Proc. Nati. Acad. Sci. USAVol. 88, pp. 3431-3435, April 1991Biochemistry

Characterization of tissue-specific transcription by the humansynapsin I gene promoterGERALD THIEL*tt, PAUL GREENGARD*, AND THOMAS C. SUDHOFt§*The Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021; and tHoward Hughes Medical Institute and§Department of Molecular Genetics, University of Texas Southwestern Medical School, Dallas, TX 75235

Contributed by Paul Greengard, December 28, 1990

ABSTRACT Synapsin Ia and synapsin lb are abundantsynaptic vesicle proteins that are derived by differential splic-ing from a single gene. To identify control elements directingthe neuronal expression of synapsins Ia/b, we functionallyanalyzed the promoter region of the human synapsin I gene. Ahybrid gene was constructed containing 2 kilobases of 5'flanking sequence from the synapsin I gene fused to thebacterial gene chloramphenicol acetyltransferase and trans-fected into 12 different neuronal and nonneuronal cell lines. Ingeneral, expression of the chimeric reporter gene showedexcellent correlation with endogenous expression of synapsin Iin different neuronal cell lines, whereas transcription was lowin all nonneuronal cell lines examined. The addition of thesimian virus 40 enhancer promoted non-tissue-specific expres-sion. Deletion mutagenesis of the synapsin I promoter revealedthe presence of positive and negative sequence elements. Abasal (constitutive) promoter that directs reporter gene expres-sion in neuronal and nonneuronal cell lines was mapped to theregion -115 to +47. The promoter region from -422 to -22contains positive elements that upon fusion with the herpessimplex virus thymidine kinase promoter potentiate its tran-scription in PC12 and neuroblastoma cells but not in Chinesehamster ovary cells.

Synapsins Ia and Ib (derived from a single gene and collec-tively referred to as synapsin I) are peripheral membraneproteins that are localized on small synaptic vesicles in thenerve terminal (1, 2). A probable function of synapsin Iconsists in the linking of synaptic vesicles to elements of thecytoskeleton (3, 4), a function that is controlled by thephosphorylation state of synapsin I. Synaptic vesicles con-tain two other members of the synapsin family (2, 5), syn-apsin Ila and synapsin Ilb, which are encoded by a differentgene (2).The synapsin I gene is a good candidate for an investigation

of neuron-specific gene expression. Synapsin I has a wide-spread distribution in the central and peripheral nervoussystems and is not expressed in nonneuronal cells. Further-more, synapsin I is a marker of terminal neuronal differen-tiation in that the expression of synapsin I reflects the finalmaturation of a neuroblast to a neuron. An investigation ofthe transcriptional regulation of the synapsins has two goals.First, the regulation of synapsin gene expression controls theamount and types of synapsins in the nerve terminal and isimportant for synaptic function. Second, an understanding ofthe regulation of synapsin I gene expression may help in theunderstanding of gene expression in the nervous system ingeneral.The 5' flanking regions of the human and rat synapsin I

genes were recently cloned (6, 7). In the present study, weanalyzed the upstream region of the human synapsin I geneand found that 2 kilobases (kb) of the 5' upstream region are

sufficient for tissue-specific expression. The constitutive andthe core promoter sequences were mapped, as well aspositive and negative regions within the promoter.

MATERIALS AND METHODSPlasmids. A plasmid containing the human synapsin I

promoter sequence from -1952 to +47 in pBluescript (Strat-agene) was used to construct the chimeric synapsin I-chlor-amphenicol acetyltransferase (CAT) expression plasmidspSyCAT-1 to pSyCAT-8. Using restriction sites 5' in thepromoter sequence and 3' in the pBluescript polylinker, wecloned the following restriction fragments upstream of apromoterless CAT gene in pCAThasic (Promega): a 1998-base-pair (bp) Xho I/Sma I fragment (pSyCAT-1), a 1230-bpAsp7l8/Xba I fragment (pSyCAT-2), a 1016-bp Bgl II/Sma Ifragment (pSyCAT-3), an 893-bp Sph I/Sma I fragment(pSyCAT-4), a 469-bp Pst I/EcoRI fragment (pSyCAT-5), a281-bpAlu I/Xba I fragment (pSyCAT-6), a 162-bp Sty IlXbaI fragment (pSyCAT-7), and a 70-bp Nar I/Xba I fragment(pSyCAT-8). To construct an expression plasmid containingthe simian virus 40 (SV40) enhancer downstream of the CATgene (pSyCAT-SV40), we inserted the 1998-bp Xho I/Sma Ifragment in the polylinker ofpCATenhancer (Promega). Theplasmid pTK-CAT has been described (8). To constructchimeric synapsin 1/thymidine kinase (TK) promoters weinserted restriction fragments or annealed oligonucleotidescontaining synapsin I promoter sequences upstream of theTK promoter. The plasmids pTK1, pTK2, and pTK3 containthe sequences -115/-22, -234/-22, and -422/-22, re-spectively, which were excised from pSyCAT-7, pSyCAT-6,and pSyCAT-5 as HindIII/Nar I fragments. Plasmid pTK4contains a HindIII/Sty I fragment derived from pSyCAT-6(promoter sequence -234/-113). The plasmids pTK5 andpTK6 contain annealed oligonucleotides of the synapsin Ipromoter sequences -187/-116 and -234/-188, respec-tively. The plasmid pICP4CAT, which contains the herpessimplex virus immediate early gene 3 regulatory region from-550 to +40, was a kind gift of Rozanne Sandri-Goldin(University of California, Irvine). The plasmids pCATcontroland pCMV/3 were purchased from Promega and Clontech,respectively.

Cell Culture. PC12 pheochromocytoma cells (provided byJ. Buxbaum, The Rockefeller University) were cultured in85% Dulbecco's modified Eagle medium (DMEM), 10% fetalbovine serum (FBS), 5% horse serum, 100 units of penicillinper ml, and 100 ,zg of streptomycin per ml. The mouseneuroblastoma cell lines NS20Y and NS26 (a gift of M.Nirenberg, National Institutes ofHealth), the monkey kidneyfibroblast cell line CV-1, Chinese hamster ovary (CHO) cells(kindly provided by S. E. Gandy, The Rockefeller Univer-

Abbreviations: CAT, chloramphenicol acetyltransferase; TK, thy-midine kinase; SV40, simian virus 40; CHO, Chinese hamster ovary.tTo whom reprint requests should be addressed at: The RockefellerUniversity, 1230 York Avenue, New York, NY 10021.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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sity), and the rat muscle cell line L6 (obtained from K. Miles,Downstate Medical School, Brooklyn, NY) were maintainedin 90% DMEM, 10% FBS, 100 units of penicillin per ml, and100 ug of streptomycin per ml. The human neuroblastomacell line SH-SY5Y (9), a gift of J. Biedler and B. A. Sprenger(Sloan-Kettering Institute for Cancer Research, New York),was grown in 85% DMEM, 15% FBS, 100 units of penicillinper ml, and 100 ,ug of streptomycin per ml. The mouseneuroblastoma cell line N18TG2 and the immortalized celllines SN48, SN56, and HN25, derived from somatic fusion ofseptal or hippocampal neurons with N18TG2 neuroblastomacells (refs. 10-12; kindly provided by Bruce Wainer, TheUniversity of Chicago), were cultured in DMEM containing10% FBS, 2 mM L-glutamine, and 60 mg of gentamicin perliter. The human hepatoma cell line HepG2 (ATCC no. HB8065) was grown in F12 medium supplemented with 1oFBS, 100 units of penicillin per ml, and 100 ,ug of strepto-mycin per ml.

Transfections and CAT Assays. Plasmids were bandedtwice in CsCl prior to transfection. Transfections were doneaccording to ref. 13. Cells were incubated with calciumphosphate/DNA precipitate containing 8 ,g of CAT-plasmidand 2 ,ug ofpCMVP for 6 hr, followed by a glycerol shock of2 min (15% glycerol in Opti-MEM, GIBCO). PC12 andSH-SY5Y cells were lipofected (ref. 14, protocol A), using 16,ug of CAT-plasmid, 4 ,ug ofpCMVP3, and 50 ,ug of Lipofectin(GIBCO). Cells were harvested after 48-60 hr and lysed bythree cycles offreeze-thaw. The cellular debris was removedby centrifugation and the supernatant was used to measure/B-galactosidase activity (15). The remaining cell extract washeated for 10 min at 65°C (16) and then used to measure CATactivity following method 1 in ref. 15 using butyryl coenzymeA (Sigma). For quantification the reaction products wereextracted with xylene (Aldrich) according to ref. 17.

Miscellaneous Techniques. Total rat brain RNA was ex-tracted and purified according to ref. 18. Primer extensionanalysis was performed according to ref. 19. The endogenoussynapsin I message was mapped with a primer that wascomplementary to nucleotides + 127 to + 154 of the ratmRNA. Western blots were probed with an antipeptideantibody to phosphorylation site 3 in synapsin I or with amonoclonal antibody specific for synapsin II (kind gifts ofA. J. Czernik and Y.-L. Siow, The Rockfeller University)and a horseradish peroxidase-coupled secondary antibody(Amersham). Blots were developed using enhanced chemilu-minescence (ECL, Amersham).

RESULTSPrimer Extension. Primer extension analysis using rat brain

total RNA and a primer complementary to synapsin I mRNAshowed a single extension product (data not shown). Se-quences are numbered with + 1 being the first transcribednucleotide corresponding to nucleotide 1953 in ref. 6. Thesequence surrounding the transcript start was identical to thefunctional initiator sequence 5'-CTCANTCT-3' (20), whereA is the transcript start site.

Expression of a Synapsin I-CAT Hybrid Gene in DifferentCell Lines. A plasmid was constructed containing a 2-kbfragment of the 5' flanking region of the human synapsin Igene (sequence from -1951 to +47) fused to a promoterlessCAT gene (Fig. 1A). This plasmid was transiently transfectedinto different neuronal and nonneuronal cell lines. In allexperiments a plasmid that contained the f3-galactosidasegene under control of the cytomegalovirus promoter/enhancer was cotransfected. CAT activities were then nor-malized to p-galactosidase activity of each cell extract tocorrect for transfection efficiencies. Fig. 1B shows a repre-sentative CAT assay. The CAT gene under control of thesynapsin I promoter/regulatory region was strongly ex-

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Structure of the fusion gene pSyCAT-1. (B) Expression of synapsinI-CAT fusion gene in NS26 and L6 cells: a representative autorad-iograph showing thin-layer chromatographic separation of butyry-lated chloramphenicol from chloramphenicol. As a negative control,we transfected a promoterless CAT gene (plasmid pCATbasic) andas a positive control we used a plasmid that contained the CAT geneunder control ofthe herpes simplex virus IE3 gene promoter (plasmidpICP4CAT). (C) Cell-type-specific expression of synapsin I-CATfusion genes in various neuronal and nonneuronal cell lines. CATactivity was normalized for transfection efficiency, dividing CATactivity by ,B-galactosidase activity, and is expressed as a percentageof the herpes virus IE3 gene promoter activity. At least fourexperiments were done with each cell type and the mean ± SEM isdepicted.

pressed in the neuroblastoma cell line NS26 but not in themuscle cell line L6. The promoterless CAT plasmid pCAT-basic was inactive in both cell lines. The plasmid pICP4CATwas transfected as a positive control and it was stronglyexpressed in both cell lines.To analyze the tissue-specific expression of the synapsin

I-CAT fusion gene in more detail, we introduced this con-struct into several neuronal and nonneuronal cell lines. Eachcell line was transfected in parallel with a plasmid containingthe CAT gene under the control of the herpes virus IE3 genepromoter/regulatory region (pICP4CAT). This promoter wasstrongly active in all cell lines examined. The activity of thispromoter was arbitrarily set at 100% and the transcription ofthe synapsin I-CAT gene was expressed as a percentage ofthe herpes virus IE3 gene promoter activity. Fig. 1C showsthat the human synapsin I promoter is most active in PC12cells and in the mouse neuroblastoma cell lines NS20Y andNS26. The fusion gene is either not transcribed or transcribedonly at low levels in the neuroblastoma cell lines SH-SY5Y

3432 Biochemistry: Thiel et al.

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and N18TG2 and in the immortalized cell line HN25. Thisindicates that in this aspect the HN25 cells resemble theneuroblastoma parent more than the hippocampal neuron.However, the transcription is increased 8.8-fold and 10.2-foldin the immortalized cell lines SN48 and SN56, respectively,in comparison to the parent N18TG2 cells, providing furtherevidence that these cell lines reached a higher differentiationlevel than the neuroblastoma cells. The synapsin I-CAT genewas either not expressed or only poorly expressed in allnonneuronal cell lines, suggesting that the 2-kb promoter/regulatory region of the human synapsin I gene containssignals for cell-selective gene expression.Immunoblot analysis of the endogenous synapsins in the

neuronal cell lines used here reveals that synapsins I and IIare both expressed in neuroblastoma cell lines NS20Y andNS26 and in the immortalized septal cells SN48 and SN56(Fig. 2). In all four cell lines the synapsin I-CAT gene wasexpressed. The cell lines that did not transcribe the synapsinI promoter, the neuroblastomas SH-SY5Y and N18TG2 andthe immortalized hippocampal line HN25, showed little or noendogenous synapsin I. In PC12 cells there was only a smallamount of endogenous synapsin I though the transfectedsynapsin I-CAT gene was very actively transcribed. In gen-eral, the level ofendogenous synapsins I and II in all neuronalcell lines was very low compared to that of brain.The SV40 Enhancer Promotes Non-Tissue-Specific Expres-

sion. The SV40 enhancer is promiscuously active in numer-ous rodent and mammalian cells. To test if the enhancerinfluences tissue-specific expression of the human synapsinI promoter, we constructed a fusion gene containing thesynapsin I promoter/regulatory region, the CAT codingsequence, and the SV40 enhancer (Fig. 3A, plasmid pSyCAT-SV40). Fig. 3B shows a thin-layer chromatographic separa-tion of butyrylated chloramphenicol from chloramphenicol.The plasmid pCATcontrol, containing the CAT gene undercontrol of the SV40 promoter and enhancer, was weaklyexpressed in NS26 neuroblastoma cells. In L6 cells, how-ever, a good signal was seen. The plasmid pCATenhancer,which contains a promoterless CAT gene and the SV40enhancer, was used as a negative control. No CAT activitywas seen. Finally, the plasmid pSyCAT-SV40 is expressed inneuronal and nonneuronal cell lines. A quantitative analysis(depicted in Fig. 3C) revealed that the SV40 enhancer doesnot increase expression ofthe hybrid gene in the neuronal celllines PC12 and NS26. However, in the nonneuronal cell linesL6, CHO, and HepG2 the enhancer increased CAT activity58-fold, 52-fold, and 21-fold, respectively.

Deletion Analysis of the Human Synapsin I Promoter. TolocalizeDNA sequences responsible for neuron-specific geneexpression, we made progressive 5' deletions of the humansynapsin I promoter/regulatory region (Fig. 4A). The dele-

Synapsin Ha -

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FIG. 2. Expression of endogenous synapsin I and synapsin II indifferent neuronal cell lines. Cell homogenates (150 ,g per lane) weresubjected to 7.5% SDS/polyacrylamide gel electrophoresis and im-munoblotted. As a positive control 15 ,ug of rat brain cortex homog-enate was loaded into the left lane.

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FIG. 3. The SV40 enhancer promotes non-tissue-specific expres-sion. (A) Structure ofthe fusion gene pSyCAT-SV40. (B) Expressionof the fusion gene pSyCAT-SV40 in NS26 and L6 cells. A repre-sentative autoradiograph is shown. The negative and positive con-trols were a promoterless CAT gene that contained the SV40enhancer (pCATenhancer) and a CAT gene under control of SV40promoter and enhancer sequences (pCATcontrol). (C) Non-tissue-specific expression ofsynapsin I/CAT/SV40-fusion gene in neuronaland nonneuronal cell lines. The values are expressed as described inthe legend to Fig. 1.

tion mutants were introduced into PC12, NS26, SH-SY5Y,CV-1, and CHO cells. Fig. 4B shows that a deletion from-1951 to -1184 resulted in a 4.9- and 5.5-fold decrease inCAT activity in NS26 and PC12 cells, respectively. Furtherdeletions from -1183 to -970 and from -969 to -847 did noteffect the activity of the synapsin-CAT fusion gene in neu-ronal cell lines. However, a larger deletion from -846 to-423 increased the expression in neuronal and nonneuronalcell lines, suggesting the presence of negative cis-actingsequence elements between -1183 and -423. In PC12 cellsthe synapsin I-CAT hybrid gene containing the promoter/regulatory region from -422 to +47 (pSyCAT-5) was even2.4-fold more active than the initial construct pSyCAT-1.

In the nonneuronal cell lines tested, the most active hybridgene contained synapsin promoter sequences from -115 to+47 (plasmid pSyCAT-7). This sequence represents the basal(constitutive) promoter that contains no tissue-specific ele-ment. However, in comparison to pSyCAT-1, CAT activitiesincreased in CV-1 and CHO cells 3.0- and 3.6-fold, respec-tively, after deletion of sequences upstream from -422,suggesting that some cell-specific restriction had been de-leted. The hybrid gene pSyCAT-8, including the synapsin Ipromoter sequence from -23 to +47, shows only low expres-

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FIG. 4. Deletion analysis of the human synapsin I promoter. (A) Representation of synapsin I-CAT plasmids containing progressive 5'deletions of the synapsin I promoter/regulatory region; a diagram of the 5' upstream region of the human synapsin I gene promoter is shownon top, including restriction sites used to construct the expression plasmids pSyCAT-1, pSyCAT-2, pSyCAT-3, pSyCAT-4, pSyCAT-5,pSyCAT-6, pSyCAT-7, and pSyCAT-8. (B) Histogram showing the CAT activity of extracts of PC12, NS26, SH-SY5Y, CV-1, and CHO cellstransfected with the plasmids in A. The values are expressed as described in the legend to Fig. 1. At least four separate experiments were doneand the results show mean ± SEM.

sion in all cell lines and therefore is believed to represent thecore promoter.

Effect of Synapsin I Promoter Fragments on HeterologousPromoters. The hybrid gene containing the synapsin I pro-moter/regulatory region from -422 to +47 (pSyCAT-5)showed increased expression of the reporter gene in CV-1and CHO cells compared to the synapsin I-CAT hybrid genespSyCAT-1 to pSyCAT-4. To test if this sequence contains atissue-specific element, we fused the entire region or frag-ments in front of a herpes simplex virus TK promoter (Fig.5A), introduced the constructs in PC12, NS20Y, NS26, andCHO cells, and examined promoter strength by measuringthe CAT activity of the extracts. Fig. 5B reveals that none ofthe synapsin I/TK promoter fusions increased the strength ofthe TK promoter in CHO cells. In the neuronal cell lines thelongest synapsin I promoter insert (-422/-22; plasmidpTK3) increased CAT activity 2.5-fold in PC12 and NS26 and3.1-fold in NS20Y cells, suggesting that this fragment in-cludes one or more elements that increase transcriptionparticularly in neuronal cells. Some shorter synapsin I pro-moter sequences increased the transcription slightly. Thesedata reveal that the synapsin I promoter/regulatory sequence-422/-22 must contain more than one cell-selective ele-ment, because (with the exception of pTK4 in NS26 cells)none of the shorter fragments tested reached the stimulatingeffect on transcription as the entire 401-bp fragment.

DISCUSSIONWe report here results of an analysis of the cis-acting DNAsequences that control transcription of the human synapsin Igene. We have found that a 2-kb fragment of the 5' upstreamregion of the synapsin I gene directed tissue-specific expres-sion of a reporter gene in various neuronal cell lines but notin cell lines derived from nonneuronal tissue. However, not

all neuronal cell lines tested expressed the hybrid synapsinI-CAT gene. Only cells that represent a higher differentiationlevel expressed synapsin I. We found that immortalizedseptal cells have the ability to transcribe the introducedsynapsin I-CAT hybrid gene. In contrast to the neuroblas-toma N18TG2 cells, these cells appear to contain the trans-acting factors necessary for the expression of the synapsin Igene. A hybrid gene containing 2 kb of the synapsin Ipromoter/regulatory region, the CAT open reading frameand the SV40 enhancer, was active in neuronal and nonneu-ronal cell lines. The SV40 enhancer has been shown tocontain binding sites for several transcription factors (21).These factors might interact with ubiquitous factors bound tothe proximal region of the synapsin I promoter and therebystimulate transcription in nonneuronal cells without the re-quirement for specific neuronal factors.To identify DNA sequences responsible for tissue-specific

expression we carried out deletion mutagenesis ofthe humansynapsin I promoter. We showed that the region from -115to +47 represents a basal (constitutive) promoter that directsexpression in neuronal and nonneuronal cells. Experimentsusing hybrid synapsin I/TK promoters revealed that thesynapsin promoter region from -422 to -22 increased thestrength of the TK promoter in PC12 and neuroblastoma cellsbut not in CHO cells. This suggests that this fragmentincludes cis-acting sequences that direct increased expres-sion in neuronal cells.

Recently, a study analyzing the rat synapsin I promoter inNS20Y cells (7) indicated that the region from -225 to + 105may be sufficient for cell-type-specific transcription. Resultsobtained with 12 different cell lines as with multiple promoterfragments suggest that the situation may be more complex.The synapsin I promoter region from -422 to -235 andprobably further upstream regions are necessary to establishtight tissue specificity. Sauerwald et al. (7) discussed the

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involvement of two sequence elements called SNN (region-214/-200, sequence 5'-CCTTCGCCCCCGC-3', and-164/-156, sequence 5'-CGCGCTGAC-3', in the humansynapsin I gene). However, when we fused a restrictionfragment of the synapsin I promoter containing both SNNsequences to a herpes virus TK promoter, the activityincreased only slightly in PC12 and NS20Y cells (plasmidpTK4). Only in NS26 cells were we able to measure asignificant increase of 2.8-fold over the expression of pTK-CAT. Oligonucleotides containing only one of the SNNsequences inserted upstream of the TK promoter (plasmidspTK5 and pTK6) increased the expression only minimally inneuronal cell lines. Therefore, the role of the SNN elementsin tissue-specific gene expression is not yet clear. Furtheranalysis of the synapsin I promoter should increase ourunderstanding of long-term regulation of synapsin I, specif-ically, and of regulation of neuronal gene expression, ingeneral.

We are grateful to June Biedler, Joseph Buxbaum, Samuel Gandy,Marshall Nirenberg, Kathryn Miles, B. A. Spengler, and BruceWainer for providing cell lines, to Andrew J. Czernik and Y.-L. Siowfor providing antibodies, and to Rozanne Sandri-Goldin for theplasmid pICP4CAT. We thank Michelle E. Ehrlich and Piera Cic-chetti for a critical reading of the manuscript. This study wassupported by U.S. Public Health Service Grant MH39327.

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21. Jones, N. C., Rigby, P. W. & Ziff, E. B. (1988) Genes Dev. 2,267-281.

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