©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Analysis of Acetylcholine Receptor 3 Gene Promoter Motifs That Bind Sp1 and AP2 (*)

Xiangdong Yang , Dmitry Fyodorov , Evan S. Deneris (§)

From the (1) Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4975

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this study, we performed an analysis of the neuronal nicotinic acetylcholine receptor 3 subunit gene promoter region, -238/+47, to identify cis and trans elements that are important for basal activity in PC12 cells. Sequence analyses of the 3 promoter and footprint assays revealed an Sp1 binding site between -79 and -57 (termed the 3 GA motif) and an AP2 binding site between -30 and -7. Using mobility shift analysis, we found that PC12 cell extracts contain proteins that specifically bind to the 3 GA motif and are immunologically related to Sp1. Mutation of the 3 GA motif, which prevented binding of Sp1, resulted in a 75% decrease in promoter activity. Mutation of the AP2 site resulted in only a minor loss of promoter activity, which is consistent with the lack of AP2 binding activity in PC12 extracts. In Drosophila Schneider line 2 (S2) cell cotransfection assays, Sp1 activated the 3 promoter in a GA motif-dependent manner. Furthermore, multimerization of the GA motif upstream of the -globin TATA box conferred Sp1 responsiveness. Our results indicate that Sp1 can activate transcription through direct interaction with the 3 GA motif and that this motif plays a major role in 3 promoter basal activity in PC12 cells.


INTRODUCTION

Nicotinic acetylcholine receptors (nAchRs)() are excitatory ligand-gated ion channels encoded by a family of at least 15 differentially expressed genes (1, 2, 3, 4) . These genes encode subunits that can be assembled into different heteromeric subtypes, depending on the particular cell-restricted pattern of subunit expression and undefined intracellular rules of permissive subunit-subunit interactions (5) . The nAchR subunit genes 4, 3, and 5 are clustered over an 60-kilobase region of the vertebrate genome (6, 7, 8) . The clustered organization may reflect a mechanism for coordinating cell type and temporal expression of these genes as has been demonstrated for the vertebrate globin genes (9) . Indeed, these clustered genes are coexpressed in many ganglionic neuronal cell populations and the chromaffin tumor line PC12 (6, 7, 10, 11, 12) . In the central nervous system, however, the expression patterns of these genes overlap to some extent but are not entirely concordant (7, 13, 14, 15, 16) . This suggests that within the cluster, gene-specific cis elements also play a role in cell type-specific expression of these genes. We are interested in investigating the mechanisms that coordinately regulate nAchR genes to permit the assembly of different neural restricted subtype heteromers and are focusing on transcriptional regulation of the clustered genes. Because of the clustered organization, the first question we addressed was whether individual promoters are associated with these genes. Analysis of RNA isolated from PC12 cells and sympathetic ganglia indicated that the 3 gene initiates transcription at multiple sites positioned within the 4/3 intergenic region. Transient transfection analysis in these cells indicated a promoter that maps to the 3 start site region (17) . In this report, we have investigated the 3 promoter to identify important cis elements and interacting trans factors in PC12 cells. An Sp1 binding site (referred to as a GA motif) positioned near the start site region was found to play a major role for 3 promoter activity in these cells. In contrast, an AP2 binding site positioned within the start site region had only a small effect on reporter activity. The expression of brain-enriched regulatory proteins that interact with Sp1 binding motifs (18, 19) raises the possibility that the 3 GA motif plays a role in establishing the precise cell-specific pattern of 3 expression in vivo.


EXPERIMENTAL PROCEDURES

Luciferase Reporter Plasmids

The 3 promoter 5` deletion constructs used in this study were prepared with the ExoIII deletion kit from Promega. The resulting 5` boundaries of these deletion constructs were determined by dideoxy sequence analysis (20) .

A site-specific Sp1 binding site mutation was introduced by recombinant polymerase chain reaction into 3 reporter, -1607/+47-luc, to generate mutSp1-luc. A mutagenic oligonucleotide carrying base substitutions 5`-GGT CCG ATG CAT ATG CCA CGA CGG GCG AAC GTC C-3` (residues -78 to -44) was used in a reaction with a downstream primer positioned within the luciferase gene to generate a 385-bp fragment. The second mutagenic oligonucleotide, 5`-TCG TGG CAT ATG CAT CGG ACC ACG GTC TCC-3` (residues -57 to -87), was used in a separate polymerase chain reaction with an upstream primer to generate an 874-bp fragment. Products of the two reactions were gel-purified, heat-denatured, and then annealed by the overlapping complementary 5` ends. The 3` ends were extended, and then products were amplified using upstream and downstream primers. Amplified DNA was cut with Bpu1102I and XhoI and then used in a ligation reaction to replace the Bpu1102I/ XhoI fragment of the -1607/+47-luc vector. Similarly, a site-specific AP2 mutation was introduced into -1607/+47-luc with the following mutagenic oligonucleotides: upper, 5`-CGC CTC CAT GCA TTA CAG CTG CTT GCA GCG CTT G-3` (residues -30 to +4); lower, 5`-GCA GCT GTA ATG CAT GGA GGC GGG GAG GCC-3` (residues -8 to -38). The resulting construct is called mutAP2-luc. To introduce the Sp1 and AP2 double mutation (mutSp1/AP2-luc), an AP2 mutation was introduced into the mutSp1-luc construct using the same mutagenic oligonucleotides described above. Underlined residues above indicate base change sites. All constructs carrying polymerase chain reaction-generated mutations were sequenced across the -238/+47 region to ensure introduction of base changes at only the intended positions.

To prepare a reporter construct, in which the GA motif within the 3 promoter was replaced with SV40 21-bp repeats, the Sp1 footprint region in -1607/+47-luc was substituted with an SV40 promoter Sp1 footprint region by polymerase chain reaction-based mutagenesis. Specifically, ACG CAG GAG ACC GTG GTC CGA CGC CCC TCC CAC GAC GGG CGA A (residues -92 to -50 of 3 promoter) were substituted with CCG CGG CCC CTA ACT CCG CCC ATC CCG CCC CTA ACT CCG CCC A, which contains two 21-bp repeats (one of which is an 18 out of 21 bp match) of the SV40 promoter. The mutagenic primers were the following: upper, 5`-CTC CGC CCA TCC CGC CCC TAA CTC CGC CCA CGT CCC GCC TCG GC-3`; lower, 5`-GGG GCG GGA TGG GCG GAG TTA GGC CCC GCG GCG ATG CCT CGC C-3`. The upstream and downstream primers were the same as those used to prepare mutSp1-luc.

To prepare a promoter construct (4XSp1-TATA-luc) containing multiple copies of the 3 GA motif positioned upstream of the -globin TATA box, two 53-mer oligonucleotides that contain four copies of the -75 to -65 portion of the Sp1 footprint region within the 3 gene were prepared and annealed: upper, 5`-CCG ACG CCC CTC CCA CGC CCC TCC CAC GCC CCC TCC CAC GCC CTC CCA CGT AC-3`; lower, 5`-GTG GGA GGG GCG TGG GAG GGG CGT GGG AGG GGC GTG GGA GGG GCG TCG GGT AC-3`. The resulting double-stranded oligonucleotide was inserted into a luciferase reporter, 20 bp upstream of the -globin TATA box.

Cell Culture

PC12 cells were grown using conditions previously described (17) . Schneider line 2 (S2) cells were grown at 26 °C in Shields and Sang M3 insect medium (Sigma) containing 10% fetal bovine serum (HyClone) that was first heat-inactivated at 56 °C for 30 min.

Transfection

PC12 cells were transfected by electroporation as described previously (17) . S2 cells were transfected with Lipofectin reagent (Life Technologies, Inc.). S2 cells were pelleted and washed twice with M3 medium. 1 10cells were then resuspended in 0.5 ml of M3 medium. For each transfection, 30 µl of Lipofectin and 25 µg of plasmid DNA were aliquoted separately into tubes containing 1 ml of M3 medium. Lipofectin and DNA in M3 medium were then mixed and immediately added to resuspended S2 cells. After 2-4 h of incubation at room temperature, cells were precipitated and washed twice with M3 medium. Cells were then resuspended in 10 ml of complete M3 medium, plated, and kept at 26 °C. After 48 h, cells were precipitated and washed twice with phosphate-buffered saline. Cell extracts were prepared using luciferase cell lysis reagent (Promega). Luciferase assays, -galactosidase assays, and protein assays were performed as described previously (17) .

DNase I Footprinting

Footprinting assays were performed as described previously (17) with 1-6 footprint units of either Sp1 or AP2 protein (Promega).

Electrophoretic Mobility Shift Assay (EMSA)

PC12 cell extracts were prepared essentially as described (21) . Cells were grown to near confluence, harvested by centrifugation, and washed twice with phosphate-buffered saline. All of the following steps were performed at 4 °C. The cells were resuspended in four packed cell volumes of 10 m M Tris-HCl (pH 7.9), 1 m M EDTA, and 5 m M dithiothreitol. After 20 min, the cells were lysed by homogenization in a Dounce homogenizer with eight strokes using a ``B'' pestle. Four packed cell volumes of 50 m M Tris-HCl (pH 7.9), 10 m M MgCl, 2 m M dithiothreitol, 25% sucrose, 50% glycerol were added, and the suspension was gently mixed. With continued gentle stirring, 1 packed cell volume of saturated (NH)SOwas added dropwise. The lysate was gently stirred for an additional 30 min, followed by centrifugation at 45,000 rpm in a Beckman L7-55 rotor for 3 h. Protein was precipitated from the supernatant by the addition of ammonium sulfate (0.3 g/ml) and neutralized with 1 M NaOH (0.1 ml/10 g of solid (NH)SO). The precipitate was resuspended (5% of the volume of the high speed supernatant) in 25 m M HEPES (pH 7.9), 100 m M KCl, 12 m M MgCl, 0.5 m M EDTA, 2 m M dithiothreitol, 17% glycerol and dialyzed against resuspension buffer for 8-12 h. The dialyzed material was then aliquoted and stored at -80 °C until needed.

EMSA was performed with P-end-labeled double-stranded oligonucleotides that were incubated with PC12 cell extract or either Sp1 or AP2 protein in EMSA buffer (15 m M HEPES (pH 7.1), 60 m M KCl, 1.2 m M dithiothreitol, 7.2 m M MgCl, 0.3 m M EDTA, 10% glycerol, 0.1 mg/ml poly(dIdC)). For oligonucleotide competition analysis, a 150-600-fold molar excess of competitor oligonucleotides was also added to the mixture. After 30 min at 30 °C, the mixture was chilled on ice for 5 min before analysis on polyacrylamide gels. For some EMSAs, 1-3 µl of anti-Sp1 or anti-AP2 antibody (Santa Cruz Biotechnology, Inc.) was added to the mixture. The reaction was then incubated at 4 °C for 2 h. DNA-protein complexes were fractionated on 5% polyacrylamide gels in 1 Tris-glycine buffer (50 m M Tris base, 380 m M glycine, and 2 m M EDTA) at 4 °C. Free probe and DNA-protein complexes were visualized by autoradiography using Kodak XAR-5 film.


RESULTS

We showed previously that the activities of 3 promoter constructs -1607/+47-luc and -238/+47-luc did not differ significantly when the constructs were transfected into PC12 cells (17) . Luciferase activity of each of these reporters was 20% of that obtained from the SV40 promoter. Because the -238/+47 contains the entire 3 multistart site region, these results together suggest that 3 promoter elements important for activity in PC12 cells are positioned within this G + C-rich fragment. To determine the position of these elements we quantitated the reporter activity of a set of deletions spanning the -238/+47 segment of the 3 gene and upstream region. As shown in Fig. 1, deletion to -77 caused only a small decrease in reporter activity. However, a large decrease (80% of -238/+47) in promoter activity was observed in a reporter in which sequences upstream of -36 were absent. Deletion of sequences to +30 resulted in background reporter activity. These results indicate the presence of an important cis element within segment -77/-36.


Figure 1: Analysis of 3 promoter deletions. Deletion constructs are shown relative to the -238/+47 portion of the 3 upstream region. The dashed line indicates the transcription initiation region. Relative luciferase activities were obtained after correction for transfection efficiency with a co-transfected RSV-gal plasmid. Error bars represent mean ± the range from duplicate transfections.



Transcription Factors Sp1 and AP2 Bind to the 3 Promoter

Positioned within segment -77/+30 are sequence motifs that are either perfect or single base mismatches to the consensus binding sites for transcription factors Sp1 (22) and AP2 (23) . To determine whether or not any of these consensus sequences were genuine Sp1 or AP2 binding sites, we performed footprint analyses using pure Sp1 and AP2 proteins, respectively (Promega). As shown in Fig. 2, these proteins produced single nonoverlapping footprints near the multistart site region. The strong footprint produced by Sp1 was positioned between -79 and -57, which is adjacent to the 5` boundary of the start site region. Inspection of the Sp1-protected segment revealed a core sequence, 5`-CCCCTCCC-3`, that is a perfect match to the previously determined Sp1 consensus binding motif (24) . Moreover, this motif is reminiscent of an inverted GA box, which was previously characterized as an Sp1 binding motif in various promoters such as JC virus early promoter (25) and insulin-like growth factor-binding protein-2 promoter (26) . Thus, we refer to this Sp1 binding site as an 3 GA motif. AP2 protected a segment, -30/-7, positioned within the transcription start site region (Fig. 2 B). This footprint included a core sequence with a single base mismatch to the AP2 consensus site derived from analysis of the SV40 promoter (23) .

Sp1 but Not AP2 Binding Activity Is Detectable in PC12 Cells

The zinc-finger protein, Sp1, has been implicated as an important trans-acting factor for numerous genes (24) . Recent studies indicate that Sp1 is one of several members of a differentially expressed gene family (18, 27, 28) . Therefore, we were interested in determining whether PC12 cells express Sp1 or Sp1-like proteins that bind the 3 GA motif. We prepared PC12 cell extracts to perform EMSA with an oligonucleotide probe (Fig. 3 A) containing the entire Sp1 footprinted region (Sp1). As shown in Fig. 3 B, two major protein complexes (C1 and C2) were formed on Sp1. C1 had the same mobility as the complex formed by recombinant Sp1 protein (Fig. 3 C). These complexes were specific because unlabeled Sp1inhibited complex formation, but an oligonucleotide in which the 3 GA motif was mutated (mutSp1) did not. The formation of two major complexes, C1 and C2, on an Sp1 binding site is similar to Sp1-containing complexes reported by others (26, 29, 30, 31) .


Figure 3: Detection of GA motif binding activity in PC12 cell extracts. A, the middle strand represents a portion of the 3 promoter, which contains the GA motif ( overline). The upper and lower sequences represent Sp1probe and mutant Sp1oligonucleotide (mutSp1), respectively, used in EMSAs presented in B and C. Substituted bases of mutSp1are indicated by asterisks. B, PC12 cell extracts were incubated with Sp1probe in the absence or presence of increasing amounts of unlabeled double-stranded competitors. Lane 1, probe alone; lanes 2-8, 8 µg of PC12 cell extract; lanes 3-5, 150-, 300-, and 600-fold molar excess of Sp1competitor; lanes 6-8, 150-, 300-, and 600-fold molar excess of mutSp1competitor. Major complexes are indicated as C1 and C2. C, Sp1probe was incubated with either 48 ( lanes 2-4) or 8 µg ( lanes 8-10) of PC12 cell extract ( CE) or 1 footprint unit of human Sp1 protein ( lanes 6-7) in the presence of 0.1 µg ( lanes 3, 7, and 9), 0.2 µg ( lane 4), or 0.3 µg ( lane 10) of anti-Sp1 polyclonal antibody ( Ab Sp1). As discussed under ``Results'', lower molecular weight complexes indicated by asterisk in B and C may represent degradation products of C1 and/or C2 or uncharacterized intact proteins.



To determine whether these complexes contained Sp1 and/or Sp1-related proteins, anti-Sp1 antibody was incubated with the extract and probe mixture. Addition of anti-Sp1 antibody reduced the intensity of both C1 and C2 and resulted in the appearance of a larger complex that did not enter the gel, suggesting that Sp1 is present in C1 and C2. (Fig. 3 C, lanes 1- 4). The inability to completely supershift C1 and C2 could result from insufficient anti-Sp1 antibody or the presence of other Sp1-related molecules in addition to Sp1 in the complexes. To distinguish between these two possibilities, we adjusted the amount of extract so that the level of C1 formed would be similar to the level of the complex containing recombinant Sp1. As shown in Fig. 3 C ( lanes 5-10), 0.1 µg of the antibody completely supershifted the complex formed by pure Sp1, but only a partial supershift of C1 and C2 was observed with 0.3 µg of the antibody. Therefore, C1 and C2 are likely to contain an additional Sp1-related protein(s). We also noted the presence of additional higher mobility complexes (Fig. 3, B and C, asterisks) that were specifically competed by unlabeled Sp1but were not supershifted (Fig. 3 C, asterisk); therefore, these represent either degradation products of Sp1 that lack the epitope recognized by anti-Sp1 antibody or distinct proteins unrelated to Sp1.

The transcription factor AP2 has been shown to be widely expressed in neural crest cell lineages during mouse embryogenesis (32) . Because the PC12 line is derived from cells arising from neural crest and because we identified an AP2 binding motif in the 3 promoter, we next investigated whether PC12 cells express proteins that bind this motif. We radiolabeled an oligonucleotide (AP2probe) corresponding to the AP2 footprint region and incubated it with pure AP2 in the absence or presence of either unlabeled competitor AP2or a mutated AP2(mutAP2) oligonucleotide (Fig. 4 A). As shown in Fig. 4 B, formation of AP2 complexes was almost completely inhibited by incubation with excess unlabeled AP2oligonucleotides but not with an equivalent amount of mutAP2oligonucleotides. These oligonucleotides were then used to determine whether we could detect AP2 binding activity in PC12 cell extracts. Unexpectedly, we did not detect AP2 immunoreactive binding activity to the AP2 motif in these extracts.


Figure 4: AP2 protein is not detectable in PC12 cell extracts. A, middle strand represents a portion of the 3 promoter that contains the AP2 motif ( overline). The upper and lower sequences represent AP2probe and mutant AP2oligonucleotide (mutAP2), respectively, used in EMSAs presented in B and C. Substituted bases of mutAP2are indicated by asterisks. B, EMSAs were performed with AP2probe and AP2 protein in the absence or presence of increasing amounts of indicated competitors. Lane 1, probe alone; lanes 2-8, 0.5 footprint units of AP2 protein; lanes 3-5, 150-, 300-, and 600-fold molar excess of unlabeled AP2competitor; lanes 6-8, 150-, 300-, and 600-fold molar excess of unlabeled mutAP2competitor. C, AP2probe was incubated with either AP2 protein (1 footprint unit) or PC12 cell extract (48 µg) in the presence of 0.1 ( lanes 3 and 5) or 0.3 µg ( lane 6) of anti-AP2 antibody. The asterisk indicates a very low abundance of uncharacterized complexes that migrate much faster than the AP2-containing complexes.



Transfection Analysis of GA and AP2 Motif Mutations in the 3 Promoter

To determine whether the 3 GA motif constitutes an important cis element in the 3 promoter, we prepared mutated 3 reporters in which wild type GA motif sequences were changed to those of mutSp1(Fig. 3 A). As shown in Fig. 5, this mutated construct (mutSp1-luc) directed luciferase activity that was 25% of that obtained from the wild type construct (-1607/+47-luc). This result, which is consistent with deletion analysis (Fig. 1), demonstrates that the GA motif is critical for 3 basal activity in PC12 cells. We also replaced the Sp1 footprinted region of 3 with two copies of the SV40 early promoter 21-bp repeats, which contain multiple Sp1-binding GC boxes. Transfection of this construct into cells resulted in an 2-fold increase in promoter activity (Fig. 5, SV40-Sp1-luc), presumably because more Sp1 can bind to the promoter. Thus, 3 promoter function may be rate-limited by the level of GA motif binding activity in PC12 cells.


Figure 5: Transfection analysis of GA and AP2 motif mutations. PC12 cells were transfected with 10 µg of luciferase reporters driven by either 3 wild type promoter (-1607/+47-luc) or reporter constructs containing mutated GA motif (mutSp1), mutated AP2 site (mutAP2), or both (mutSp1/AP2). SV40-Sp1 represents a reporter construct in which the 3 Sp1 footprint region was substituted with the 21-bp repeat region of the SV40 promoter. no insert, pGL2 basic vector. PC12 cell extracts were prepared 2 days after electroporation. Relative luciferase activities were obtained as described in the legend to Fig. 1. Error bars represent mean ± the range from duplicate experiments.



Given the data presented in Figs. 1 and 4, a mutation within the AP2 binding site would not be expected to substantially alter 3 promoter activity. To test this idea, we mutated the AP2 binding site to generate mutAP2-luc and compared its activity with that of the wild type 3 promoter. As shown in Fig. 5the activity of mutAP2-luc was 30% less than the activity obtained with -1607/+47-luc. To confirm the results of the individual mutations, we prepared an 3 reporter (mutSp1/AP2-luc) in which both the GA motif and AP2 motif were mutated. The activity of this construct was slightly lower than that of mutSp1-luc. These results, together, provide strong evidence for a major contribution of the GA motif to 3 promoter activity and only a minor, if any, contribution of the AP2 motif (Fig. 5). Because the AP2 site is positioned within the 3 transcription start site region, the small but reproducible decrease in 3 promoter activity observed with mutations in the AP2 motif may have resulted from mutation of a subset of basal elements.

Sp1 Can Transactivate the 3 Promoter and a Heterologous Promoter via Direct Interaction with 3 GA Motifs

We have shown that Sp1 and/or an Sp1-related protein can bind to a GA motif in the 3 promoter. Furthermore, the GA motif was found to be critical for 3 promoter activity, which suggests that Sp1 transactivates the 3 promoter upon binding to this motif. To demonstrate this directly, we performed cotransfection assays in the Drosophila Schneider line, S2, which does not express endogenous Sp1. Different quantities of an expression vector, pPacSp1 (33) , driving the synthesis of Sp1 RNA were introduced into these cells together with either -1607/+47-luc or mutSp1-luc. As shown in Fig. 6 A, mutation of the 3 GA motif reduced transactivation by Sp1, which shows that Sp1 can modulate the 3 promoter upon binding to this motif. Activation by coexpressed Sp1, however, was not completely abolished by mutation of the GA motif. The residual activation may result from binding of overexpressed Sp1 to low affinity sites within the G + C-rich 3 promoter.


Figure 6: GA motif-dependent transactivation by Sp1. A, Drosophila Schneider S2 cells were co-transfected with 10 µg of either -1607/+47-luc ( open squares) or mutSp1-luc ( solid triangles), 5 µg of RSV-gal plasmid, and indicated amounts of pPacSp1. Cell extracts were prepared 48 h later. Luciferase activities were corrected for -galactosidase activity. Fold activation was calculated as the activity of each reporter in the presence of the indicated amount of pPacSp1 divided by the activity of the reporter without effector. B, Sp1 transactivation of a luciferase reporter containing zero ( solid triangles) or four copies (open squares) of the 3 Sp1 footprinted region positioned 20 bp upstream of the -globin TATA box. Cell extracts and luciferase activities were obtained, and -fold activation was calculated as described in A.



To determine whether Sp1 can transactivate a heterologous minimal promoter via the 3 GA motif, we prepared a luciferase reporter in which four copies of the GA motif were positioned upstream of a -globin TATA box. The resulting construct (4XSp1-TATA-luc) was then cotransfected into S2 cells with various amounts of pPacSp1 effector. The results shown in Fig. 6 B indicate that the activity of 4XSp1-TATA-luc was increased in a dose-dependent manner by coexpression of Sp1. Transactivation was entirely dependent upon the presence of the GA motifs, because no transactivation was observed from a -globin TATA reporter in which GA motifs were absent. These results demonstrate that the GA motif can mediate transactivation by direct interaction with Sp1 within the context of the 3 promoter and a heterologous promoter.


DISCUSSION

In this and a previous report (17) we identified cis and trans elements important for nAchR 3 gene promoter activity in PC12 cells. Positioned adjacent to or within the start site region are high affinity binding sites for transcription factors Sp1 and AP2, respectively. The Sp1 footprinted region contains an upper strand core sequence, 5`-CCCCTCCC-3`, that is reminiscent of Sp1 binding motifs (termed GA boxes) that are present within the promoters of several other genes (25) . Transient transfection analysis of deletions and point mutations downstream of -1607 indicated that the 3 GA motif was the only major 3 cis element we could detect outside the transcription start site region. It is possible, however, that closely positioned positive and negative elements went undetected by our set of deletions. Cotransfections into Schneider S2 cells demonstrated that the 3 GA motif is clearly capable of mediating transactivation by Sp1, because mutation of the GA motif reduced Sp1 transactivation of 3 reporters, and multimerization of the GA motif upstream of the -globin TATA box resulted in GA motif-dependent and dose-dependent activation by Sp1. Analysis of the AP2 motif indicated that its contribution to 3 promoter activity in PC12 cells is small. Based on the analyses in PC12 cells presented here and previously (17) , we propose that the 4/3 intergenic region contains a single 3 TATA-less promoter composed of a multistart site region, a GA motif that mediates transactivation by Sp1 and/or Sp1-related proteins, and an AP2 motif of undefined function (Fig. 7). Further analyses will be required to determine the role of these elements for tissue-specific expression of 3 in vivo and also the position of additional 3 cis elements outside the -1607/+47 region.


Figure 7: Schematic of rat nAchR 3 promoter elements. Transcriptional orientation and relative positions of clustered neuronal nAchR genes are indicated by large arrows. Position and partial sequence of the 3 promoter in the rat 4/3 intergenic region are shown. Cis elements that bind Sp1 and AP2 are overlined. Dashed line indicates transcription start site region relative to the 5` end of cDNA, PCA48 (17).



Mobility shift assays were used to identify proteins in PC12 cells that could bind the 3 GA motif. Using the entire Sp1 footprinted region as probe (Sp1), we detected the formation of two major complexes, C1 and C2, that were specifically competed by unlabeled Sp1and that were both supershifted with a polyclonal antibody raised against a human Sp1 epitope. The mobility of C1 was indistinguishable from that formed by pure human Sp1 on Sp1, suggesting that C1 contains Sp1. Because Sp1 is known to be post-translationally modified (34) , C2 may represent a modified form of proteins present in C1 or a degradation product.

Neither C1 nor C2 was completely supershifted by anti-Sp1 antibody. The inefficient recognition of C1 and C2 by anti-Sp1 antibody is not likely to result from species-specific differences in epitopes, because the peptide used to prepare the polyclonal antibody is perfectly conserved between rats and humans. A more likely possibility is that C1 and C2 contain additional Sp1-related proteins that are very similar in size to Sp1. This idea is supported by the discovery of a family of differentially expressed genes encoding Sp1 and the related proteins, Sp2, Sp3, and Sp4 (18, 27, 28) . These proteins bind to DNA with similar specificities and affinities. The molecular weights of Sp1, Sp3, and Sp4 are also very similar, and thus complexes formed by these proteins on DNA are not readily distinguishable by electrophoresis (18, 27) . Sp1, Sp2, and Sp3 are is considered to be ubiquitously expressed (18, 35) , although the level of Sp1 expression varies at least 100-fold in different tissues (35) . In contrast, expression of Sp4 is much more limited; Sp4 RNA is abundant in brain but cannot be detected in several other tissues (18) .

Promoters for a number of other neural restricted ligand-gated ion channel genes have been characterized, and comparison of these to the 3 promoter reveals notable similarities (36, 37, 38, 39, 40, 41) . First, at least two transcription initiation sites have been found in the promoter regions of the GABAR 3 and , nAchR 2 and 7, and NMDAR1 genes. For most of these promoters initiation occurs at multiple nonadjacent sites that are positioned within a G + C-rich region several tens of nucleotides in length. Second, consistent with multiple start sites none of these promoters contains sequences that would indicate a TATA box. Third, Sp1 consensus motifs are often found within these promoter regions, although a direct interaction of Sp1 with these motifs has been demonstrated only for nAchR 3 and the GABAR 3 (36) promoter. These common features are often referred to as those of housekeeping genes, because many ubiquitously expressed genes contain similarly structured promoters. However, as is apparent from the genes listed above, these are also features of neural restricted genes. This suggests that these genes are controlled by constitutive promoters whose activity is modulated by sequence-specific enhancer and/or silencer binding proteins to produce restricted patterns of expression. Indeed, evidence for this type of mechanism has been demonstrated for the neural restricted SCG10 and type II sodium channel genes (42, 43, 44, 45, 46) . These genes contain housekeeping-type minimal promoters that by themselves lack the cis information required for proper cell type-specific activity. To achieve appropriate cell type-specific control, silencer motifs that are positioned upstream of the promoters and that bind factors present in SCG10-negative and type II sodium channel-negative tissues are required. Thus, based on the structural features of the 3 promoter it is expected that additional cis elements, such as silencers, influence 3 promoter activity to restrict its in vivo pattern of transcription.

Although appropriate expression of SCG10 and type II sodium channel genes is critically dependent on interaction of distant silencer elements with repressors, the expression of brain-enriched factors that interact with common Sp1 binding motifs ( e.g. GA motif) raises the possibility that the minimal promoters of neural restricted genes that contain these motifs contribute to tissue-specific expression. One such example of a brain-enriched factor, noted above, is the Sp1-related factor, Sp4 (18) . Another example is a factor that interacts with the promoter of the tissue plasminogen activator gene. The tissue plasminogen activator gene is abundantly expressed in brain and is controlled by a promoter that is more active in the presence of brain-derived nuclear extracts than in those obtained from liver or kidney. Analysis of tissue-specific elements in the tissue plasminogen activator promoter revealed GC boxes that bind both an Sp1-like factor and a brain-enriched factor (19) . If 3 promoter activity can be modulated by the level of Sp1 factors, as is indicated here by substitution of the GA motif with the SV40 promoter 21-bp repeats and cotransfection assays in S2 cells, then increased neuronal expression of brain-enriched factors that bind Sp1 motifs may modulate cell-specific transcription of the 3 gene. Given the apparent diversity of brain-enriched regulatory proteins that interact with Sp1 binding motifs and the low level of Sp1 expression in the brain relative to other tissues (35) it will be important to determine the neuronal transcription factors that interact with the 3 GA motif and perhaps play a role in establishing the precise pattern of 3 expression in vivo.


FOOTNOTES

*
This work was supported by Grant RO1 NS29123 from the National Institutes of Health, Grant 3090R1 from the Council for Tobacco Research, and Grant 91014080 from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Neuroscience, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4975. Tel.: 216-368-8725; Fax: 216-368-4650; E-mail: esd@po.cwru.edu.

The abbreviations used are: nAchR, nicotinic acetylcholine receptor; bp, base pair(s); EMSA, electrophoretic mobility shift assay.


ACKNOWLEDGEMENTS

We thank John Incardona and Dr. Terry Rosenberry for providing S2 cells and a Lipofectin transfection method for S2 cells, and we thank Dr. R. Tjian for pPacSp1.


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