Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3431-3435, April 1991 Characterization of tissue-specific transcription by the human synapsin I promoter GERALD 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 abundant sufficient for tissue-specific expression. The constitutive and synaptic vesicle that are derived by differential splic- the core promoter sequences were mapped, as well as ing from a single gene. To identify control elements directing positive and negative regions within the promoter. the neuronal expression of synapsins Ia/b, we functionally analyzed the promoter region of the human synapsin I gene. A MATERIALS AND METHODS gene was constructed containing 2 kilobases of 5' flanking sequence from the synapsin I gene fused to the Plasmids. A plasmid containing the human synapsin I bacterial gene chloramphenicol acetyltransferase and trans- promoter sequence from -1952 to +47 in pBluescript (Strat- fected into 12 different neuronal and nonneuronal cell lines. In agene) was used to construct the chimeric synapsin I-chlor- general, expression of the chimeric reporter gene showed amphenicol acetyltransferase (CAT) expression plasmids excellent correlation with endogenous expression of synapsin I pSyCAT-1 to pSyCAT-8. Using restriction sites 5' in the in different neuronal cell lines, whereas transcription was low promoter sequence and 3' in the pBluescript polylinker, we in all nonneuronal cell lines examined. The addition of the cloned the following restriction fragments upstream of a simian virus 40 enhancer promoted non-tissue-specific expres- promoterless CAT gene in pCAThasic (Promega): a 1998- sion. Deletion mutagenesis of the synapsin I promoter revealed base-pair (bp) Xho I/Sma I fragment (pSyCAT-1), a 1230-bp the presence of positive and negative sequence elements. A Asp7l8/Xba I fragment (pSyCAT-2), a 1016-bp Bgl II/Sma I basal (constitutive) promoter that directs reporter gene expres- fragment (pSyCAT-3), an 893-bp Sph I/Sma I fragment sion in neuronal and nonneuronal cell lines was mapped to the (pSyCAT-4), a 469-bp Pst I/EcoRI fragment (pSyCAT-5), a region -115 to +47. The promoter region from -422 to -22 281-bpAlu I/Xba I fragment (pSyCAT-6), a 162-bp Sty IlXba contains positive elements that upon fusion with the herpes I fragment (pSyCAT-7), and a 70-bp Nar I/Xba I fragment simplex virus kinase promoter potentiate its tran- (pSyCAT-8). To construct an expression plasmid containing scription in PC12 and neuroblastoma cells but not in Chinese the simian virus 40 (SV40) enhancer downstream of the CAT hamster ovary cells. gene (pSyCAT-SV40), we inserted the 1998-bp Xho I/Sma I fragment in the polylinker ofpCATenhancer (Promega). The Synapsins Ia and Ib (derived from a single gene and collec- plasmid pTK-CAT has been described (8). To construct tively referred to as synapsin I) are peripheral membrane chimeric synapsin 1/ (TK) promoters we proteins that are localized on small synaptic vesicles in the inserted restriction fragments or annealed oligonucleotides nerve terminal (1, 2). A probable function of synapsin I containing synapsin I promoter sequences upstream of the consists in the linking of synaptic vesicles to elements of the TK promoter. The plasmids pTK1, pTK2, and pTK3 contain cytoskeleton (3, 4), a function that is controlled by the the sequences -115/-22, -234/-22, and -422/-22, re- phosphorylation state of synapsin I. Synaptic vesicles con- spectively, which were excised from pSyCAT-7, pSyCAT-6, tain two other members of the synapsin family (2, 5), syn- and pSyCAT-5 as HindIII/Nar I fragments. Plasmid pTK4 apsin Ila and synapsin Ilb, which are encoded by a different contains a HindIII/Sty I fragment derived from pSyCAT-6 gene (2). (promoter sequence -234/-113). The plasmids pTK5 and The synapsin I gene is a good candidate for an investigation pTK6 contain annealed oligonucleotides of the synapsin I of neuron-specific . Synapsin I has a wide- promoter sequences -187/-116 and -234/-188, respec- spread distribution in the central and peripheral nervous tively. The plasmid pICP4CAT, which contains the herpes systems and is not expressed in nonneuronal cells. Further- simplex virus immediate early gene 3 regulatory region from more, synapsin I is a marker of terminal neuronal differen- -550 to +40, was a kind gift of Rozanne Sandri-Goldin tiation in that the expression of synapsin I reflects the final (University ofCalifornia, Irvine). The plasmids pCATcontrol maturation of a neuroblast to a neuron. An investigation of and pCMV/3 were purchased from Promega and Clontech, the transcriptional regulation ofthe synapsins has two goals. respectively. First, the regulation of synapsin gene expression controls the Cell Culture. PC12 pheochromocytoma cells (provided by amount and types of synapsins in the nerve terminal and is J. Buxbaum, The Rockefeller University) were cultured in important for synaptic function. Second, an understanding of 85% Dulbecco's modified Eagle medium (DMEM), 10% fetal the regulation of synapsin I gene expression may help in the bovine serum (FBS), 5% horse serum, 100 units of penicillin understanding of gene expression in the nervous system in per ml, and 100 ,zg of streptomycin per ml. The mouse general. neuroblastoma cell lines NS20Y and NS26 (a gift of M. The 5' flanking regions of the human and rat synapsin I Nirenberg, National Institutes ofHealth), the monkey kidney were recently cloned (6, 7). In the present study, we fibroblast cell line CV-1, Chinese hamster ovary (CHO) cells analyzed the upstream region of the human synapsin I gene (kindly provided by S. E. Gandy, The Rockefeller Univer- and found that 2 kilobases (kb) of the 5' upstream region are Abbreviations: CAT, chloramphenicol acetyltransferase; TK, thy- The publication costs of this article were defrayed in part by page charge midine kinase; SV40, simian virus 40; CHO, Chinese hamster ovary. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be addressed at: The Rockefeller in accordance with 18 U.S.C. §1734 solely to indicate this fact. University, 1230 York Avenue, New York, NY 10021. 3431 Downloaded by guest on September 25, 2021 3432 Biochemistry: Thiel et al. Proc. Natl. Acad. Sci. USA 88 (1991) sity), and the rat muscle cell line L6 (obtained from K. Miles, xoI Pw I I Downstate Medical School, Brooklyn, NY) were maintained human in 90% DMEM, 10% FBS, 100 units of penicillin per ml, and A a...... r...r...... CAT 1 100 ug of streptomycin per ml. The human neuroblastoma synapsin I promoter cell line SH-SY5Y (9), a gift ofJ. Biedler and B. A. Sprenger (Sloan-Kettering Institute for Cancer Research, New York), was grown in 85% DMEM, 15% FBS, 100 units of penicillin B CELL LINES per ml, and 100 ,ug of streptomycin per ml. The mouse - -PLASMIDS neuroblastoma cell line N18TG2 and the immortalized cell NS26 L6 lines SN48, SN56, and HN25, derived from somatic fusion of pCAThasic septal or hippocampal neurons with N18TG2 neuroblastoma .4 * . _ # pSyCAT- cells (refs. 10-12; kindly provided by Bruce Wainer, The _.4 *| * 0 plCP4CAT University of Chicago), were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and 60 mg of gentamicin per liter. The human hepatoma cell line HepG2 (ATCC no. HB 8065) was grown in F12 medium supplemented with 1o C FBS, 100 units of penicillin per ml, and 100 ,ug of strepto- mycin per ml. Transfections and CAT Assays. Plasmids were banded twice in CsCl prior to transfection. Transfections were done .Y, according to ref. 13. Cells were incubated with calcium phosphate/DNA precipitate containing 8 ,g ofCAT-plasmid 7g and 2 ,ug of pCMVP for 6 hr, followed by a glycerol shock of 2 min (15% glycerol in Opti-MEM, GIBCO). PC12 and la SH-SY5Y cells were lipofected (ref. 14, protocol A), using 16 ,ug ofCAT-plasmid, 4 ,ug ofpCMVP3, and 50 ,ug ofLipofectin (GIBCO). Cells were harvested after 48-60 hr and lysed by three cycles offreeze-thaw. The cellular debris was removed by centrifugation and the supernatant was used to measure /B-galactosidase activity (15). The remaining cell extract was heated for 10 min at 65°C (16) and then used to measure CAT activity following method 1 in ref. 15 using butyryl coenzyme A (Sigma). For quantification the reaction products were extracted with xylene (Aldrich) according to ref. 17. nonneuronal Miscellaneous Techniques. Total rat brain RNA was ex- neuronaloceli les tracted and purified according to ref. 18. Primer extension cen lines was to ref. 19. The endogenous analysis performed according FIG. 1. Neuron-specific expression of synapsin I-CAT gene. (A) synapsin I message was mapped with a primer that was Structure of the fusion gene pSyCAT-1. (B) Expression of synapsin complementary to nucleotides + 127 to + 154 of the rat I-CAT fusion gene in NS26 and L6 cells: a representative autorad- mRNA. Western blots were probed with an antipeptide iograph showing thin-layer chromatographic separation of butyry- to phosphorylation site 3 in synapsin I or with a lated chloramphenicol from chloramphenicol. As a negative control, specific for synapsin II (kind gifts of we transfected a promoterless CAT gene (plasmid pCATbasic) and A. J. Czernik and Y.-L. Siow, The Rockfeller University) as a positive control we used a plasmid that contained the CAT gene and a horseradish peroxidase-coupled secondary antibody under control ofthe herpes simplex virus IE3 gene promoter (plasmid (Amersham). Blots were developed using enhanced chemilu- pICP4CAT). (C) Cell-type-specific expression of synapsin I-CAT minescence Amersham). fusion genes in various neuronal and nonneuronal cell lines. CAT (ECL, activity was normalized for transfection efficiency, dividing CAT activity by ,B-galactosidase activity, and is expressed as a percentage RESULTS of the herpes virus IE3 gene promoter activity. At least four experiments were done with each cell type and the mean ± SEM is Primer Extension. Primer extension analysis using rat brain depicted. total RNA and a primer complementary to synapsin I mRNA showed a single extension product (data not shown). Se- pressed in the neuroblastoma cell line NS26 but not in the quences are numbered with + 1 being the first transcribed muscle cell line L6. The promoterless CAT plasmid pCAT- nucleotide corresponding to nucleotide 1953 in ref. 6. The basic was inactive in both cell lines. The plasmid pICP4CAT sequence surrounding the transcript start was identical to the was transfected as a positive control and it was strongly functional initiator sequence 5'-CTCANTCT-3' (20), where expressed in both cell lines. A is the transcript start site. To analyze the tissue-specific expression of the synapsin Expression of a Synapsin I-CAT Hybrid Gene in Different I-CAT fusion gene in more detail, we introduced this con- Cell Lines. A plasmid was constructed containing a 2-kb struct into several neuronal and nonneuronal cell lines. Each fragment of the 5' flanking region of the human synapsin I cell line was transfected in parallel with a plasmid containing gene (sequence from -1951 to +47) fused to a promoterless the CAT gene under the control of the herpes virus IE3 gene CAT gene (Fig. 1A). This plasmid was transiently transfected promoter/regulatory region (pICP4CAT). This promoter was into different neuronal and nonneuronal cell lines. In all strongly active in all cell lines examined. The activity of this experiments a plasmid that contained the f3-galactosidase promoter was arbitrarily set at 100% and the transcription of gene under control of the cytomegalovirus promoter/ the synapsin I-CAT gene was expressed as a percentage of enhancer was cotransfected. CAT activities were then nor- the herpes virus IE3 gene promoter activity. Fig. 1C shows malized to p-galactosidase activity of each cell extract to that the human synapsin I promoter is most active in PC12 correct for transfection efficiencies. Fig. 1B shows a repre- cells and in the mouse neuroblastoma cell lines NS20Y and sentative CAT assay. The CAT gene under control of the NS26. The fusion gene is either not transcribed or transcribed synapsin I promoter/regulatory region was strongly ex- only at low levels in the neuroblastoma cell lines SH-SY5Y Downloaded by guest on September 25, 2021 Biochemistry: Thiel et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3433

and N18TG2 and in the immortalized cell line HN25. This ho I human indicates that in this aspect the HN25 cells resemble the A / SV40 neuroblastoma parent more than the hippocampal neuron. - CAT However, the transcription is increased 8.8-fold and 10.2-fold synapsin I promoter enhancer in the immortalized cell lines SN48 and SN56, respectively, in comparison to the parent N18TG2 cells, providing further B evidence that these cell lines reached a higher differentiation CELL LINES level than the neuroblastoma cells. The synapsin I-CAT gene NS26 L6 PLASMIDS was either not expressed or only poorly expressed in all nonneuronal cell lines, suggesting that the 2-kb promoter/ _ | A4I.0* * pCATcontrol regulatory region of the human synapsin I gene contains _o _1 pCATenhancer signals for cell-selective gene expression. .4_ * pSyCAT-SV40 Immunoblot analysis of the endogenous synapsins in the

neuronal cell lines used here reveals that synapsins I and II PI are both expressed in neuroblastoma cell lines NS20Y and C NS26 and in the immortalized septal cells SN48 and SN56 (Fig. 2). In all four cell lines the synapsin I-CAT gene was 120- expressed. The cell lines that did not transcribe the synapsin I promoter, the neuroblastomas SH-SY5Y and N18TG2 and 100- the immortalized hippocampal line HN25, showed little or no endogenous synapsin I. In PC12 cells there was only a small 40 - amount of endogenous synapsin I though the transfected vC ) synapsin I-CAT gene was very actively transcribed. In gen- w C eral, the level ofendogenous synapsins I and II in all neuronal 30- cell lines was very low compared to that of brain. V4. The SV40 Enhancer Promotes Non-Tissue-Specific Expres- sion. The SV40 enhancer is promiscuously active in numer- 20- ous rodent and mammalian cells. To test if the enhancer

influences tissue-specific expression of the human synapsin 10- I promoter, we constructed a fusion gene containing the synapsin I promoter/regulatory region, the CAT coding sequence, and the SV40 enhancer (Fig. 3A, plasmid pSyCAT- CY Z I SV40). Fig. 3B shows a thin-layer chromatographic separa- tion of butyrylated chloramphenicol from chloramphenicol. The plasmid pCATcontrol, containing the CAT gene under neuronal nonneuronal control of the SV40 promoter and enhancer, was weakly cell lines cell lines expressed in NS26 neuroblastoma cells. In L6 cells, how- ever, a good signal was seen. The plasmid pCATenhancer, FIG. 3. The SV40 enhancer promotes non-tissue-specific expres- which contains a promoterless CAT gene and the SV40 sion. (A) Structure ofthe fusion gene pSyCAT-SV40. (B) Expression enhancer, was used as a negative control. No CAT activity of the fusion gene pSyCAT-SV40 in NS26 and L6 cells. A repre- sentative autoradiograph is shown. The negative and positive con- was seen. Finally, the plasmid pSyCAT-SV40 is expressed in trols were a promoterless CAT gene that contained the SV40 neuronal and nonneuronal cell lines. A quantitative analysis enhancer (pCATenhancer) and a CAT gene under control of SV40 (depicted in Fig. 3C) revealed that the SV40 enhancer does promoter and enhancer sequences (pCATcontrol). (C) Non-tissue- not increase expression ofthe hybrid gene in the neuronal cell specific expression ofsynapsin I/CAT/SV40-fusion gene in neuronal lines PC12 and NS26. However, in the nonneuronal cell lines and nonneuronal cell lines. The values are expressed as described in L6, CHO, and HepG2 the enhancer increased CAT activity the legend to Fig. 1. 58-fold, 52-fold, and 21-fold, respectively. Deletion Analysis of the Human Synapsin I Promoter. To tion mutants were introduced into PC12, NS26, SH-SY5Y, localize DNA sequences responsible for neuron-specific gene CV-1, and CHO cells. Fig. 4B shows that a deletion from expression, we made progressive 5' deletions of the human -1951 to -1184 resulted in a 4.9- and 5.5-fold decrease in synapsin I promoter/regulatory region (Fig. 4A). The dele- CAT activity in NS26 and PC12 cells, respectively. Further deletions from -1183 to -970 and from -969 to -847 did not effect 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 nonneuronal cell lines, suggesting the presence of negative cis-acting sequence elements between -1183 and -423. In PC12 cells the synapsin I-CAT hybrid gene containing the promoter/ regulatory region from -422 to +47 (pSyCAT-5) was even 2.4-fold more active than the initial construct pSyCAT-1. In the nonneuronal cell lines tested, the most active hybrid gene contained synapsin promoter sequences from -115 to Ha - 41mb Synapsin +47 (plasmid pSyCAT-7). This sequence represents the basal Synapsin Ilb_- am 4.NWM. (constitutive) promoter that contains no tissue-specific ele- I ment. However, in comparison to pSyCAT-1, CAT activities increased in CV-1 and CHO cells 3.0- and 3.6-fold, respec- FIG. 2. Expression of endogenous synapsin I and synapsin II in different neuronal cell lines. Cell homogenates (150 ,g per lane) were tively, after deletion of sequences upstream from -422, subjected to 7.5% SDS/polyacrylamide gel electrophoresis and im- suggesting that some cell-specific restriction had been de- munoblotted. As a positive control 15 ,ug of rat brain cortex homog- leted. The hybrid gene pSyCAT-8, including the synapsin I enate was loaded into the left lane. promoter sequence from -23 to +47, shows only low expres- Downloaded by guest on September 25, 2021 3434 Biochemistry: Thiel et al. Proc. Natl. Acad. Sci. USA 88 (1991)

BglII Alul NarI Xho Asp 7 8 Sph Pst jSty I Pvu II ,I synaRsin B Butyrylated Chloramphenicol (%of pICP4CAT) human synapsin promoter 10 20 3,0 40 4 i _4i A_ t -200C 00 _ LX-L. 77.-,- : ;a:.:.;..;.; .m- ..-.....I ------CAT pSyCAT-1

ED PC12 CAT ] LnJ NS26 ------__ PSyCAT-2 4 EIS SH-SY5Y ... 7- ":' '7j -i Cv--1 pSyCAT-3 CAT - CHO

.--- CAT pSyCAT- 4 _~~~~~~~~~~~~~~~~~~~~~~~~~2 52% CAT pSyCAT-5 52 % ICAT PSYCAT-6

CAT pSyCAT-7

L 3 w Gema ATPSyCAT A. .)

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 shown on 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 cells transfected with the plasmids in A. The values are expressed as described in the legend to Fig. 1. At least four separate experiments were done and the results show mean ± SEM. sion in all cell lines and therefore is believed to represent the all neuronal cell lines tested expressed the hybrid synapsin core promoter. I-CAT gene. Only cells that represent a higher differentiation Effect of Synapsin I Promoter Fragments on Heterologous level expressed synapsin I. We found that immortalized Promoters. The hybrid gene containing the synapsin I pro- septal cells have the ability to transcribe the introduced moter/regulatory region from -422 to +47 (pSyCAT-5) synapsin I-CAT hybrid gene. In contrast to the neuroblas- showed increased expression of the reporter gene in CV-1 toma N18TG2 cells, these cells appear to contain the trans- and CHO cells compared to the synapsin I-CAT hybrid genes acting factors necessary for the expression of the synapsin I pSyCAT-1 to pSyCAT-4. To test if this sequence contains a gene. A hybrid gene containing 2 kb of the synapsin I tissue-specific element, we fused the entire region or frag- promoter/regulatory region, the CAT open reading frame ments in front of a herpes simplex virus TK promoter (Fig. and the SV40 enhancer, was active in neuronal and nonneu- 5A), introduced the constructs in PC12, NS20Y, NS26, and ronal cell lines. The SV40 enhancer has been shown to CHO cells, and examined promoter strength by measuring contain binding sites for several transcription factors (21). the CAT activity of the extracts. Fig. 5B reveals that none of These factors might interact with ubiquitous factors bound to the synapsin I/TK promoter fusions increased the strength of the proximal region of the synapsin I promoter and thereby the TK promoter in CHO cells. In the neuronal cell lines the stimulate transcription in nonneuronal cells without the re- longest synapsin I promoter insert (-422/-22; plasmid quirement for specific neuronal factors. pTK3) increased CAT activity 2.5-fold in PC12 and NS26 and To identify DNA sequences responsible for tissue-specific 3.1-fold in NS20Y cells, suggesting that this fragment in- expression we carried out deletion mutagenesis ofthe human cludes one or more elements that increase transcription synapsin I promoter. We showed that the region from -115 particularly in neuronal cells. Some shorter synapsin I pro- to +47 represents a basal (constitutive) promoter that directs moter sequences increased the transcription slightly. These expression in neuronal and nonneuronal cells. Experiments data reveal that the synapsin I promoter/regulatory sequence using hybrid synapsin I/TK promoters revealed that the -422/-22 must contain more than one cell-selective ele- synapsin promoter region from -422 to -22 increased the ment, because (with the exception of pTK4 in NS26 cells) strength ofthe TK promoter in PC12 and neuroblastoma cells none of the shorter fragments tested reached the stimulating but not in CHO cells. This suggests that this fragment effect on transcription as the entire 401-bp fragment. includes cis-acting sequences that direct increased expres- sion in neuronal cells. Recently, a study analyzing the rat synapsin I promoter in DISCUSSION NS20Y cells (7) indicated that the region from -225 to + 105 We report here results of an analysis of the cis-acting DNA may be sufficient for cell-type-specific transcription. Results sequences that control transcription of the human synapsin I obtained with 12 different cell lines as with multiple promoter gene. We have found that a 2-kb fragment of the 5' upstream fragments suggest that the situation may be more complex. region of the synapsin I gene directed tissue-specific expres- The synapsin I promoter region from -422 to -235 and sion of a reporter gene in various neuronal cell lines but not probably further upstream regions are necessary to establish in cell lines derived from nonneuronal tissue. However, not tight tissue specificity. Sauerwald et al. (7) discussed the Downloaded by guest on September 25, 2021 Biochemistry: Thiel et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3435

PstI Auul Styl NarI PvuII f;~~~~~~~ ~~----- synapsin I A B relative CAT activity human synapsin I promoter 1 2 3

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TK v .!-, .-,, 11 __t ~~ffCAT IpTK-3 Z . , .77777,ZZ , 77,z,,,,z,,, zI'll .11111111=11 . D TK F -7 - ::- F W D p I pTK-4 - ;-!, lls...... M5777770- IMU,21MCAT 0

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FIG. 5. Expression of hybrid synapsin I/TK promoters in PC12, neuroblastoma, and CHO cells. (A) Schematic diagram of constructs containing synapsin I promoter fragments cloned in front of a herpes virus TK promoter. (B) Histogram showing CAT activities of extracts of PC12, neuroblastoma (NS20Y and NS26), and CHO cells transfected with hybrid synapsin I/TK promoter plasmids shown in A. The amount of CAT activity detected when pTK-CAT was transfected is arbitrarily set at 1 for each cell line.

involvement of two sequence elements called SNN (region 3. Bahler, M. & Greengard, P. (1987) Nature (London) 326, -214/-200, sequence 5'-CCTTCGCCCCCGC-3', and 704-707. sequence 5'-CGCGCTGAC-3', in the human 4. Petrucci, T. C. & Morrow, J. S. (1987) J. Cell Biol. 105, -164/-156, 1355-1363. synapsin I gene). However, when we fused a restriction 5. Thiel, G., Sudhof, T. C. & Greengard, P. (1990) J. Biol. Chem. fragment of the synapsin I promoter containing both SNN 265, 16527-16533. sequences to a herpes virus TK promoter, the activity 6. Sudhof, T. C. (1990) J. Biol. Chem. 265, 7849-7852. increased only slightly in PC12 and NS20Y cells (plasmid 7. Sauerwald, A., Hoesche, C., Oschwald, R. & Killimann, pTK4). Only in NS26 cells were we able to measure a M. W. (1990) J. Biol. Chem. 265, 14932-14937. significant increase of 2.8-fold over the of 8. Cato, A. C. B., Miksicek, R., Schutz, G., Arnemann, J. & expression pTK- Beato, M. (1986) EMBO J. 5, 2237-2240. CAT. Oligonucleotides containing only one of the SNN 9. Biedler, J. L., Helson, L. & Spengler, B. A. (1973) CancerRes. sequences inserted upstream of the TK promoter (plasmids 33, 2643-2652. pTK5 and pTK6) increased the expression only minimally in 10. Hammond, D. N., Wainer, B. H., Tonsgard, J. H. & Heller, A. neuronal cell lines. Therefore, the role of the SNN elements (1986) Science 234, 1237-1240. in tissue-specific gene expression is not yet clear. Further 11. Lee, H. J., Hammond, D. N., Large, T. H. & Wainer, B. H. (1990) Dev. Brain. Res. 52, 219-228. analysis of the synapsin I promoter should increase our 12. Lee, H. J., Hammond, D. N., Large, T. H., Roback, J. D., understanding of long-term regulation of synapsin I, specif- Sim, J. A., Brown, D. A., Otten, U. H. & Wainer, B. H. (1990) ically, and of regulation of neuronal gene expression, in J. Neurosci. 10, 1779-1787. general. 13. van der Eb, A. J. & Graham, F. L. (1980) Methods Enzymol. 65, 826-839. We are grateful to June Biedler, Joseph Buxbaum, Samuel Gandy, 14. FeIgner, P. L. & Holms, M. (1989) Focus 11, 21-25. Marshall Nirenberg, Kathryn Miles, B. A. Spengler, and Bruce 15. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Wainer for providing cell lines, to Andrew J. Czernik and Y.-L. Siow Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold for providing , and to Rozanne Sandri-Goldin for the Spring Harbor, NY). plasmid We thank Michelle E. Ehrlich and Piera Cic- 16. Sleigh, M. (1986) Anal. Biochem. 156, 251-256. pICP4CAT. 17. Seed, B. & Sheen, J.-Y. (1988) Gene 67, 271-277. chetti for a critical reading of the manuscript. This study was 18. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, supported by U.S. Public Health Service Grant MH39327. W. J. (1979) Biochemistry 18, 5294-5299. 19. Sudhof, T. C., Van der Westhuyzen, D. R., Goldstein, J. L., 1. De Camilli, P., Benfenati, P., Valtorta, F. & Greengard, P. Brown, M. S. & Russell, D. W. (1987) J. Biol. Chem. 262, (1991) Annu. Rev. Cell Biol. 6, 433-460. 10773-10779. 2. Sudhof, T. C., Czernik, A. J., Kao, H.-T., Takai, K., 20. Smale, S. T., Schmidt, M. C., Berk, A. J. & Baltimore, D. Johnston, P. A., Horiuchi, A., Kanazir, S. D., Wagner, S. D., (1990) Proc. Natl. Acad. Sci. USA 87, 4509-4515. Perin, M. S., De Camilli, P. & Greengard, P. (1989) Science 21. Jones, N. C., Rigby, P. W. & Ziff, E. B. (1988) Genes Dev. 2, 245, 1474-1480. 267-281. Downloaded by guest on September 25, 2021