, 289–301.

21. Thiel, G., Greengard, P. & Su¨ dhof, T.C. (1991) Characterization of tissue-specific transcription by the human synapsin I gene promoter. Proc. Natl Acad. Sci. USA 88, 3431–3435.

34. Roopra, A., Huang, Y. & Dingledine, R. (2001) Neurological disease: listening to gene silencers. Mol. Interv. 1, 219–228. 35. Kallunki, P., Jenkinson, S., Edelman, G.M. & Jones, F.S. (1995) Silencer elements modulate the expression of the gene for the neuron-glia cell adhesion molecule, Ng-CAM. J. Biol. Chem. 138, 1343–1354.

22. Kaufmann, K., Bach, K. & Thiel, G. (2001) Extracellular signal- regulated protein kinases Erk1/Erk2 stimulate expression and biological activity of the transcriptional regulator Egr-1. Biol. Chem. 382, 1077–1081.

23. Thiel, G., Schoch, S. & Petersohn, D. (1994) Regulation of synapsin I gene expression by the zinc finger transcription factor zif268/egr-1. J. Biol. Chem. 269, 15294–15301.

36. Kallunki, P., Edelman, G.M. & Jones, F.S. (1997) Tissue-specific expression of the L1 cell adhesion molecule is modulated by the neural restrictive silencer element. J. Cell Biol. 270, 21291–21298. 37. Thomas-Reetz, A., Hell, J.W., During, M.J., Walch-Solimena, C., Jahn, R. & De Camilli, P. (1993) A c-aminobutyric acid trans- porter driven by a proton pump is present in synaptic-like microvesicles of pancreatic b cells. Proc. Natl Acad. Sci. USA 90, 5317–5321.

24. McBurney, M.W., Reuhl, K.R., Ally, A.I., Nasipuri, S., Bell, J.C. & Craig, J. (1988) Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture. J. Neurosci. 8, 1063– 1073.

38. Thomas-Reetz, A. & De Camilli, P. (1994) A role for synaptic vesicles in non-neuronal cells: clues from panceatic b cells and from chromaffin cells. FASEB J. 8, 209–216.

25. Schoch, S., Cibelli, G. & Thiel, G. (1996) Neuron-specific gene expression of synapsin I. Major role of a negative regulatory mechanism. J. Biol. Chem. 271, 3317–3323.

26. Huang, Y., Myers, S.J. & Dingledine, R. (1999) Transcriptional repression by REST: recruitment of Sin3A and histone deacetylase to neuronal genes. Nature Neurosci. 2, 867–872.

39. Atouf, F., Czernichow, P. & Scharfmann, R. (1997) Expression traits in pancreatic beta cells. Implication of of neuronal neuron-restrictive silencing factor/repressor element silencing transcription factor, a neuron-restrictive silencer. J. Biol. Chem. 272, 1929–1934.

27. Naruse, Y., Aoki, T., Kojima, T. & Mori, N. (1999) Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes. Proc. Natl Acad. Sci. USA 96, 13691–13696.

40. Cibelli, G., Schoch, S., Pajunk, H., Brand, I.A. & Thiel, G. (1996) A (G+C) -rich motif in the aldolase C promoter functions as a constitutive transcriptional enhancer element. Eur. J. Biochem. 237, 311–317.

41. Ross, S., Tienhaara, A., Lee, M.-S., Tsai, L.-H. & Gill, G. (2002) GC box transcription factors control the neuronal specific tran- scription of the cyclin-dependent kinase 5 regulator p35. J. Biol. Chem. 277, 4455–4464.

28. Ballas, N., Battaglioli, E., Atouf, F., Andres, M.E., Chenoweth, J., Anderson, M.E., Burger, C., Moniwa, M., Davie, J.R., Bowers, W.J., Federoff, H.J., Rose, D.W., Rosenfeld, M.G., Brehm, P. & Mandel, G. (2001) Regulation of neuronal traits by a novel transcriptional complex. Neuron 31, 353–365.