Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3431-3435, April 1991

Biochemistry

Characterization of tissue-specific transcription by the human synapsin I gene 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 synaptic vesicle proteins that are derived by differential splicing from a single gene. To identify control elements directing the neuronal expression of synapsins Ia/b, we functionally analyzed the promoter region of the human synapsin I gene. A hybrid gene was constructed containing 2 kilobases of 5' flanking sequence from the synapsin I gene fused to the bacterial gene chloramphenicol acetyltransferase and transfected into 12 different neuronal and nonneuronal cell lines. In general, expression of the chimeric reporter gene showed excellent correlation with endogenous expression of synapsin I in different neuronal cell lines, whereas transcription was low in all nonneuronal cell lines examined. The addition of the simian virus 40 enhancer promoted non-tissue-specific expression. Deletion mutagenesis of the synapsin I promoter revealed the presence of positive and negative sequence elements. A basal (constitutive) promoter that directs reporter gene expression in neuronal and nonneuronal cell lines was mapped to the region -115 to +47. The promoter region from -422 to -22 contains positive elements that upon fusion with the herpes simplex virus thymidine kinase promoter potentiate its transcription in PC12 and neuroblastoma cells but not in Chinese hamster ovary cells.

Synapsins Ia and Ib (derived from a single gene and collectively referred to as synapsin I) are peripheral membrane proteins that are localized on small synaptic vesicles in the nerve terminal (1, 2). A probable function of synapsin I consists in the linking of synaptic vesicles to elements of the cytoskeleton (3, 4), a function that is controlled by the phosphorylation state of synapsin I. Synaptic vesicles contain two other members of the synapsin family (2, 5), synapsin Ila and synapsin Ilb, which are encoded by a different gene (2). The synapsin I gene is a good candidate for an investigation of neuron-specific gene expression. Synapsin I has a widespread distribution in the central and peripheral nervous systems and is not expressed in nonneuronal cells. Furthermore, synapsin I is a marker of terminal neuronal differentiation in that the expression of synapsin I reflects the final maturation of a neuroblast to a neuron. An investigation of the transcriptional regulation of the synapsins has two goals. First, the regulation of synapsin gene expression controls the amount and types of synapsins in the nerve terminal and is important for synaptic function. Second, an understanding of the regulation of synapsin I gene expression may help in the understanding of gene expression in the nervous system in general. The 5' flanking regions of the human and rat synapsin I genes were recently cloned (6, 7). In the present study, we analyzed the upstream region of the human synapsin I gene and found that 2 kilobases (kb) of the 5' upstream region are

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

MATERIALS AND METHODS Plasmids. A plasmid containing the human synapsin I promoter sequence from -1952 to +47 in pBluescript (Stratagene) was used to construct the chimeric synapsin I-chloramphenicol acetyltransferase (CAT) expression plasmids pSyCAT-1 to pSyCAT-8. Using restriction sites 5' in the promoter sequence and 3' in the pBluescript polylinker, we cloned the following restriction fragments upstream of a promoterless CAT gene in pCAThasic (Promega): a 1998base-pair (bp) Xho I/Sma I fragment (pSyCAT-1), a 1230-bp Asp7l8/Xba I fragment (pSyCAT-2), a 1016-bp Bgl II/Sma I fragment (pSyCAT-3), an 893-bp Sph I/Sma I fragment (pSyCAT-4), a 469-bp Pst I/EcoRI fragment (pSyCAT-5), a 281-bpAlu I/Xba I fragment (pSyCAT-6), a 162-bp Sty IlXba I fragment (pSyCAT-7), and a 70-bp Nar I/Xba I fragment (pSyCAT-8). To construct an expression plasmid containing the simian virus 40 (SV40) enhancer downstream of the CAT gene (pSyCAT-SV40), we inserted the 1998-bp Xho I/Sma I fragment in the polylinker of pCATenhancer (Promega). The plasmid pTK-CAT has been described (8). To construct chimeric synapsin 1/thymidine kinase (TK) promoters we inserted restriction fragments or annealed oligonucleotides containing synapsin I promoter sequences upstream of the TK promoter. The plasmids pTK1, pTK2, and pTK3 contain the sequences -115/-22, -234/-22, and -422/-22, respectively, which were excised from pSyCAT-7, pSyCAT-6, and pSyCAT-5 as HindIII/Nar I fragments. Plasmid pTK4 contains a HindIII/Sty I fragment derived from pSyCAT-6 (promoter sequence -234/-113). The plasmids pTK5 and pTK6 contain annealed oligonucleotides of the synapsin I promoter sequences -187/-116 and -234/-188, respectively. The plasmid pICP4CAT, which contains the herpes simplex 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 pCATcontrol and pCMV/3 were purchased from Promega and Clontech, respectively. Cell Culture. PC12 pheochromocytoma cells (provided by J. Buxbaum, The Rockefeller University) were cultured in 85% Dulbecco's modified Eagle medium (DMEM), 10% fetal bovine serum (FBS), 5% horse serum, 100 units of penicillin per ml, and 100 ,zg of streptomycin per ml. The mouse neuroblastoma cell lines NS20Y and NS26 (a gift of M. Nirenberg, National Institutes of Health), the monkey kidney fibroblast cell line CV-1, Chinese hamster ovary (CHO) cells (kindly provided by S. E. Gandy, The Rockefeller UniverAbbreviations: CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; SV40, simian virus 40; CHO, Chinese hamster ovary. tTo whom reprint requests should be addressed at: The Rockefeller University, 1230 York Avenue, New York, NY 10021.

The publication costs of this article were defrayed in part by page charge payment. 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 maintained in 90% DMEM, 10% FBS, 100 units of penicillin per ml, and 100 ug of streptomycin per ml. The human neuroblastoma cell 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 penicillin per ml, and 100 ,ug of streptomycin per ml. The mouse neuroblastoma cell line N18TG2 and the immortalized cell lines SN48, SN56, and HN25, derived from somatic fusion of septal or hippocampal neurons with N18TG2 neuroblastoma cells (refs. 10-12; kindly provided by Bruce Wainer, The 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 FBS, 100 units of penicillin per ml, and 100 ,ug of streptomycin per ml. Transfections and CAT Assays. Plasmids were banded twice in CsCl prior to transfection. Transfections were done according to ref. 13. Cells were incubated with calcium phosphate/DNA precipitate containing 8 ,g of CAT-plasmid and 2 ,ug of pCMVP for 6 hr, followed by a glycerol shock of 2 min (15% glycerol in Opti-MEM, GIBCO). PC12 and SH-SY5Y cells were lipofected (ref. 14, protocol A), using 16 ,ug of CAT-plasmid, 4 ,ug of pCMVP3, and 50 ,ug of Lipofectin (GIBCO). Cells were harvested after 48-60 hr and lysed by three cycles of freeze-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. Miscellaneous Techniques. Total rat brain RNA was extracted and purified according to ref. 18. Primer extension analysis was performed according to ref. 19. The endogenous synapsin I message was mapped with a primer that was complementary to nucleotides + 127 to + 154 of the rat mRNA. Western blots were probed with an antipeptide antibody to phosphorylation site 3 in synapsin I or with a monoclonal antibody specific for synapsin II (kind gifts of A. J. Czernik and Y.-L. Siow, The Rockfeller University) and a horseradish peroxidase-coupled secondary antibody (Amersham). Blots were developed using enhanced chemiluminescence (ECL, Amersham).

RESULTS Primer Extension. Primer extension analysis using rat brain total RNA and a primer complementary to synapsin I mRNA showed a single extension product (data not shown). Sequences are numbered with + 1 being the first transcribed nucleotide corresponding to nucleotide 1953 in ref. 6. The sequence surrounding the transcript start was identical to the functional initiator sequence 5'-CTCANTCT-3' (20), where A is the transcript start site. Expression of a Synapsin I-CAT Hybrid Gene in Different Cell Lines. A plasmid was constructed containing a 2-kb fragment of the 5' flanking region of the human synapsin I gene (sequence from -1951 to +47) fused to a promoterless CAT gene (Fig. 1A). This plasmid was transiently transfected into different neuronal and nonneuronal cell lines. In all experiments a plasmid that contained the f3-galactosidase gene under control of the cytomegalovirus promoter/ enhancer was cotransfected. CAT activities were then normalized to p-galactosidase activity of each cell extract to correct for transfection efficiencies. Fig. 1B shows a representative CAT assay. The CAT gene under control of the synapsin I promoter/regulatory region was strongly ex-

Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 1. Neuron-specific expression of synapsin I-CAT gene. (A) Structure of the fusion gene pSyCAT-1. (B) Expression of synapsin I-CAT fusion gene in NS26 and L6 cells: a representative autoradiograph showing thin-layer chromatographic separation of butyrylated chloramphenicol from chloramphenicol. As a negative control, we transfected a promoterless CAT gene (plasmid pCATbasic) and as a positive control we used a plasmid that contained the CAT gene under control ofthe herpes simplex virus IE3 gene promoter (plasmid pICP4CAT). (C) Cell-type-specific expression of synapsin I-CAT fusion genes in various neuronal and nonneuronal cell lines. CAT activity was normalized for transfection efficiency, dividing CAT activity by ,B-galactosidase activity, and is expressed as a percentage of the herpes virus IE3 gene promoter activity. At least four experiments were done with each cell type and the mean ± SEM is depicted.

pressed in the neuroblastoma cell line NS26 but not in the muscle cell line L6. The promoterless CAT plasmid pCATbasic was inactive in both cell lines. The plasmid pICP4CAT was transfected as a positive control and it was strongly expressed in both cell lines. To analyze the tissue-specific expression of the synapsin I-CAT fusion gene in more detail, we introduced this construct into several neuronal and nonneuronal cell lines. Each cell line was transfected in parallel with a plasmid containing the CAT gene under the control of the herpes virus IE3 gene promoter/regulatory region (pICP4CAT). This promoter was strongly active in all cell lines examined. The activity of this promoter was arbitrarily set at 100% and the transcription of the synapsin I-CAT gene was expressed as a percentage of the herpes virus IE3 gene promoter activity. Fig. 1C shows that the human synapsin I promoter is most active in PC12 cells and in the mouse neuroblastoma cell lines NS20Y and NS26. The fusion gene is either not transcribed or transcribed only at low levels in the neuroblastoma cell lines SH-SY5Y

Biochemistry: Thiel et al. and N18TG2 and in the immortalized cell line HN25. This indicates that in this aspect the HN25 cells resemble the neuroblastoma parent more than the hippocampal neuron. However, the transcription is increased 8.8-fold and 10.2-fold in the immortalized cell lines SN48 and SN56, respectively, in comparison to the parent N18TG2 cells, providing further evidence that these cell lines reached a higher differentiation level than the neuroblastoma cells. The synapsin I-CAT gene was either not expressed or only poorly expressed in all nonneuronal cell lines, suggesting that the 2-kb promoter/ regulatory region of the human synapsin I gene contains signals for cell-selective gene expression. Immunoblot analysis of the endogenous synapsins in the neuronal cell lines used here reveals that synapsins I and II are both expressed in neuroblastoma cell lines NS20Y and NS26 and in the immortalized septal cells SN48 and SN56 (Fig. 2). In all four cell lines the synapsin I-CAT gene was expressed. The cell lines that did not transcribe the synapsin I promoter, the neuroblastomas SH-SY5Y and N18TG2 and the immortalized hippocampal line HN25, showed little or no endogenous synapsin I. In PC12 cells there was only a small amount of endogenous synapsin I though the transfected synapsin I-CAT gene was very actively transcribed. In general, the level ofendogenous synapsins I and II in all neuronal cell lines was very low compared to that of brain. The SV40 Enhancer Promotes Non-Tissue-Specific Expression. The SV40 enhancer is promiscuously active in numerous rodent and mammalian cells. To test if the enhancer influences tissue-specific expression of the human synapsin 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 pSyCATSV40). Fig. 3B shows a thin-layer chromatographic separation of butyrylated chloramphenicol from chloramphenicol. The plasmid pCATcontrol, containing the CAT gene under control of the SV40 promoter and enhancer, was weakly expressed in NS26 neuroblastoma cells. In L6 cells, however, a good signal was seen. The plasmid pCATenhancer, which contains a promoterless CAT gene and the SV40 enhancer, was used as a negative control. No CAT activity was seen. Finally, the plasmid pSyCAT-SV40 is expressed in neuronal and nonneuronal cell lines. A quantitative analysis (depicted in Fig. 3C) revealed that the SV40 enhancer does not increase expression of the hybrid gene in the neuronal cell lines PC12 and NS26. However, in the nonneuronal cell lines L6, CHO, and HepG2 the enhancer increased CAT activity 58-fold, 52-fold, and 21-fold, respectively. Deletion Analysis of the Human Synapsin I Promoter. To localize DNA sequences responsible for neuron-specific gene expression, we made progressive 5' deletions of the human synapsin I promoter/regulatory region (Fig. 4A). The dele-

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Proc. Natl. Acad. Sci. USA 88 (1991)

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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 in 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 neuronal 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 +47 (plasmid pSyCAT-7). This sequence represents the basal (constitutive) promoter that contains no tissue-specific element. However, in comparison to pSyCAT-1, CAT activities increased in CV-1 and CHO cells 3.0- and 3.6-fold, respectively, after deletion of sequences upstream from -422, suggesting that some cell-specific restriction had been deleted. The hybrid gene pSyCAT-8, including the synapsin I promoter sequence from -23 to +47, shows only low expres-

Biochemistry: Thiel et al.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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Effect of Synapsin I Promoter Fragments on Heterologous Promoters. The hybrid gene containing the synapsin I promoter/regulatory region from -422 to +47 (pSyCAT-5) showed increased expression of the reporter gene in CV-1 and CHO cells compared to the synapsin I-CAT hybrid genes pSyCAT-1 to pSyCAT-4. To test if this sequence contains a tissue-specific element, we fused the entire region or fragments in front of a herpes simplex virus TK promoter (Fig. 5A), introduced the constructs in PC12, NS20Y, NS26, and CHO cells, and examined promoter strength by measuring the CAT activity of the extracts. Fig. 5B reveals that none of the synapsin I/TK promoter fusions increased the strength of the TK promoter in CHO cells. In the neuronal cell lines the longest synapsin I promoter insert (-422/-22; plasmid pTK3) increased CAT activity 2.5-fold in PC12 and NS26 and 3.1-fold in NS20Y cells, suggesting that this fragment includes one or more elements that increase transcription particularly in neuronal cells. Some shorter synapsin I promoter sequences increased the transcription slightly. These data reveal that the synapsin I promoter/regulatory sequence -422/-22 must contain more than one cell-selective element, because (with the exception of pTK4 in NS26 cells) none of the shorter fragments tested reached the stimulating effect on transcription as the entire 401-bp fragment.

DISCUSSION We report here results of an analysis of the cis-acting DNA sequences that control transcription of the human synapsin I gene. We have found that a 2-kb fragment of the 5' upstream region of the synapsin I gene directed tissue-specific expression of a reporter gene in various neuronal cell lines but not in cell lines derived from nonneuronal tissue. However, not

all neuronal cell lines tested expressed the hybrid synapsin I-CAT gene. Only cells that represent a higher differentiation level expressed synapsin I. We found that immortalized septal cells have the ability to transcribe the introduced synapsin I-CAT hybrid gene. In contrast to the neuroblastoma N18TG2 cells, these cells appear to contain the transacting factors necessary for the expression of the synapsin I gene. A hybrid gene containing 2 kb of the synapsin I promoter/regulatory region, the CAT open reading frame and the SV40 enhancer, was active in neuronal and nonneuronal cell lines. The SV40 enhancer has been shown to contain binding sites for several transcription factors (21). These factors might interact with ubiquitous factors bound to the proximal region of the synapsin I promoter and thereby stimulate transcription in nonneuronal cells without the requirement for specific neuronal factors. To identify DNA sequences responsible for tissue-specific expression we carried out deletion mutagenesis of the human synapsin I promoter. We showed that the region from -115 to +47 represents a basal (constitutive) promoter that directs expression in neuronal and nonneuronal cells. Experiments using hybrid synapsin I/TK promoters revealed that the synapsin promoter region from -422 to -22 increased the strength of the TK promoter in PC12 and neuroblastoma cells but not in CHO cells. This suggests that this fragment includes cis-acting sequences that direct increased expression in neuronal cells. Recently, a study analyzing the rat synapsin I promoter in NS20Y cells (7) indicated that the region from -225 to + 105 may be sufficient for cell-type-specific transcription. Results obtained with 12 different cell lines as with multiple promoter fragments suggest that the situation may be more complex. The synapsin I promoter region from -422 to -235 and probably further upstream regions are necessary to establish tight 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 human synapsin I gene). However, when we fused a restriction fragment of the synapsin I promoter containing both SNN sequences to a herpes virus TK promoter, the activity increased only slightly in PC12 and NS20Y cells (plasmid pTK4). Only in NS26 cells were we able to measure a significant increase of 2.8-fold over the expression of pTKCAT. Oligonucleotides containing only one of the SNN sequences inserted upstream of the TK promoter (plasmids pTK5 and pTK6) increased the expression only minimally in neuronal cell lines. Therefore, the role of the SNN elements in tissue-specific gene expression is not yet clear. Further analysis of the synapsin I promoter should increase our understanding of long-term regulation of synapsin I, specifically, and of regulation of neuronal gene expression, in general. We are grateful to June Biedler, Joseph Buxbaum, Samuel Gandy, Marshall Nirenberg, Kathryn Miles, B. A. Spengler, and Bruce Wainer for providing cell lines, to Andrew J. Czernik and Y.-L. Siow for providing antibodies, and to Rozanne Sandri-Goldin for the plasmid pICP4CAT. We thank Michelle E. Ehrlich and Piera Cicchetti for a critical reading of the manuscript. This study was supported by U.S. Public Health Service Grant MH39327. 1. De Camilli, P., Benfenati, P., Valtorta, F. & Greengard, P. (1991) Annu. Rev. Cell Biol. 6, 433-460. 2. Sudhof, T. C., Czernik, A. J., Kao, H.-T., Takai, K., Johnston, P. A., Horiuchi, A., Kanazir, S. D., Wagner, S. D., Perin, M. S., De Camilli, P. & Greengard, P. (1989) Science 245, 1474-1480.

3. Bahler, M. & Greengard, P. (1987) Nature (London) 326, 704-707. 4. Petrucci, T. C. & Morrow, J. S. (1987) J. Cell Biol. 105, 1355-1363. 5. Thiel, G., Sudhof, T. C. & Greengard, P. (1990) J. Biol. Chem. 265, 16527-16533. 6. Sudhof, T. C. (1990) J. Biol. Chem. 265, 7849-7852. 7. Sauerwald, A., Hoesche, C., Oschwald, R. & Killimann, M. W. (1990) J. Biol. Chem. 265, 14932-14937. 8. Cato, A. C. B., Miksicek, R., Schutz, G., Arnemann, J. & Beato, M. (1986) EMBO J. 5, 2237-2240. 9. Biedler, J. L., Helson, L. & Spengler, B. A. (1973) CancerRes. 33, 2643-2652. 10. Hammond, D. N., Wainer, B. H., Tonsgard, J. H. & Heller, A. (1986) Science 234, 1237-1240. 11. Lee, H. J., Hammond, D. N., Large, T. H. & Wainer, B. H. (1990) Dev. Brain. Res. 52, 219-228. 12. Lee, H. J., Hammond, D. N., Large, T. H., Roback, J. D., Sim, J. A., Brown, D. A., Otten, U. H. & Wainer, B. H. (1990) J. Neurosci. 10, 1779-1787. 13. van der Eb, A. J. & Graham, F. L. (1980) Methods Enzymol. 65, 826-839. 14. FeIgner, P. L. & Holms, M. (1989) Focus 11, 21-25. 15. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 16. Sleigh, M. (1986) Anal. Biochem. 156, 251-256. 17. Seed, B. & Sheen, J.-Y. (1988) Gene 67, 271-277. 18. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 19. Sudhof, T. C., Van der Westhuyzen, D. R., Goldstein, J. L., Brown, M. S. & Russell, D. W. (1987) J. Biol. Chem. 262, 10773-10779. 20. Smale, S. T., Schmidt, M. C., Berk, A. J. & Baltimore, D. (1990) Proc. Natl. Acad. Sci. USA 87, 4509-4515. 21. Jones, N. C., Rigby, P. W. & Ziff, E. B. (1988) Genes Dev. 2, 267-281.