Multiple Functional Sp1 Domains in the Minimal Promoter Region of the Neuronal Nicotinic Receptor α5 Subunit Gene
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.4693
ISSN1083-351X
AutoresAntonio Campos‐Caro, Carmen Carrasco-Serrano, Luis M. Valor, Salvador Viniegra, Juan J. Ballesta, Manuel Criado,
Tópico(s)Ion channel regulation and function
ResumoThe α5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from −111 to −40). Deletion analysis and transient transfections showed that a 266-base pair region (−111 to +155) gave rise to ∼77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the α5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased α5 promoter activity by ∼50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50–69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment −111 to −27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in α5 subunit expression, as has also been demonstrated previously for α3 and β4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits. The α5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from −111 to −40). Deletion analysis and transient transfections showed that a 266-base pair region (−111 to +155) gave rise to ∼77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the α5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased α5 promoter activity by ∼50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50–69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment −111 to −27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in α5 subunit expression, as has also been demonstrated previously for α3 and β4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits. Nicotinic acetylcholine receptors (nAChRs) 1The abbreviations nAChRsnicotinic acetylcholine receptorsbpbase pair(s)EMSAelectrophoretic mobility shift assay are members of the gene superfamily of neurotransmitter-gated ion channels (1Sargent P.B. Annu. Rev. Neurosci. 1993; 16: 403-443Crossref PubMed Scopus (935) Google Scholar, 2Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (564) Google Scholar). These multimeric receptors are heteromers or, in some cases, homomers of subunits (α2–α9 and β2–β4) that exhibit well defined and restricted expression patterns in vivo (1Sargent P.B. Annu. Rev. Neurosci. 1993; 16: 403-443Crossref PubMed Scopus (935) Google Scholar). The diversity of neuronal nAChRs arises, at least in part, from the different combinations of subunits able to form functional nAChRs (3Papke R.L. Prog. Neurobiol. (Oxf.). 1993; 41: 509-531Crossref PubMed Scopus (127) Google Scholar). Thus, it is clear that their differential expression affects the electrophysiological and pharmacological properties of the resultant receptors (4McGehee D.S. Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (898) Google Scholar). Moreover, potential changes in subunit expression in response to modulation of synaptic function might have important consequences on the signals transduced by nAChRs (5Role L.W. Berg D.K. Neuron. 1996; 16: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). nicotinic acetylcholine receptors base pair(s) electrophoretic mobility shift assay To understand how the regional and developmental expression of nAChR subunits is controlled, we have started to analyze the transcriptional mechanisms that regulate expression of the nAChR subunits expressed in chromaffin cells of the bovine adrenal gland. This cell type represents a relevant and accessible model in which to study a particular neuronal nAChR subtype with a well defined function. Previously, we cloned the bovine α3 (6Criado M. Alamo L. Navarro A. Neurochem. Res. 1992; 17: 281-287Crossref PubMed Scopus (68) Google Scholar), α5 and β4 (7Campos-Caro A. Smillie F. Domı́nguez del Toro E. Rovira J.C. Vicente-Agulló F. Chapuli J. Juı́z J.M. Sala S. Sala F. Ballesta J.J. Criado M. J. Neurochem. 1997; 68: 488-497Crossref PubMed Scopus (113) Google Scholar), and α7 (8Garcı́a-Guzmán M. Sala F. Sala S. Campos-Caro A. Stühmer W. Gutiérrez L.M. Criado M. Eur. J. Neurosci. 1995; 7: 647-655Crossref PubMed Scopus (96) Google Scholar) subunits of neuronal nAChRs, which are expressed in chromaffin cells as components of the two nAChR subtypes typically present at the peripheral nervous system (9Conroy W.G. Berg D.K. J. Biol. Chem. 1995; 270: 4424-4431Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). We have also shown that nAChRs formed by α7 subunits are differentially expressed in adrenergic cells (10Criado M. Domı́nguez del Toro E. Carrasco-Serrano C. Smillie F.I. Juı́z J.M. Viniegra S. Ballesta J.J. J. Neurosci. 1997; 17: 6554-6564Crossref PubMed Google Scholar), probably as the result of transcriptional regulation, whereas α3, α5, and β4 subunits have a less restricted distribution in adrenergic and noradrenergic cells (7Campos-Caro A. Smillie F. Domı́nguez del Toro E. Rovira J.C. Vicente-Agulló F. Chapuli J. Juı́z J.M. Sala S. Sala F. Ballesta J.J. Criado M. J. Neurochem. 1997; 68: 488-497Crossref PubMed Scopus (113) Google Scholar). Interestingly, the α3, α5, and β4 subunit genes have been found tightly clustered in the avian (11Couturier S. Erkman L. Valera S. Rungger D. Bertrand S. Boulter J. Ballivet M. Bertrand D. J. Biol. Chem. 1990; 265: 17560-17567Abstract Full Text PDF PubMed Google Scholar) and mammalian (12Boulter J. O'Shea-Greenfield A. Duvoisin R.M. Connolly J.G. Wada E. Jensen A. Gardner P.D. Ballivet M. Deneris E.S. McKinnon D. Heinemann S. Patrick J. J. Biol. Chem. 1990; 265: 4472-4482Abstract Full Text PDF PubMed Google Scholar) genomes, with the α3 and α5 genes contiguous and having opposed transcription polarity. A number of studies have concentrated on the transcriptional regulation of the α3 and β4 subunits. Deneris and co-workers (13Yang X. McDonough J. Fyodorov D. Morris M. Wang F. Deneris E.S. J. Biol. Chem. 1994; 269: 10252-10264Abstract Full Text PDF PubMed Google Scholar, 14Fyodorov D. Deneris E. Mol. Cell. Biol. 1996; 16: 5004-5014Crossref PubMed Scopus (24) Google Scholar) have shown that the POU domain factor SCIP/Tst-1 is able to activate the α3 subunit promoter, probably as a consequence of protein-protein interactions at the level of the basal transcriptional machinery. Furthermore, an enhancer located in the 3′-untranslated exon of the β4 subunit (15Yang X. Yang F. Fyodorov D. Wang F. McDonough J. Herrup K. Deneris E. J. Neurobiol. 1997; 32: 311-324Crossref PubMed Scopus (22) Google Scholar, 16McDonough J. Deneris E. J. Neurosci. 1997; 17: 2273-2283Crossref PubMed Google Scholar), at the β4/α3 intergenic region, activates transcription from the α3 and β4 subunit promoters in a cell type-specific manner, possibly via a novel ETS domain factor, Pet-1, whose expression is almost restricted to the adrenal medulla (17Fyodorov D. Nelson T. Deneris E. J. Neurobiol. 1998; 34: 151-163Crossref PubMed Scopus (50) Google Scholar). Considerable effort has also been dedicated to the transcriptional regulation of the β4 subunit. Thus, Gardner and co-workers (18Du Q. Tomkinson A.E. Gardner P.D. J. Biol. Chem. 1997; 272: 14990-14995Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) have shown that Purα interacts with a 19-bp element in the β4 promoter. In addition, Sp1 (19Bigger C.B. Casanova E.A. Gardner P.D. J. Biol. Chem. 1996; 271: 32842-32848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and Sp3 (20Bigger C.B. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1997; 272: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) transactivate the β4 promoter in a synergistic way, an effect possibly mediated by heterogeneous nuclear ribonucleoprotein K, which affects the transactivation of β4 promoter activity by Sp1 and Sp3 differentially (21Du Q. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1998; 273: 19877-19883Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). As no information has been available until now for the promoter of the α5 subunit, we have chosen to focus on it, with the aim of finding a possible link in the regulation of the three subunits. This study reports that at least five positive regulatory elements exist in the α5 promoter proximal region. These elements, all of them GC boxes, were shown to interact with Sp1. Since the α3 promoter is also the target of Sp1 (22Yang X. Fyodorov D. Deneris E.S. J. Biol. Chem. 1995; 270: 8514-8520Crossref PubMed Scopus (57) Google Scholar), we suggest the possible involvement of this transcription factor in a regulatory mechanism common to the α3, β4, and α5 subunits. A cDNA probe containing 152 bp of the 5′-end of exon 2 was used to screen a bovine genomic library in EMBL-3 SP6/T7 (CLONTECH, Heidelberg, Germany) as described previously (8Garcı́a-Guzmán M. Sala F. Sala S. Campos-Caro A. Stühmer W. Gutiérrez L.M. Criado M. Eur. J. Neurosci. 1995; 7: 647-655Crossref PubMed Scopus (96) Google Scholar). Several overlapping bacteriophage clones were purified and characterized. Poly(A)+ RNA was directly selected from a lysate of bovine adrenal medulla by oligo(dT)-Dynabeads (Dynal, Oslo, Norway) and used in the RNase protection experiments. Probes were generated with SP6 and T7 polymerases (Boehringer Mannheim, Barcelona, Spain), [α-32P]CTP (Amersham Pharmacia Biotech, Madrid, Spain), and the corresponding linearized templates (in the pSPT18 vector, Boehringer Mannheim). A 431-bpBglII-PvuII fragment of the α5 gene that included 163 bp 5′ to exon 1 and 249 bp downstream in the same exon was subcloned into the BamHI and HincII sites of pSPT18. After linearization of the plasmid with EcoRI, a probe of 479 nucleotides was synthesized with SP6 polymerase. As control, a cRNA sense fragment of 368 nucleotides was synthesized by linearizing the same template with HincII and using T7 polymerase. This cRNA protected a fragment of 358 nucleotides upon RNase treatment. Parallel experiments were carried out with a smaller probe that overlapped the first one. For this purpose, a 341-bp fragment of the α5 gene that included 319 bp of the 5′-end of the first probe (downstream of the HincII site mentioned above) was also subcloned into pSPT18. After linearization of the plasmid withEcoRI, a probe of 403 nucleotides was synthesized with SP6 polymerase. The same control sense cRNA used above then produced a protected fragment of 328 nucleotides when used instead of adrenal medulla RNA (see Fig. 2 for further explanations). RNase protection experiments were performed using an RNase protection kit (Boehringer Mannheim) as indicated by the manufacturer. Protected fragments were separated on a 7 m urea and 6% acrylamide gel along with several other labeled RNAs of known size, which were also synthesized and used for calibration. All α5 promoter-luciferase gene fusions were made in the pGL2-Basic vector (Promega, Madison, WI), introducing in its polylinker, upstream of the luciferase gene, the suitable α5 promoter fragments. These fragments were generated with restriction enzymes and directly cloned into pGL2-Basic or subcloned first in pBluescript and then transferred to pGL2-Basic. The vector pGL2-Control, which expresses the luciferase gene under the regulation of the SV40 promoter and enhancer sequences, was used to check luciferase activity. Deletion analysis of the most promoter-proximal region was performed by generating polymerase chain reaction fragments with suitable sense oligonucleotides and an antisense primer (5′-CTTTATGTTTTTGGCGTCTTCC-3′) that anneals to the pGL2-Basic vector downstream of the site of transcription initiation. The basic strategy for site-directed mutagenesis of the different elements in region −111 to −40 of the α5 promoter (see Fig. 6) consisted of the following steps. (a) We performed polymerase chain reaction (25 cycles at 94 °C for 10 s, 62 °C for 30 s, and 68 °C for 45 s) amplification of p111α5LUC (or its single or double mutants when double or quadruple mutants were desired, respectively) with appropriate mutagenic primers in the sense orientation, which generated restriction sites useful for further mutant constructions and to confirm mutagenesis. We used the same oligonucleotide mentioned above as antisense primer. The introduced mutations are indicated in lowercase letters in Fig.6 A (sites 1–4) and Fig. 10 A (site 5). The mutant sequences did not create any known binding site for transcription factors as predicted by the MatInspector data base search (23Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2424) Google Scholar). (b) Polymerase chain reaction products were cloned into pBluescript, sequenced, and transferred to the appropriate construct into the pGL2-Basic vector.Figure 1The 5′-region of the bovine α5 subunit gene. A, the nucleotide sequence of a fragment of genomic clone λα5–21 carrying exon 1 (with the protein sequence indicated underneath initalics), the 5′-end of intron 1 (denoted by anarrowhead), and ∼1400 bp of 5′-flanking sequence is indicated. The translation start codon is underlined, and the major transcription initiation site (position +1) is denoted by thearrow. B, shown is the alignment of the 52- and 42-bp direct repeats in the α5 subunit core promoter region. Potential Sp1-binding sites are underlined. The center of each of these sites is separated by ∼10–11 bp, or a multiple of this number, indicating that they are approximately located on the same side of the DNA, as each turn of B-DNA contains 10.5 bp (see Ref. 37Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 200-203Crossref PubMed Scopus (374) Google Scholar).View Large Image Figure ViewerDownload (PPT)Figure 10Gel mobility shift assays of the fifth Sp1-binding site. A, region –80 and –71 contains an additional putative binding site for Sp1 (underlined andlabeled as 5 in the upper sequence). Several nucleotides of this potential element were mutated, as indicated in the lower sequence, in combination with the previously mutated sites and used as probe (Quintuple Mutant). B, labeled wild-type DNA fragment −111 to −27 and the corresponding quintuple mutant were used as gel mobility shift probes in the presence of 2 μg of crude chromaffin cell nuclear extracts. Competitor (Comp.) DNA fragments corresponding to the wild type (WT; lanes 3 and 4) and the quintuple mutant (QM; lanes 5 and6) were added in 25- and 100-fold excesses. Lanes 1 and 7 were run in the absence of protein (−). • corresponds to the band that was supershifted by Sp1 antibodies (see Fig. 7), whereas the band indicated by ▪ has not been identified.C, the same probes from B were used in the presence of 0.05 (lanes 10 and 13) and 0.2 (lanes 11 and 14) footprint units of recombinant Sp1. Lanes 9 and 12 contained probe run in the absence of protein (−).View Large Image Figure ViewerDownload (PPT) Chromaffin cells were isolated from bovine adrenal glands as described (24Gandı́a L. Casado L.F. López M.G. Garcı́a A.G. Br. J. Pharmacol. 1991; 103: 1073-1078Crossref PubMed Scopus (42) Google Scholar) and cultured in 90% Dulbecco's modified Eagle's medium (Sigma, Madrid), 10% fetal calf serum, 10 μm cytosine arabinoside, and 10 μm 5-fluoro-2′-deoxyuridine (Sigma) to prevent fibroblast proliferation. SHSY-5Y human neuroblastoma cells were grown in 90% Eagle's minimal essential medium with Glutamax-1 (Gibco-BRL, Barcelona) and 10% fetal calf serum. COS cells were grown in 90% Dulbecco's modified Eagle's medium and 10% fetal calf serum. Plasmids were banded in two gradients of CsCl. Both cell types were transfected by the calcium phosphate procedure (25Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6493) Google Scholar). Chromaffin cells on 48-well plates (5 × 105 cells/well) were incubated with 0.75 μg of pGL2 vector or an equivalent amount (in molar terms) of the different constructs derived from this vector and with 0.75 μg of β-galactosidase expression vector pCH110 (Amersham Pharmacia Biotech, Barcelona) as a control of transfection efficiency. SHSY-5Y cells (105 cells/well) or COS cells (5 × 104 cells/well) on 24-well plates were incubated with 1.5 μg of the different α7 constructs and 1.5 μg of pCH110 per well. Cells were harvested after 48 h and lysed with reporter lysis buffer (Promega). β-Galactosidase and luciferase were then determined in the lysates with the corresponding assay systems (Promega). Luciferase activity was normalized to values obtained with the p163α5LUC (see Fig. 3) or p111α5LUC (see Fig. 6) plasmid in the same cell type. When comparing α5, α7, and β4 promoters (see Fig.4), representative constructs for each of the subunits, giving the maximal promoter activity, were chosen. They are indicated in the corresponding figure legends.Figure 4Comparison of the α5 subunit promoter with the SV40 viral promoter and the nAChR α7 and β4 subunit promoters. SHSY-5Y and chromaffin cells were transfected with p111α5LUC, p199α7LUC (see Ref. 50Carrasco-Serrano C. Campos-Caro A. Viniegra S. Ballesta J.J. Criado M. J. Biol. Chem. 1998; 273: 20021-20028Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), p(19)β4 (a deletion construct giving the maximal activity for the bovine β4 promoter (L. M. Valor, A. Campos-Caro, C. Carrasco-Serrano, M. Criado, S. Viniegra, J. J. Ballesta, unpublished results)), and pGL2-Control to compare the activity of the α5, α7, and β4 promoters relative to the SV40 viral promoter. Promoter activity was normalized to values obtained with the pGL2-Control plasmid. Data are expressed as described in the legend to Fig. 3.View Large Image Figure ViewerDownload (PPT) Crude nuclear extracts were prepared from chromaffin and SHSY-5Y cells as described by Schreiber et al. (26Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar). The DNA fragment corresponding to region −111 to −27 was obtained by digesting pBluescript subclones with EcoRI-KpnI and end-labeled by Klenow filling with [α-32P]dATP. The DNA-protein binding reaction volumes were 20 μl containing 10 mm Tris, pH 7.5, 50 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 10% glycerol, 5 μg of bovine serum albumin, 2 μg of poly(dA-dT)·(dA-dT) (Amersham Pharmacia Biotech), 2 μg of nuclear extract protein, and 20,000 cpm of 32P-labeled probe. Reactions were incubated for 10 min at room temperature; the labeled probe was added; and the incubation was continued for an additional 20-min period. For competition studies, the nuclear extract was incubated with the competing probe prior to the labeled probe during 20 min. Supershift assays were performed by preincubating nuclear extracts with 2 μl of antibodies against different transcription factors (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit IgG (Sigma) for 3 h on ice before probe addition. Ten μg of nuclear proteins/lane was separated by 10% SDS-polyacrylamide gel electrophoresis. Western blotting was carried out as described by Towbin et al. (27Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44919) Google Scholar). After the transfer, nitrocellulose membranes were blocked overnight at 4 °C with 5% dry milk in phosphate-buffered saline and incubated in the same way with the anti-Sp1 antibody (1:500) in phosphate-buffered saline and 5% dry milk. After incubation with the secondary antibody at room temperature for 2 h, the bands were visualized by a chromogenic reaction (Sigma Fast, nitro blue tetrazolium, Sigma). The sense strand corresponding to region −111 to −27 of the α5 promoter was end-labeled by Klenow filling with [α-32P]dATP. Assays were performed with the Sure Track Footprinting kit (Pharmacia) according to the manufacturer's instructions. Recombinant Sp1 was incubated with the radiolabeled double-stranded fragment (∼25,000 cpm) using the binding reaction conditions described above in the EMSA experiments (except for the absence of EDTA and the presence of 2.5 mmMgCl2). Immediately following the 30-min incubation at room temperature, 0.5 mm CaCl2 and 1 mmMgCl2 were added to the reactions. This was followed by the addition of 1 unit of DNase I. The reactions were incubated at room temperature for 1 min and stopped with the addition of stop solution (SDS/EDTA). The DNA was prepared for loading onto a 7 murea and 8% polyacrylamide sequencing gel (∼15,000 cpm/lane), and the Maxam-Gilbert A/G chemical sequencing reaction was included as reference ladder. To examine the requirements for α5 subunit transcription, its promoter region was isolated and analyzed. A bovine genomic library was screened, and several overlapping clones were isolated. Clone λα5–21 contained ∼16 kilobases of bovine genomic sequence including exons 1 and 2 and ∼1.6 kilobases of 5′-flanking region. This region was further subcloned and sequenced (Fig.1 A). Comparison of this sequence to a data base of binding sequences of known transcription factors revealed the main features of the promoter/regulatory region of the α5 gene: the lack of a TATA box and the presence of several GC boxes, all of them concentrated into ∼110 bases located 5′ to the transcription initiation site. Two perfect Sp1 consensus sites ((G/T)GGGCGGGGC) were present within this GC-rich region. Additional sites with one mismatch, regarding perfect consensus sequences, were also observed for Egr-1, Ap-1, Oct-1, and Ap-2. It is interesting to note the presence of a contiguous direct repeat of two 52- and 42-bp monomers (Fig. 1 B), each of them containing one of the perfect Sp1 sites and two other elements to which this transcription factor could potentially bind. The 5′-end of α5 mRNA was mapped by RNase protection analyses (Fig. 2). A 479-residue antisense riboprobe (Fig. 2, Probe 1) yielded two main protected fragments of ∼250 and 249 bases. The major one mapped transcription initiation to an adenosine present in a group of three (position +1, black arrow in Fig. 1) and located ∼40 bp downstream of the GC-rich repeats. To improve precision in the determination of the transcription initiation site, a second overlapping probe was used (Fig. 2, Probe 2). In this case, two protected fragments were also observed, which were 157 and 156 bp long and mapped transcription initiation to the same place. Therefore, these were tentatively considered the main transcription initiation sites. Other initiation sites may exist, as this is a typical feature of promoters without TATA boxes, and in fact, minor protected fragments of smaller size and intensity were also observed. A series of constructs was generated to determine the regions of the α5 subunit promoter that contributed to its maximal activity (Fig.3). These constructs were introduced into SHSY-5Y and chromaffin cells, a neuroblastoma cell line and a primary cell culture, respectively, that express the α5 subunit endogenously (7Campos-Caro A. Smillie F. Domı́nguez del Toro E. Rovira J.C. Vicente-Agulló F. Chapuli J. Juı́z J.M. Sala S. Sala F. Ballesta J.J. Criado M. J. Neurochem. 1997; 68: 488-497Crossref PubMed Scopus (113) Google Scholar, 28Lukas R. Norman S. Lucero L. Mol. Cell. Neurosci. 1993; 4: 1-12Crossref PubMed Scopus (156) Google Scholar). In SHSY-5Y cells, the construct containing 111 bp of α5 promoter sequence plus 155 bp of 5′-noncoding region (p111α5LUC) showed the maximal activity. This construct covered all Sp1 sites already mentioned. A shorter construct (p65α5LUC) with only two Sp1 sites showed a 50% decrease in promoter activity, whereas further 5′-deletions that removed all the Sp1 sites (p39α5LUC, p+7α5LUC, and p+56α5LUC) were inactive. In chromaffin cells, results were similar, suggesting that sequences in the minimal promoter, between 111 and 39 bp upstream of the start site of transcription, appear to be critical for basal transcription of the α5 subunit gene in transient transfection assays. Reporter constructs larger than p111α5LUC did not show significant changes in relative luciferase activity when expressed in chromaffin cells. However, in SHSY-5Y cells, a small but constant decrease was observed from one construct to the next larger one, being maximal with p752α5LUC, which showed a marked decrease in activity (36%). The largest construct tested (p1412α5LUC), however, exhibited increased activity (73%). Therefore, in SHSY-5Y but not chromaffin cells, elements predominantly located between –600 and –750 with respect to the transcription initiation site have a negative effect on α5 promoter activity. We next compared the α5 promoter with the SV40 viral promoter, present in the vector pGL2-Control, and the nAChR α7 and β4 subunit promoters (Fig. 4). These subunits are also expressed in chromaffin and SHSY-5Y cells (7Campos-Caro A. Smillie F. Domı́nguez del Toro E. Rovira J.C. Vicente-Agulló F. Chapuli J. Juı́z J.M. Sala S. Sala F. Ballesta J.J. Criado M. J. Neurochem. 1997; 68: 488-497Crossref PubMed Scopus (113) Google Scholar, 8Garcı́a-Guzmán M. Sala F. Sala S. Campos-Caro A. Stühmer W. Gutiérrez L.M. Criado M. Eur. J. Neurosci. 1995; 7: 647-655Crossref PubMed Scopus (96) Google Scholar, 28Lukas R. Norman S. Lucero L. Mol. Cell. Neurosci. 1993; 4: 1-12Crossref PubMed Scopus (156) Google Scholar), probably forming part of the same (β4) or different (α7) nAChR subtype in which α5 is present. In addition, the β4 subunit gene is located in the same gene cluster as the α5 subunit gene (11Couturier S. Erkman L. Valera S. Rungger D. Bertrand S. Boulter J. Ballivet M. Bertrand D. J. Biol. Chem. 1990; 265: 17560-17567Abstract Full Text PDF PubMed Google Scholar, 12Boulter J. O'Shea-Greenfield A. Duvoisin R.M. Connolly J.G. Wada E. Jensen A. Gardner P.D. Ballivet M. Deneris E.S. McKinnon D. Heinemann S. Patrick J. J. Biol. Chem. 1990; 265: 4472-4482Abstract Full Text PDF PubMed Google Scholar), as indicated previously. In chromaffin cells, the maximal nAChR subunit promoter activity corresponded to the α5 subunit, which accounted for about one-half of the activity shown by the SV40 promoter and was 10- and 2-fold higher that the activities of the β4 and α7 promoters, respectively. By contrast, in SHSY-5Y cells, the activities of the three subunit promoters were similar and about one-fifth the activity of the SV40 promoter. Therefore, it appears that some cell-specific differences exist among the three nAChR subunit promoters, which in any case are weaker than the SV40 viral promoter. The α5 promoter constructs already tested in chromaffin and SHSY-5Y cells were also transfected into COS cells in an attempt to find elements that would confer cell specificity. Although this cell line does not express nAChRs, the pattern of promoter activity (Fig.5) was similar to the one found in chromaffin cells. Thus, p111α5LUC showed the maximal activity. A shorter construct (p65α5LUC) with only two Sp1 sites showed a 44% decrease in promoter activity, whereas further 5′-deletions that removed all the Sp1 sites (p39α5LUC, p+7α5LUC, and p+56α5LUC) were inactive. Constructs larger than p111α5LUC did not show significant changes in luciferase activity relative to this construct. Interestingly, whereas in SHSY-5Y and chromaffin cells, the α5 promoter was clearly weaker than the SV40 promoter, present in the pGL2-Control vector, in COS cells, it was ∼2-fold stronger than the viral promoter. Therefore, no cell-specific elements were
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