Is RE1/NRSE a Common cis-Regulatory Sequence for ChAT and VAChT Genes?
2000; Elsevier BV; Volume: 275; Issue: 47 Linguagem: Inglês
10.1074/jbc.m006895200
ISSN1083-351X
AutoresStéphanie De Gois, Leı̈la Houhou, Yoshio Oda, Marilys Corbex, Fabrice Pajak, Etienne Thévenot, Guilan Vodjdani, Jacques Mallet, Sylvie Berrard,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoCholine acetyltransferase (ChAT), the biosynthetic enzyme of acetylcholine, and the vesicular acetylcholine transporter (VAChT) are both required for cholinergic neurotransmission. These proteins are encoded by two embedded genes, the VAChT gene lying within the first intron of the ChAT gene. In the nervous system, both ChAT and VAChT are synthesized only in cholinergic neurons, and it is therefore likely that the cell type-specific expression of their genes is coordinately regulated. It has been suggested that a 2336-base pair genomic region upstream from the ChAT and VAChT coding sequences drives ChAT gene expression in cholinergic structures. We investigated whether this region also regulates VAChT gene transcription. Transfection assays showed that this region strongly represses the activity of the native VAChT promoters in non-neuronal cells, but has no major effect in neuronal cells whether or not they express the endogenous ChAT and VAChT genes. The silencer activity of this region is mediated solely by a repressor element 1 or neuron-restrictive silencer element (RE1/NRSE). Moreover, several proteins, including RE1-silencing transcription factor or neuron-restrictive silencer factor, are recruited by this regulatory sequence. These data suggest that this upstream region and RE1/NRSE co-regulate the expression of the ChAT and VAChT genes. Choline acetyltransferase (ChAT), the biosynthetic enzyme of acetylcholine, and the vesicular acetylcholine transporter (VAChT) are both required for cholinergic neurotransmission. These proteins are encoded by two embedded genes, the VAChT gene lying within the first intron of the ChAT gene. In the nervous system, both ChAT and VAChT are synthesized only in cholinergic neurons, and it is therefore likely that the cell type-specific expression of their genes is coordinately regulated. It has been suggested that a 2336-base pair genomic region upstream from the ChAT and VAChT coding sequences drives ChAT gene expression in cholinergic structures. We investigated whether this region also regulates VAChT gene transcription. Transfection assays showed that this region strongly represses the activity of the native VAChT promoters in non-neuronal cells, but has no major effect in neuronal cells whether or not they express the endogenous ChAT and VAChT genes. The silencer activity of this region is mediated solely by a repressor element 1 or neuron-restrictive silencer element (RE1/NRSE). Moreover, several proteins, including RE1-silencing transcription factor or neuron-restrictive silencer factor, are recruited by this regulatory sequence. These data suggest that this upstream region and RE1/NRSE co-regulate the expression of the ChAT and VAChT genes. choline acetyltransferase electrophoretic mobility shift assay glyceraldehyde-3-phosphate dehydrogenase phosphoglycerate kinase repressor element 1/neuron-restrictive silencer element RE1-silencing transcription factor/neuron-restrictive silencer factor thymidine kinase vesicular acetylcholine transporter base pair(s) reverse transcription polymerase chain reaction In cholinergic neurons, the neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT)1 and is then translocated into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The expression of ChAT and VAChT proteins is tightly linked. Both ChAT and VAChT are absolutely required for cholinergic neurotransmission (1Rand J.B. Russell R.L. Genetics. 1984; 106: 227-248Crossref PubMed Google Scholar), and, in the nervous system, these proteins are only synthesized in cholinergic cells. Moreover, ChAT and VAChT are both expressed from the same gene locus, referred to as “cholinergic gene locus.” Indeed, in nematode,Drosophila, and mammals, the VAChT gene resides within the first intron of the ChAT gene and in the same transcriptional orientation (Fig. 1; Refs. 2Alfonso A. Grundahl K. McManus J.R. Asbury J.M. Rand J.B. J. Mol. Biol. 1994; 241: 627-630Crossref PubMed Scopus (85) Google Scholar, 3Bejanin S. Cervini R. Mallet J. Berrard S. J. Biol. Chem. 1994; 269: 21944-21947Abstract Full Text PDF PubMed Google Scholar, 4Erickson J.D. Varoqui H. Schafer M.K. Modi W. Diebler M.F. Weihe E. Rand J. Eiden L.E. Bonner T.I. Usdin T.B. J. Biol. Chem. 1994; 269: 21929-21932Abstract Full Text PDF PubMed Google Scholar, 5Naciff J.M. Misawa H. Dedman J.R. Neuroreport. 1997; 8: 3467-3473Crossref PubMed Scopus (42) Google Scholar, 6Kitamoto T. Wang W. Salvaterra P.M. J. Biol. Chem. 1998; 273: 2706-2713Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).Figure 2Schematic representation of the rat VAChT gene (top line) and of the reporter gene constructs used for transfections that contain fragments of the VAChT gene promoter region. Boxes represent exon R and the VAChT coding region. Arrows indicate the transcription initiation sites of the VAChT gene. Regions 1, 2, and 3 were defined as follows: region 1, from −5605 to −3269 (0 corresponds to the position of the translation initiation site of VAChT), between EcoRI and HindIII restriction sites; region 2, from −3268 to −1373 (restriction sites HindIII and HindIII); region 3, from −1372 to −346 (restriction sites HindIII and SphI). Region 2 contains the promoter region common to the ChAT and VAChT genes, and region 3 the two specific promoters of the VAChT gene. The open circle represents the RE1/NRSE homologous sequence. The crossed circleand m indicate that the RE1/NRSE is mutated. These VAChT gene 5′ regions were inserted into the plasmid KSluc upstream from the reporter gene encoding firefly luciferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Schematic representation of the rat cholinergic gene locus (top line) and of the various rat ChAT and VAChT mRNA species (7Kengaku M. Misawa H. Deguchi T. Brain Res. Mol. Brain Res. 1993; 18: 71-76Crossref PubMed Scopus (78) Google Scholar, 11Cervini R. Houhou L. Pradat P.F. Bejanin S. Mallet J. Berrard S. J. Biol. Chem. 1995; 270: 24654-24657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Black filled boxes indicate coding sequences; open and gray filled boxes represent noncoding sequences of the VAChT and ChAT genes, respectively. R, N, and M are the three non-coding exons of the ChAT gene. Exon R, which is shared by both ChAT and VAChT genes, is shown in gray andwhite. Vertical bars within the exons represent splice sites. Arrows indicate the transcriptional start sites identified for the ChAT and/or VAChT genes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The mode of transcription of these two unconventionally organized genes has been elucidated in the rat. In this species, the 5′ end of the ChAT gene is composed of three non-coding exons, designated R, N, and M, that are alternatively contained into five different mRNAs (Fig. 1; Ref. 7Kengaku M. Misawa H. Deguchi T. Brain Res. Mol. Brain Res. 1993; 18: 71-76Crossref PubMed Scopus (78) Google Scholar). These transcripts are generated from several promoters, two of them lying upstream from exons R and M (Fig. 1; Refs. 8Ibanez C. Persson H. Eur. J. Neurosci. 1991; 3: 1309-1315Crossref PubMed Scopus (49) Google Scholar, 9Bejanin S. Habert E. Berrard S. Edwards J.B. Loeffler J.P. Mallet J. J. Neurochem. 1992; 58: 1580-1583Crossref PubMed Scopus (49) Google Scholar, 10Misawa H. Ishii K. Deguchi T. J. Biol. Chem. 1992; 267: 20392-20399Abstract Full Text PDF PubMed Google Scholar). Multiple promoter regions have also been identified for the VAChT gene and give rise to five VAChT mRNAs with different 5′-untranslated sequences. One promoter, located upstream from the exon R, may be common to the ChAT gene, whereas two VAChT-specific promoters lie downstream from this exon (Fig. 1; Refs. 3Bejanin S. Cervini R. Mallet J. Berrard S. J. Biol. Chem. 1994; 269: 21944-21947Abstract Full Text PDF PubMed Google Scholar and 11Cervini R. Houhou L. Pradat P.F. Bejanin S. Mallet J. Berrard S. J. Biol. Chem. 1995; 270: 24654-24657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These various transcripts each encode single ChAT or VAChT proteins. This unusual organization for two functionally related mammalian genes suggests that the ChAT and VAChT genes may be co-regulated. Indeed, coordinated up-regulation of ChAT and VAChT transcripts by various extracellular factors such as leukemia inhibitory factor, ciliary neurotrophic factor, retinoic acid, cAMP, glucocorticoids, nerve growth factor, and even stress has been demonstrated (12Berrard S. Varoqui H. Cervini R. Israël M. Mallet J. Diebler M.F. J. Neurochem. 1995; 65: 939-942Crossref PubMed Scopus (130) Google Scholar, 13Berse B. Blusztajn J.K. J. Biol. Chem. 1995; 270: 22101-22104Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 14Berse B. Blusztajn J.K. FEBS Lett. 1997; 410: 175-179Crossref PubMed Scopus (41) Google Scholar, 15Misawa H. Takahashi R. Deguchi T. Neuroreport. 1995; 6: 965-968Crossref PubMed Scopus (74) Google Scholar, 16Tian X. Sun X. Suszkiw J.B. Neurosci Lett. 1996; 209: 134-136Crossref PubMed Scopus (32) Google Scholar, 17Kaufer D. Friedman A. Seidman S. Soreq H. Nature. 1998; 393: 373-377Crossref PubMed Scopus (531) Google Scholar, 18Shimojo M. Wu D. Hersh L.B. J. Neurochem. 1998; 71: 1118-1126Crossref PubMed Scopus (36) Google Scholar, 19Oosawa H. Fujii T. Kawashima K. J. Neurosci. Res. 1999; 57: 381-387Crossref PubMed Scopus (58) Google Scholar). Moreover, several studies have reported that, in most regions of the central nervous system, ChAT and VAChT mRNAs and proteins have a comparable anatomical distribution and are co-localized within the same neurons (20Schäfer M.K. Weihe E. Varoqui H. Eiden L.E. Erickson J.D. J. Mol. Neurosci. 1994; 5: 1-26Crossref PubMed Scopus (98) Google Scholar, 21Schäfer M.K. Weihe E. Erickson J.D. Eiden L.E. J. Mol. Neurosci. 1995; 6: 225-235Crossref PubMed Scopus (77) Google Scholar, 22Arvidsson U. Riedl M. Elde R. Meister B. J. Comp. Neurol. 1997; 378: 454-467Crossref PubMed Scopus (342) Google Scholar, 23Ichikawa T. Ajiki K. Matsuura J. Misawa H. J. Chem. Neuroanat. 1997; 13: 23-39Crossref PubMed Scopus (199) Google Scholar). Therefore, the ChAT and VAChT genes may share transcriptional regulatory sequences that restrict their expression to cholinergic neurons. Transient transfection experiments previously revealed that the 3-kilobase pair genomic sequence upstream from the translation initiation site of VAChT (regions 2 and 3, Fig. 2) does not contain such regulatory DNA sequences (8Ibanez C. Persson H. Eur. J. Neurosci. 1991; 3: 1309-1315Crossref PubMed Scopus (49) Google Scholar, 11Cervini R. Houhou L. Pradat P.F. Bejanin S. Mallet J. Berrard S. J. Biol. Chem. 1995; 270: 24654-24657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In contrast, various data suggest that a 2336-bp region just upstream and designated region 1 (Fig. 2) regulates the cholinergic neuron-specific expression of the ChAT gene. (i) In non-neuronal cells, in vitro, this region is able to repress the activity of the ChAT/VAChT promoter region 2, as well as that of a heterologous promoter (8Ibanez C. Persson H. Eur. J. Neurosci. 1991; 3: 1309-1315Crossref PubMed Scopus (49) Google Scholar, 24Lönnerberg P. Schoenherr C.J. Anderson D.J. Ibanez C.F. J. Biol. Chem. 1996; 271: 33358-33365Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar); (ii) in transgenic mice, region 1 drove the expression of a downstream heterologous promoter in various cholinergic structures (25Lönnerberg P. Lendahl U. Funakoshi H. Arhlund-Richter L. Persson H. Ibanez C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4046-4050Crossref PubMed Scopus (26) Google Scholar); (iii) the pattern of transgene expression in the brain of these mice paralleled both qualitatively and quantitatively that of endogenous ChAT mRNAs; and (iv) in spinal cord, transgene expression was developmentally regulated, similarly to the endogenous ChAT gene. Region 1 contains a 21-bp sequence homologous to the neuron-restrictive silencer element (NRSE, Ref. 26Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full Text PDF PubMed Scopus (342) Google Scholar) of the SCG10 gene or repressor element 1 (RE1, Ref. 27Kraner S.D. Chong J.A. Tsay H.J. Mandel G. Neuron. 1992; 9: 37-44Abstract Full Text PDF PubMed Scopus (281) Google Scholar) of the type II sodium channel gene. A similar motif is present in the regulatory regions of several other genes whose expression is restricted to neurons (28Schoenherr C.J. Paquette A.J. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9881-9886Crossref PubMed Scopus (355) Google Scholar). The transcription factor that binds RE1/NRSE is a zinc-finger repressor protein designated REST (for RE1-silencing transcription factor, Ref. 29Chong J.A. Tapia-Ramirez J. Kim S. Toledo-Aral J.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (906) Google Scholar) or NRSF (for neuron-restrictive silencer factor, Ref. 30Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (910) Google Scholar). REST/NRSF mRNA and protein have been detected in a majority of non-neuronal cell types and in undifferentiated neuronal progenitors (29Chong J.A. Tapia-Ramirez J. Kim S. Toledo-Aral J.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (906) Google Scholar, 30Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (910) Google Scholar), suggesting that REST/NRSF prevents full expression of the neuronal phenotype during early neurogenesis. In addition, REST/NRSF mRNA is differentially expressed in mature neurons of the adult brain (31Palm K. Belluardo N. Metsis M. Timmusk T. J. Neurosci. 1998; 18: 1280-1296Crossref PubMed Google Scholar). REST/NRSF may thus be also required for the differential expression of target genes in different neuronal subpopulations. The RE1/NRSE of region 1, in vitro, represses the expression of a heterologous downstream promoter in non-neuronal cells by binding REST/NRSF (24Lönnerberg P. Schoenherr C.J. Anderson D.J. Ibanez C.F. J. Biol. Chem. 1996; 271: 33358-33365Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We investigated whether region 1 and more specifically RE1/NRSE are also involved in the cell-specific regulation of the VAChT gene expression in the rat. Transient transfections of non-neuronal and neuronal-like cells revealed that region 1 and RE1/NRSE are parts of a regulatory mechanism that strongly represses VAChT gene expression in non-neuronal cells, but not in neuronal cells. We also demonstrated the existence in non-neuronal cells of several nuclear proteins or protein complexes that interact specifically with this silencer element. Region 1 and RE1/NRSE may thus co-regulate both ChAT and VAChT gene expression. Moreover, the cholinergic specific expression of VAChT gene requires the contribution of additional control elements of the cholinergic gene locus. Total RNA was purified from indicated cell lines by the RNable method (Eurobio) and quantified spectrophotometrically. Single-stranded cDNAs were synthesized with avian myeloblastosis virus reverse transcriptase (Promega) using 1 μg of RNA as the template and random hexamers (pd(N)6, Amersham Pharmacia Biotech) as primers. Reactions without RNA or without reverse transcriptase were used as controls. A Prolabo thermal cycler was used for PCR with one eighth of the reverse transcription reaction mix as template and Taq DNA polymerase (Promega), as described previously (12Berrard S. Varoqui H. Cervini R. Israël M. Mallet J. Diebler M.F. J. Neurochem. 1995; 65: 939-942Crossref PubMed Scopus (130) Google Scholar). The number of PCR cycles was 22 for G3PDH, 32 for VAChT, and 35 for ChAT and REST/NRSF cDNA amplifications. The sequences of the primers used are as follows: ChAT (+), GTCTTGGATGTTGTCATTAATTTC; ChAT (−), TCTCTGGTAAAGCCTGTAGTAAGC; VAChT (+), CACCAAACTGTCGGAAGCGGTG; VAChT(−), GCAGCGAAGAGCGTGGCATAGTC; REST/NRSF (+), GAGTCTGAAGAACAGTTCGTACAT; REST/NRSF (−), CTTGAAGTTGCTCCTATCGGCTGT; G3PDH (+), ACCACAGTCCATGCCATCAC; G3PDH (−), TCCACCACCCTGTTGCTGTA. Various fragments of the promoter region of the rat VAChT gene (Fig. 2) were subcloned into the promoterless plasmid KSluc, upstream from the luciferase reporter gene and downstream from a transcription terminator. The KSluc was constructed by W. Faust and A. M. Catherin. 2W. Faust and A. M. Catherin, unpublished results. To simplify the plasmid nomenclature, we designated as 1, 2, and 3 the 2336-bpEcoRI/HindIII fragment upstream from the VAChT gene promoters, the 1895-bp HindIII/HindIII fragment containing the ChAT/VAChT promoter upstream from exon R, and the 1026-bpHindIII/SphI fragment containing the two VAChT promoters downstream from exon R, respectively (Fig. 2). DNA fragments 2, 3, 23, and 123 were prepared by appropriate digestion of a largerEcoRI/EcoRI genomic fragment of 7334 bp, obtained from a rat ChAT/VAChT genomic λEMBL3 clone isolated previously (9Bejanin S. Habert E. Berrard S. Edwards J.B. Loeffler J.P. Mallet J. J. Neurochem. 1992; 58: 1580-1583Crossref PubMed Scopus (49) Google Scholar) and subcloned into pBSKS+ (Bluescript; Stratagene, La Jolla, CA). These fragments were inserted into the uniqueHindIII site of KSluc to generate the reporter plasmids designated pl-2, pl-3, pl-23, and pl-123, respectively. The plasmid pl-m123, which contains a two-base substitution mutation in the RE1/NRSE sequence, was generated from pl-123 by mutagenesis using theCLONTECH Transformer site-directed mutagenesis kit. The plasmids pl-13 and pl-m13 were obtained from pl-123 and pl-m123, respectively, by excision of fragment 2 by HindIII digestion, followed by self-ligation of the plasmids. Cloning junctions and the RE1/NRSE mutation were confirmed by DNA sequence analysis. Two complementary oligonucleotides (cgcgTCCAGCACCACGGACAGTTCCa and cgcgtGGAACTGTCCGTGGTGCTGGA) containing the sequence of the region 1 RE1/NRSE flanked by MluI restriction sites, were annealed. The resulting double-stranded oligonucleotide was inserted into theMluI site of the pPGK-luc vector, described in Ref. 32Millecamps S. Kiefer H. Navarro V. Geoffroy M.C. Robert J.J. Finiels F. Mallet J. Barkats M. Nat. Biotechnol. 1999; 17: 865-869Crossref PubMed Scopus (44) Google Scholar, upstream from the phosphoglycerate kinase (PGK) promoter, to generate pPGK-luc-1, or oligomerized four times in the same orientation to create pPGK-luc-4. The number and orientation of the RE1/NRSE were checked by PCR and sequencing. Plasmid DNA was purified either by two successive equilibrium centrifugations in CsCl gradients followed by a 48-h dialysis against water, or by using the Jetstar plasmid purification system (Genomed). DNA was quantified spectrophotometrically and by scanning pictures of ethidium bromide-stained DNA after migration on 1% agarose gel. The cell lines FR3T3 (Fisher rat fibroblast), N2A (mouse neuroblastoma), and NG108-15 (mouse neuroblastoma N18TG-2 × rat glioma C6BU-1) were grown at 37 °C in a 7% (NG108-15) or 5% (N2A, FR3T3) CO2 atmosphere in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum (ATGC, Inc.). For NG108-15, the culture medium was supplemented with 1 mm sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1× HAT solution (100 μm hypoxanthine, 0.4 μm aminopterin, 16 μm thymidine). Transfections were performed by the calcium phosphate precipitation method (N2A and NG108-15) or by electroporation with a single electrical pulse at 240 V using a Bio-Rad Gene Pulser (FR3T3). Cells (106) were transfected with mixtures of 1 pmol of each reporter plasmid, 4 μg of a SV40 promoter-chloramphenicol acetyltransferase vector (pCAT3-Control Vector, Promega) as an internal control to correct for differences in transfection efficiency, and a carrier DNA (pBluescript, Stratagene) to give a total amount of 12 μg of DNA. For N2A and NG108-15 cells, medium was changed 16 h after transfection. Cells were harvested 40 h after transfection. Luciferase activity was normalized with chloramphenicol acetyltransferase activity determined in the same extract (33Boularand S. Darmon M.C. Ravassard P. Mallet J. J. Biol. Chem. 1995; 270: 3757-3764Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). For each cell line, five to eight independent transfection experiments were performed. In each experiment, constructs were transfected in triplicate. For each construct, at least three independently prepared plasmid DNA samples were used. When the normalized activities driven by different constructs were compared, all the plasmids were prepared the same day. The data were analyzed by using the SAS statistical software (SAS Institute Inc., Cary, NC). Differences between the different groups (cell lines and/or constructs) were tested by analysis of variance (SAS/GLM procedure). The p-values corresponding to the two by two comparison were also computed (SAS/Ismeans and pdiff options; the test corresponds to the standardt test when no more than one explanatory factor is present). The homogeneity of the results according to the DNA preparation and the transfection experiment were tested by introducing these variables as explanatory factors into the analysis of variance models. Results obtained with small subsamples were checked by non-parametric tests. Nuclear extracts were prepared from 108 cells as already described (34Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar) with the following modifications: (i) in buffers A and C, EDTA and EGTA were omitted and ZnSO4 was added to a final concentration of 1 μm, together with an additional mixture of protease inhibitors (0.1 unit/ml aprotinin, 1 μm pepstatin, 1 μm leupeptin, 1 mm iodoacetamide); (ii) nuclear extracts were dialyzed against buffer D (20 mm Hepes, pH 7.9, 250 mmKCl, 2.5 mm MgCl2, 1 mmdithiothreitol, 1 μm ZnSO4, 25% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1 mmiodoacetamide). Protein concentrations were determined using the Bio-Rad assay system. The probe was prepared using a 49-mer double-stranded oligonucleotide, labeled with [α-32P]dCTP by Klenow filling-in, and purified following electrophoresis on a 8% polyacrylamide gel. The binding reaction was performed in a final volume of 20 μl in buffer D containing 10% glycerol. Nuclear extracts (10 μg) were preincubated for 10 min on ice in buffer D complemented with 0.1% Nonidet P-40, 2 μg of poly(dI-dC), and 50 ng of sonicated single-stranded herring sperm DNA. The 32P-end-labeled probe (10 fmol) was added either with or without a molar excess of a competitor oligonucleotide as indicated, and the binding reaction was continued for another 20 min at room temperature. For supershift experiments, either preimmune serum (1 μg) or 1 or 2 μg of an anti-REST/NRSF polyclonal antibody (a generous gift from Dr. Gail Mandel, State University of New York, Stony Brook, NY) was preincubated with the binding reaction prior to addition of the probe. The DNA-protein complexes were resolved by electrophoresis on pre-run 6% native polyacrylamide gels in 0.25× Tris borate/EDTA buffer. The following chemically synthesized double-stranded oligonucleotides with SalI overhangs were used as probes or competitors (unrelated nucleotides are shown in lowercase letters, and mutated nucleotides are underlined): wtRE1/NRSE, tcgacTAGGAACTGTCCAGCACCACGGACAGTTCCGGGAGCCGCg; mRE1/NRSE, tcgacTAGGAACTGTCCAGCACCACTTACAGTTCCGGGAGCCGCg; Y-TPH, tcgacCCTTCTCATTGGCCGCTGCCCAGCTg. Our previous studies have shown that the rat VAChT gene can be transcribed from two distinct domains of the cholinergic gene locus: (i) one upstream from exon R (region 2, Fig.2), which also promotes the transcription of the ChAT gene (3Bejanin S. Cervini R. Mallet J. Berrard S. J. Biol. Chem. 1994; 269: 21944-21947Abstract Full Text PDF PubMed Google Scholar, 11Cervini R. Houhou L. Pradat P.F. Bejanin S. Mallet J. Berrard S. J. Biol. Chem. 1995; 270: 24654-24657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar); (ii) one downstream from exon R (region 3, Fig. 2), which contains two distinct promoters for the VAChT gene. Both promoter regions 2 and 3 are constitutively functional in cholinergic cells, non-cholinergic neurons, and non-neuronal cells (11Cervini R. Houhou L. Pradat P.F. Bejanin S. Mallet J. Berrard S. J. Biol. Chem. 1995; 270: 24654-24657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Here, we used transient transfections to examine the potential involvement of a 5′ upstream domain, region 1, in the regulation of the cell type-specific expression of the VAChT gene. Reporter plasmids were constructed, in which different combinations of the VAChT promoter regions 2 and 3, with or without region 1, were fused to the firefly luciferase reporter gene (Fig. 2). These constructs were used to transfect three murine cell lines chosen for their different properties: NG108-15 (a cholinergic hybrid line), N2A (a non-cholinergic neuroblastoma line), and FR3T3 (a non-neuronal fibroblast line). The presence of ChAT and VAChT mRNAs in NG108-15 was confirmed by RT-PCR experiments. These two transcripts were not detectable in N2A or FR3T3 (Fig. 3). These cell lines were also tested for the presence of REST/NRSF transcripts. In our PCR conditions, REST/NRSF mRNA was only detected in FR3T3 (Fig. 3), indicating that NG108-15 and N2A do not normally express high amounts of REST/NRSF and thus behave as neuronal cells. Numerous independent transfection experiments were performed in NG108-15, N2A, and FR3T3 with different sets of DNA preparations. When cells from the same line were transfected simultaneously with different plasmid preparations of a given construction, the normalized luciferase activities obtained were variable and thus dependent on the DNA preparation, whatever the cell line. Therefore, to compare the transcriptional activities of different constructs, each transfection experiment was carried out with plasmid DNAs that were all prepared at the same time and by the same protocol. However, in each cell line, the order of magnitude of normalized activities of the constructs varied from one experiment to another, whereas the ratio values between the normalized activities of two constructs were similar in the different experiments. We thus present the results of the normalized luciferase activities measured in several independent transient transfection experiments as ratio values obtained with different constructs On the basis of these results, two different statistical analysis were performed (Fig. 4). We first verified in NG108-15, N2A, and FR3T3 our previous observation in other cells (11Cervini R. Houhou L. Pradat P.F. Bejanin S. Mallet J. Berrard S. J. Biol. Chem. 1995; 270: 24654-24657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) that the VAChT promoter regions 2 and 3 can be constitutively transcribed. This confirmed that both regions lack the regulatory motifs responsible for the cholinergic cell-specific expression of the VAChT gene. We next examined the influence of region 1 on the transcriptional activities of the VAChT promoter regions 2 and 3. As shown on Fig. 4(A and D) (ratios pl-123/pl-23 and pl-13/pl-3), the effect of region 1 in neuronal cells was significantly different from that in fibroblastic cells, whereas there was no major difference between its effects in cholinergic and non-cholinergic neuronal cells. In the NG108-15 cell line, transcription from the VAChT gene promoters of regions 2 and 3 together was slightly lower (25%) in the presence than absence of region 1 (Fig. 4 A). This reduction was not observed when region 1 was placed directly adjacent to region 3 (Fig.4 D). In N2A cell line, region 1 had no significant effect on the transcriptional activity of the whole VAChT promoter region 23 (Fig. 4 A), or on that of region 3 alone (Fig.4 D). Region 1 is thus ineffective in these cells. In contrast, region 1 substantially down-regulated the downstream VAChT promoters in fibroblastic cells FR3T3; the promoter activities of regions 23 and 3 were about 4-fold lower in the presence than the absence of region 1 (Fig. 4, A and D). Thus, this regulatory region exhibits a silencer function on the VAChT gene promoters, but only in non-neuronal cells. Note, however, that region 1 by itself was not sufficient to completely abolish these promoter activities in FR3T3 cells in our experimental conditions. Region 1 contains a sequence homologous to RE1/NRSE (24Lönnerberg P. Schoenherr C.J. Anderson D.J. Ibanez C.F. J. Biol. Chem. 1996; 271: 33358-33365Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and this sequence may be responsible for repression of VAChT gene transcription in non-neuronal cells. The contribution of RE1/NRSE was explored by introducing point mutations into its sequence in the construct pl-123 and the chimeric construct pl-13. Two juxtaposing guanines near the center of RE1/NRSE were replaced by thymines. This mutation was previously shown to inactivate the RE1/NRSE motif of the SCG10 gene by abolishing the DNA binding of the repressor protein and thus its silencing activity (26Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full
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